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department of energy 's ( doe 's ) complex ; uranium contamination evokes particular environmental concern due to the high solubility and mobility of its oxidized form , u(vi ) . as physically removing the contaminated material is financially prohibitive , we need innovative , cost - effective in situ stabilization technologies that exploit the processes of natural attenuation . recently , researchers at some doe sites have assessed the microbial stabilization of actinides ( u , pu , and np ) and fission products ( tc ) in subsurface environments , as in the uranium mill tailing remedial action ( umtra ) site in rifle , co ( http://www.gjem.energy.gov/moab/ ) and at the oak ridge field research centre ( orfrc ) at oak ridge , tn ( http://www.esd.ornl.gov/orifrc/ ) . a wide variety of bacteria , including desulfovibrio , geobacter , and shewanella , can couple the oxidation of organic compounds to the reduction of u(vi ) and thus reductively precipitate uranium in its reduced form u(iv ) [ 13 ] . investigators have explored the mechanisms of uranium(vi ) reduction by anaerobic bacteria [ 46 ] . they are gram - positive , spore - forming bacilli found in soils , sediments , wastes , and in the normal intestinal flora of humans and animals . the fermentation industry uses them widely to produce solvents and biofuels , including acetone , ethanol , butanol , and hydrogen [ 8 , 9 ] . some members of the class also are capable of non symbiotic nitrogen ( n2 ) fixation , and others can reductively synthesize acetate from carbon dioxide ( co2 ) via the wood - ljungdahl pathway . unlike those anaerobic respiratory bacteria , clostridia classically were viewed as obligate anaerobic , fermentative bacteria , although recent evidence showed that some strains of this group are tolerant of oxygen to some extent [ 12 , 13 ] . previously , it was showed that clostridium sp . bc1 ( atcc 53464 ) and c. sphenoides ( atcc 19403 ) can reduce uranyl - nitrate , -acetate , and -citrate complexes , as well as complexes of other metals [ 1416 ] . we then argued that if the reduction of uranyl compounds could be a general property of clostridia . we have further found that not only is this ability common among more clostridia species , but that also the strains differed in the extent of their capability . the ph of the culture significantly affected uranium(vi ) reduction , with ph 5 - 6 being the optimal one in most cases . among the strains tested , clostridium sp . apparently , the evidence suggested that clostridia is one of the major players in uranium reduction in situ in an acidic ( ph = 4 ) uranium - mine pit water [ 18 , 19 ] , at a military facility near chesapeake bay , maryland , united states , as well as at the orfrc at oak ridge , tn . in this study , using hplc and gc techniques , we have continually characterized metabolic properties of those clostridial strains active in uranium reduction under fermentation conditions and discussed their effects on performance of uranium reduction . we also demonstrated that hydrogen metabolism could play an important role in uranium(vi ) and iron(iii ) reduction by clostridia . in the end , we proposed a comprehensive model to explain molecular mechanisms of uranium reduction by clostridia and its relationship to the overall metabolism especially hydrogen ( h2 ) production . we purchased c. sphenoides ( atcc 19403 ) , c. acetobutylicum ( atcc 824 ) , and c. pasteurianum ( atcc 7040 ) from the american type culture center ( atcc ) . all strains can ferment glucose , but only c. sphenoides can metabolize citric acid as the sole carbon and energy source . all strains were grown anaerobically and maintained in the mineral salts medium , herein called msm medium , containing per liter : glucose 5.0 g , nh4cl 0.5 g , glycerol phosphate 0.3 g , mgso47h2o 0.2 g , cacl22h2o 0.5 g , peptone 0.1 g , and yeast extracts 0.1 g , 6.25 ml of 1.6 mm feso47h2o , at ph 6.8 . we dissolved all the ingredients except feso4 to about 1000 ml in deionized water in a 2000 erlenmeyer flask and prereduced by boiling and purging with nitrogen gas for 20 minutes . after allowing the solution to cool , we placed the flask in an anaerobic glove box ( 95% n2 , 5% h2 ) and then added to the medium 6.25 ml of 1.6 mm prereduced feso47h2o ( prepared by dissolving 90 mg ferrous sulfate in 200 ml of prereduced deionized water and adding 0.5 ml concentrated hcl ) and made up the total volume to 1000 ml with prereduced deionized water . the ph was adjusted to 6.8 before dispensing 40 ml aliquots of the medium into 60 ml serum bottles , fitted with butyl rubber stoppers , then crimp - sealing them with aluminum caps before autoclaving . c. sphenoids ( atcc19403 ) was also cultured in simmons citrate medium ( scm ) ; all materials and their preparation were exactly the same as for the msm medium , except that we substituted glucose with 8.2 g sodium citrate . to stabilize the ph of the culture , as needed , we added 50 mm pipes ( ph 6.8 ) or 50 mm mes ( ph 6.2 ) to the medium . the bacterial cultures were grown at 26c in the dark on a rotary shaker agitated at 100 rpm . total gas production in the headspace of the sample was measured by a pressure transducer with a needle ( model 665-d/030 , wallace and tiernan ) . we removed four - milliliter aliquots of the culture to determine bacterial growth , change in ph , and production of organic acid metabolites . the growth of the bacterium was measured by recording the turbidity of the medium at 600 nm , using a bausch and lomb spectronic 20 spectrophotometer . the u(vi)-nitrate ( uranyl nitrate ) stock solution ( 0.5 m ) was made by dissolving solid uo2(no3)2 into prereduced deionized water . the exact concentration of uranium(vi ) stock was calibrated using a kpa machine ( kinetic phosphorescence analyzer , chemchek instruments , inc . ) that was manipulated according to the manufacturer 's instruction . for the 10 mm u(vi)-nitrate stock solution , 5 ml of prereduced deionized water was added to a beaker along with 0.25 ml of 0.5 m u(vi)-nitrate , the ph adjusted to 6.1 with naoh , and the solution diluted to 10 ml for a final concentration of 10 mm . the complexes were stored in the dark and then readjusted to ph 6.1 after 24 hours , and filter - sterilized ( 0.45 m ) into a vacutainer tube . 10 mm u(vi)-citrate stock solution was made by mixing the 0.5 m u(vi)-nitrate solution with citrate acid at the molar ratio of 1 : 1 as described below . briefly , 0.5 ml of prereduced citric acid solution ( 200 mm ) was added to a beaker in the gloved box along with 0.2 ml of 0.5 m u(vi)-nitrate . the ph was adjusted to 6.1 with prereduced naoh and diluted to 10 ml for a final concentration of 10 mm . ten milliliters of the culture at the late - log phase was transferred to anaerobic preautoclaved serum bottle ( 20 ml ) , and then 0.1 ml of 10 mm u(vi)-nitrate solution added via a 1-ml syringe with a needle . to determine the u(vi ) concentration over time , aliquots of 0.1 to 0.2-ml of the bacterial culture were taken from the serum bottle , 5 l of the culture diluted in 2 ml deionized water and immediately analyzed for u(vi ) by kpa . to assess the effect of ph on u(vi ) reduction by clostridia , ten milliliter aliquots of the late - log growth phase of the bacterial culture were adjusted to the required ph values using 1 n naoh or hcl . the uv - vis ( ultraviolet - visible ) spectrophotometry was used for obtaining absorption spectra of both uranium(vi ) and uranium(iv ) . a hewlett packard model the sample was prepared as follows : after completing the uranium reduction assay described above , the total leftover culture in serum bottle was collected into a centrifuge tube in the glove box . then , its ph was adjusted to 11 using 1 n naoh , and the culture centrifuged at 10000 rpm for 10 minutes , the supernatant discarded , and the pellet was resuspended in 2-ml 10 mm citric acid solution to extract the uranium species . the solution was filtered through a 0.45 m membrane and then the filtrate was analyzed by uv - vis spectrophotometry to determine the absorption spectra of both uranium(vi ) and uranium(iv ) . the fermentation products , including organic acids and gases , were analyzed by high performance liquid chromatography ( hplc ) and gas chromatography ( gc ) . the hplc unit consisted of a shimadzu scl-10a system controller , a sil-10a autoinjector , and a lc-10as liquid chromatograph . the culture sample , filtered through a 0.45 m filter , was analyzed by hplc for organic acids using an spd-10a uv - vis detector at 210 nm ; for glucose , we employed a rid-6a refractive index detector ( shimadzu ) . a sri 8610c gas chromatograph fitted with a thermal conductivity detector was used to analyze the h2 produced through fermentation . we determined the rate of growth , changes in ph of the media , and gas production of the four clostridia strains cultured in msm or scm . we also analyzed the extent of consumption of the carbon source and the metabolic products from the strains . figure 1 shows the highest cell density ( i.e. , rate of growth ) in clostridium sp . ; this strain also generated the most gas , even after entering stationary phase , while all other strains stopped producing gas at the stationary phase . this drop probably reflected the production of organic acids ( the drop might be caused also by changing the activity of h - atpase ) . gc analysis of the headspace gas showed that the maximum percentage of hydrogen ( h2 ) produced by clostridium sp . , c. acetobutylicum , c. pasteurianum , and c. sphenoides , respectively , was about 75% , 60% , 45% , and 25% ( figure 1 ) . growth of c. sphenoides in scm medium containing citrate as the carbon source and comparing it with that in msm medium revealed no significant differences in its maximum cell density and gas production ( figure 1 ) . however , the medium 's ph slightly increased from near neutral to around ph 7.8 , due to the concurrent consumption of citric acid ( figure 1 ) . we tested alternative carbon sources including sucrose and glycerol and found that neither improved the growth of these strains ( data not shown ) . however , growing the clostridia in media supplemented with a ph buffer to stabilize ph alleviated the drop in ph due to the production of organic acid during glucose fermentation ( figure 2 ) . when cultured in msm medium supplemented with 50 mm pipes ( ph 6.8 ) , ph of the medium reached around 5 to 6 by the late - log phase of growth for clostridium sp . , c. acetobutylicum , and c. pasteurianum ; supplementation with 50 mm mes ( ph 6.2 ) caused the final ph of these cultures to reach 4 to 5 . furthermore , the ph - buffered medium affected the growth kinetics and final cell density of the strains . compared to growth in msm medium without ph buffer , clostridium sp . grew much slower initially when the medium was buffered to ph 6.8 , although growth later accelerated and it reached the stationary phase at the same time ( 40 hours ) and at the same optimal density ( od600 nm = 0.8 ) . in contrast , clostridium sp . grew much faster in the medium buffered to ph 6.2 , reaching the stationary phase 20 hours earlier at the same optimal density ( od600 nm = 0.8 ) ( figure 2 ) . the growth of c. acetobutylicum was higher in medium buffered at ph 6.2 , and it attained the stationary stage in about 10 hours earlier than that of unbuffered medium . by contrast , in msm buffered to ph 6.8 , like clostridium sp . , its growth was much lower at the first 15 hours ; thereafter , it reached the same level as that in unbuffered culture . addition of buffers did not result in an increase in final cell density ( figure 2 ) . the growth of c. pasteurianum increased in the ph 6.8 and 6.2 adjusted buffered medium ( figure 2 ) . the similar trends also occurred in the production of total gas by these strains ( figure 2 ) . analysis of culture medium by hplc showed that all these strains produced acetic acid and butyric acid ( figure 3 ) . glucose consumption and production of the organic acids were influenced by the initial ph of the medium ; buffered medium at ph 6.2 showed the rapid glucose consumption and production of acetic and butyric acids . the maximum consumption of glucose was concurrently accompanied by reaching the production peak of butyric acid in all strains assayed . in the case of clostridium sp . , glucose consumption reached 100% completion at 50 , 30 , and 40 h in unbuffered , buffered medium at ph 6.2 and 6.8 , respectively . concurrently , butyric acid production attained its peak at the same time point as that of 100% glucose consumption , and the highest concentration measured was 13.5 , 11.5 , and 10.5 mm in unbuffered , buffered medium at ph 6.2 and 6.8 , respectively . by contrast , acetic acid production reached its peak at 40 , 30 , and 30 h and the corresponding concentration measured was 8.5 , 14 , and 9.5 mm , in unbuffered , buffered medium at ph 6.2 and 6.8 , respectively . similarly , in the case of c. acetobutylicum , maximum consumption of glucose was reached at 50 , 26 , and 40 h in unbuffered , buffered medium at ph 6.2 and 6.8 , respectively . at these time point , butyric acid production attained its peak and the concentration measured was 9.5 , 13.5 , and 12 mm in unbuffered , buffered medium at ph 6.2 and 6.8 , respectively . acetic acid production reached its peak at 42 , 26 , and 30 h and the corresponding concentration measured was 6 , 8.5 , and 8 mm in unbuffered , buffered medium at ph 6.2 , and 6.8 , respectively . in the case of c. pasteurianum , maximum consumption of glucose was approached at 50 , 26 , and 30 h in unbuffered , buffered medium at ph 6.2 and 6.8 , respectively . at the same time point , butyric acid production attained its peak and the concentration measured was 7.5 , 11.5 , and 10.5 mm in unbuffered , buffered medium at ph 6.2 and 6.8 , respectively . for this strain , acetic acid production reached its peak at 42 , 26 , and 30 h , and the corresponding concentration measured was 5 , 14.5 , and 11.5 mm in unbuffered , buffered medium at ph 6.2 and 6.8 , respectively . c. sphenoides showed no increase in cell density and growth after adding ph buffers to either msn or scm medium ( data not shown ) . further , hplc measurements revealed that this strain only generated acetic acid when cultured in both scm and msm ( data not shown ) . we also observed that , when the medium was buffered at ph 7.2 and above ( 7.5 ) , clostridium sp . did not grow ; the other three strains grew slowly at ph 7.2 , but did not grow above 7.5 ( data not shown ) . we demonstrated that gaseous hydrogen , in the presence of clostridia cells , could result in both u(vi ) and fe(iii ) reduction ( figure 4 ) . cultured clostridia cells were spun down , washed , and then resuspended in carbon - free msn medium in a serum bottle under anaerobic conditions . thereafter , headspace gases were replaced with pure hydrogen or nitrogen gas . when hydrogen was provided as the headspace gas , either uranium(vi ) or iron(iii ) reduction occurred in the presence of whole cells of clostridium sp . this is in contrast to experiments which used nitrogen as the headspace gas . without whole cells , hydrogen alone did not result in either uranium(vi ) or iron(iii ) reduction , suggesting that hydrogenase indeed mediated both the uranium(vi ) and iron(iii ) reduction using hydrogen as an electron donor ( figures 4(a ) and 4(b ) ) . both the gas pressure and ph in the bottle containing bacterial cells with hydrogen decreased after overnight incubation . using other clostridia strains as testing material , similar results were also obtained ( data not shown ) . when concentration of cu(ii ) reached 20 mm , the u(vi ) reduction activity by bc1 was 100% inhibited ( figure 5 ) . the inhibition effect of cu(ii ) is not relevant with the u(vi ) form used ( uo2(no3)2 or u(vi)-citric acid complex ) ( data not shown ) . in terms of inhibition effect on uranium reduction , no difference was observed between simultaneous and stepwise addition of cu(ii ) and u(vi ) into assay ( data not shown ) , suggesting that the inhibition effect of cu(ii ) is immediate and most likely occurs at enzymatic level . cells cultured in iron deficient media compromised its capability for uranium reduction ( figures 6(a ) and 6(b ) ) . compared with iron - rich medium which contained 10 m fe(ii ) , bc1 cells cultured in iron - depleted medium containing < 0.01 m , fe(ii ) performed much poorer in u(vi ) reduction to u(iv ) during the time period of assay . previously , we demonstrated that clostridium sp . bc1 and c. sphenoides can reduce uranium [ 15 , 22 ] . later , we expanded this list to c. acetobutylicum and c. pasteurianum that were identified many years ago and have been widely used in basic and applied studies . for instance , atcc 824 , the type strain of species c. acetobutylicum , was isolated in 1924 from garden soil in connecticut and is one of the best - studied solventogenic clostridia used to develop an industrial starch - based acetone , butanol , and ethanol ( abe ) fermentation process . our study found that both c. acetobutylicum and c. pasteurianum can reduce uranium so bolstering our previous conclusion that this ability is a common phenomenon among clostridia bacteria . we demonstrated that not only all of the clostridia tested are able to reduce u(vi ) to u(iv ) , but also there are considerable differences in the extent of their ability to do so . the extent for uranium reduction varies among clostridia strains and that ph of medium strongly influences the dynamic process of uranium reduction . without a buffer supplement , our experiments showed that the ph of the cultures could drop to 3 to 4 when the late - log phase of growth was reached ; then , only the clostridium sp . culture reduced u(vi ) to u(iv ) well , while the other three strains could not do so , or performed poorly . in this study , we demonstrated that clostridium sp . was superior in terms of growth and gas production . thus , it seems reasonable to attribute the poorer performance of strains other than clostridium sp . meanwhile , our findings also exemplified that clostridium sp . has higher tolerance to harsh environmental conditions , such as low ph , as well as a stronger ability for fermentation under such conditions , especially in reducing u(vi ) in acidic environments . since this strain was isolated from an acidic metal - contaminated site , its better adaptation to acidic environments is unsurprising . however , adding the ph buffer to cultures of c. sphenoides caused no improvement in its growth . nevertheless , the reduction rate of u(vi ) by this strain was much better at a near - neutral ph , and its highest rate mostly closely approached that of clostridium sp . . thus , an optimal ph alone apparently is important to u(vi ) reduction by clostridia , even though it does not necessarily improve fermentation efficiency at the same time . we found that a suboptimal ph could compromise the ability of clostridia to reduce u(vi ) : we suggest that the underlying mechanism for this phenomenon is as follows . since most physiological reactions ideally occur at near - neutral ph , organisms developed a variety of mechanisms to maintain a near - neutral cytoplasmic ph . even those acidophilic bacteria that grow best at ~ph 3 maintain a near - neutral cytoplasmic ph and possess a membrane potential ( ) with an orientation reversed from that found in neutrophilic bacteria . for gram - positive bacteria , including clostridia , proton efflux through proton pumps , such as f1f0atpase , is the major means of raising internal ph . goodwin and zeikus demonstrated that physiological adaptations of anaerobic bacteria to low ph often are a competitive process for hydrogen production . thus , we speculate that the physiological adaptations of clostridia to suboptimal ph also compete with u(vi ) reduction . under suboptimal conditions , the bacterial response to the stressful environments becomes their priority , and more energy than normal is consumed in activities , such as amino acid metabolism , transcriptional regulation and signal transduction , transport , maintaining cell - membrane structure , and protection against oxidative stress , as was suggested by a transcriptome analysis of shewanella oneidensis cells exposed to acidic and alkaline phs . meanwhile , under the same conditions , other activities become secondary ones , such as u(vi ) reduction , and little energy is allocated to them . the mechanisms of uranium(vi ) reduction by anaerobic respiratory bacteria , including desulfovibrio , geobacter , and shewanella , are being extensively investigated [ 5 , 6 ] . however , although uranium reduction by clostridia is established , the mechanisms underlying the reaction remain unclear . our previous results showed that it was an enzymatic process since it happened only in the presence of growing or resting cells ; neither the organic - acid metabolites generated nor the extracellular components of the culture , nor heat - killed cells could reduce uranium anaerobically . previous conjectures were that the reducing power generated from fermentation , such as that of glucose , caused uranium reduction . thus , petrie at al . demonstrated that glucose amendments of the growth medium enriched the numbers of gram - positive spore - forming bacteria , and since some of the highest rates of u(vi ) reduction in situ occurred upon amendment , that fermentative processes were involved . our previous study showed that the optimizing conditions for fermentation resulted in better u(vi ) reduction support this hypothesis . indeed , improving fermentation conditions by supplementing the medium with a ph buffer increased the u(vi ) reduction rate of clostridia strains . apparently , the efficiency of fermentation is positively related to the u(vi ) reduction rate of individual strains . in this study , we demonstrate that the hydrogen metabolism could play an important role in both uranium(vi ) and iron(iii ) reduction by clostridium sp . when hydrogen gas ( h2 ) was provided in the headspace of the serum bottle , either uranium(vi ) or iron(iii ) reduction occurred in the presence of whole cells without carbon source . this is in contrast to the introduction of nitrogen gas ( n2 ) into the headspace . in the absence of whole cells , hydrogen alone could result neither uranium(vi ) nor iron(iii ) reduction , suggesting that a hydrogenase mediated both the uranium(vi ) and iron(iii ) reduction using hydrogen as the electron donor ( figure 4 ) . evidence supporting this hypothesis also came from cu(ii ) inhibition effects on uranium reduction of clostridia ( figure 5 ) . since cu(ii ) is a documented hydrogenase inhibitor , the inhibition of u(vi ) reduction is most likely through the inhibition of hydrogenase activity of clostridia cells . our experiment also showed that clostridium cells cultured in iron deficient media compromised its capability for uranium reduction ( figure 6 ) . it is also noteworthy that the deficiency in ferrous ions also affected hydrogen production ( data not shown ) . still , our recent results showing that methyl viologen ( mv ) addition affects hydrogenase activity with a significant reduction in hydrogen production also agree with this hypothesis . taken together , one scenario explaining molecular mechanisms of uranium(vi ) reduction by clostridia is emerging and is described as below . clostridia dispose of excess electrons generated during fermentation by producing hydrogen . during the fermentation process , bacteria that grow at the expense of diverse carbon sources often depend on ferredoxin or flavodoxin for essential oxidation - reduction reactions . the metabolic pathway of hydrogen production in clostridia is summarized in figure 7 . as shown in figure 7 , clostridia use ferredoxin ( fd ) to oxidize sugars and other organic matter through pyruvate to produce carbon dioxide , acetate , and butyrate . ferredoxins are acidic , low molecular weight , soluble iron - sulfur proteins found in various organisms and act as multifunctional electron carriers in diverse redox systems . flavodoxin is a flavin mononucleotide - binding redox protein with an open twisted alpha / beta structure consisting of five parallel beta - sheets connected by alpha - helices which surround the sheets in the 3d structure . ferredoxins or flavodoxins are essential components of the electron transport chains of clostridia . however , they do not themselves possess enzymatic activity in these reactions . instead , they interact with specific dehydrogenases and reductases that handle the substrates to be oxidized or reduced . hydrogenase , a molecular hydrogen evolving enzyme , receives electrons from pyruvate oxidation through up to seven fd clusters to produce hydrogen ( h2 ) by the reaction as follows : 2h + 2e h2 . in some strains of clostridia , the presence of nitrogenase complicated hydrogen metabolism . in the presence of nitrogen and absence of ammonia , nitrogenases catalyze the production of hydrogen and ammonia at the expense of atp . without nitrogen , nitrogenases can function as 100% hydrogenase to produce hydrogen in a process that requires atp and electrons from biomass . the molecular basis for biological hydrogen production is dependent upon the presence of hydrogen - producing enzymes . at present , there are three known enzymes carrying out such reactions : fe - hydrogenase , ni - fe hydrogenase , and nitrogenase . it is also found in other strict anaerobic bacteria such as desulfovibrio vulgaris [ 35 , 36 ] , as well as in some green algae such as chlamydomonas reinhardtii , and several eukaryotic protists such as trichomonas vaginalis . generally , the cytoplasmic fe - hydrogenase ( hydrogenase-1 ) functions as the generator of hydrogen by removing excess reducing equivalents during fermentations of strict anaerobic bacteria , whereas the periplasmic fe - hydrogenase ( hydrogenase-2 ) functions in hydrogen oxidation . fe - hydrogenase contains a unique complex fe - s centers in which one of the fe atoms is complexed with co and cn . compared with nitrogenase and ni - fe hydrogenase , fe - hydrogenase is highly efficient in hydrogen production . as shown in figure 7 , we postulate that both hydrogenases 1 and 2 are involved in uranium reduction of clostridia . while hydrogenase 1 functions as the generator of hydrogen by removing excess reducing equivalents during fermentations , hydrogenase 2 functions in hydrogen oxidation and more directly involved in uranium reduction . it is not clear how the hydrogenases of clostridia consume hydrogen and transfer electrons to iron(iii ) or uranium(vi ) . in addition to hydrogenase , it is not known if additional proteins are involved in this process . cytochromes are generally membrane - bound proteins that contain heme groups and carry out electron transport . many cytochromes ( c - type , b - type ) of dissimilatory metal reducing bacteria ( dmrb ) were shown to be relevant to metal reduction [ 42 , 43 ] . the presence of cytochrome in clostridia has been documented previously . from the annotated genome sequence of clostridium acetobutylicum ( atcc 824 ) , around we also postulate that iron depletion could result in the shutdown or downregulation of hydrogenase synthesis as well as other iron - containing proteins like ferredoxin . meanwhile , as a substitution , the expression of some noniron proteins such as flavodoxin could be upregulated . due to this shift in protein synthesis , clostridia compromise their metabolism of hydrogen production , as well as their capability for uranium reduction . note that our proposed model herein does not necessarily exclude possible other pathways and other components also used by clostridia for uranium reduction , such as spore - mediated metal reduction . with their widespread occurrence in soils , sediments , and low - level radioactive wastes , clostridia could play a significant role in the in situ reduction of uranium and other metals particularly at acidic ph and in nitrate - rich environments , as suggested by a number of studies [ 18 , 20 , 21 , 25 , 30 ] . however , compared with anaerobic respiratory bacteria including desulfovibrio , geobacter , and shewanella , molecular mechanisms underpinning uranium(vi ) reduction to uranium(iv ) are still not very clear . future study toward this direction should be encouraged and we expect more detailed mechanisms of uranium reduction by clostridia will be revealed in the future .

previously , it has been shown that not only is uranium reduction under fermentation condition common among clostridia species , but also the strains differed in the extent of their capability and the ph of the culture significantly affected uranium(vi ) reduction . in this study , using hplc and gc techniques , metabolic properties of those clostridial strains active in uranium reduction under fermentation conditions have been characterized and their effects on capability variance of uranium reduction discussed . then , the relationship between hydrogen metabolism and uranium reduction has been further explored and the important role played by hydrogenase in uranium(vi ) and iron(iii ) reduction by clostridia demonstrated . when hydrogen was provided as the headspace gas , uranium(vi ) reduction occurred in the presence of whole cells of clostridia . this is in contrast to that of nitrogen as the headspace gas . without clostridia cells , hydrogen alone could not result in uranium(vi ) reduction . in alignment with this observation , it was also found that either copper(ii ) addition or iron depletion in the medium could compromise uranium reduction by clostridia . in the end , a comprehensive model was proposed to explain uranium reduction by clostridia and its relationship to the overall metabolism especially hydrogen ( h2 ) production .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Final Remarks

recently , researchers at some doe sites have assessed the microbial stabilization of actinides ( u , pu , and np ) and fission products ( tc ) in subsurface environments , as in the uranium mill tailing remedial action ( umtra ) site in rifle , co ( http://www.gjem.energy.gov/moab/ ) and at the oak ridge field research centre ( orfrc ) at oak ridge , tn ( http://www.esd.ornl.gov/orifrc/ ) . a wide variety of bacteria , including desulfovibrio , geobacter , and shewanella , can couple the oxidation of organic compounds to the reduction of u(vi ) and thus reductively precipitate uranium in its reduced form u(iv ) [ 13 ] . investigators have explored the mechanisms of uranium(vi ) reduction by anaerobic bacteria [ 46 ] . previously , it was showed that clostridium sp . bc1 ( atcc 53464 ) and c. sphenoides ( atcc 19403 ) can reduce uranyl - nitrate , -acetate , and -citrate complexes , as well as complexes of other metals [ 1416 ] . we have further found that not only is this ability common among more clostridia species , but that also the strains differed in the extent of their capability . the ph of the culture significantly affected uranium(vi ) reduction , with ph 5 - 6 being the optimal one in most cases . apparently , the evidence suggested that clostridia is one of the major players in uranium reduction in situ in an acidic ( ph = 4 ) uranium - mine pit water [ 18 , 19 ] , at a military facility near chesapeake bay , maryland , united states , as well as at the orfrc at oak ridge , tn . in this study , using hplc and gc techniques , we have continually characterized metabolic properties of those clostridial strains active in uranium reduction under fermentation conditions and discussed their effects on performance of uranium reduction . we also demonstrated that hydrogen metabolism could play an important role in uranium(vi ) and iron(iii ) reduction by clostridia . in the end , we proposed a comprehensive model to explain molecular mechanisms of uranium reduction by clostridia and its relationship to the overall metabolism especially hydrogen ( h2 ) production . all strains can ferment glucose , but only c. sphenoides can metabolize citric acid as the sole carbon and energy source . all strains were grown anaerobically and maintained in the mineral salts medium , herein called msm medium , containing per liter : glucose 5.0 g , nh4cl 0.5 g , glycerol phosphate 0.3 g , mgso47h2o 0.2 g , cacl22h2o 0.5 g , peptone 0.1 g , and yeast extracts 0.1 g , 6.25 ml of 1.6 mm feso47h2o , at ph 6.8 . after allowing the solution to cool , we placed the flask in an anaerobic glove box ( 95% n2 , 5% h2 ) and then added to the medium 6.25 ml of 1.6 mm prereduced feso47h2o ( prepared by dissolving 90 mg ferrous sulfate in 200 ml of prereduced deionized water and adding 0.5 ml concentrated hcl ) and made up the total volume to 1000 ml with prereduced deionized water . the ph was adjusted to 6.8 before dispensing 40 ml aliquots of the medium into 60 ml serum bottles , fitted with butyl rubber stoppers , then crimp - sealing them with aluminum caps before autoclaving . c. sphenoids ( atcc19403 ) was also cultured in simmons citrate medium ( scm ) ; all materials and their preparation were exactly the same as for the msm medium , except that we substituted glucose with 8.2 g sodium citrate . to stabilize the ph of the culture , as needed , we added 50 mm pipes ( ph 6.8 ) or 50 mm mes ( ph 6.2 ) to the medium . total gas production in the headspace of the sample was measured by a pressure transducer with a needle ( model 665-d/030 , wallace and tiernan ) . we removed four - milliliter aliquots of the culture to determine bacterial growth , change in ph , and production of organic acid metabolites . the growth of the bacterium was measured by recording the turbidity of the medium at 600 nm , using a bausch and lomb spectronic 20 spectrophotometer . for the 10 mm u(vi)-nitrate stock solution , 5 ml of prereduced deionized water was added to a beaker along with 0.25 ml of 0.5 m u(vi)-nitrate , the ph adjusted to 6.1 with naoh , and the solution diluted to 10 ml for a final concentration of 10 mm . the complexes were stored in the dark and then readjusted to ph 6.1 after 24 hours , and filter - sterilized ( 0.45 m ) into a vacutainer tube . the ph was adjusted to 6.1 with prereduced naoh and diluted to 10 ml for a final concentration of 10 mm . ten milliliters of the culture at the late - log phase was transferred to anaerobic preautoclaved serum bottle ( 20 ml ) , and then 0.1 ml of 10 mm u(vi)-nitrate solution added via a 1-ml syringe with a needle . to determine the u(vi ) concentration over time , aliquots of 0.1 to 0.2-ml of the bacterial culture were taken from the serum bottle , 5 l of the culture diluted in 2 ml deionized water and immediately analyzed for u(vi ) by kpa . to assess the effect of ph on u(vi ) reduction by clostridia , ten milliliter aliquots of the late - log growth phase of the bacterial culture were adjusted to the required ph values using 1 n naoh or hcl . the uv - vis ( ultraviolet - visible ) spectrophotometry was used for obtaining absorption spectra of both uranium(vi ) and uranium(iv ) . a hewlett packard model the sample was prepared as follows : after completing the uranium reduction assay described above , the total leftover culture in serum bottle was collected into a centrifuge tube in the glove box . then , its ph was adjusted to 11 using 1 n naoh , and the culture centrifuged at 10000 rpm for 10 minutes , the supernatant discarded , and the pellet was resuspended in 2-ml 10 mm citric acid solution to extract the uranium species . the solution was filtered through a 0.45 m membrane and then the filtrate was analyzed by uv - vis spectrophotometry to determine the absorption spectra of both uranium(vi ) and uranium(iv ) . the hplc unit consisted of a shimadzu scl-10a system controller , a sil-10a autoinjector , and a lc-10as liquid chromatograph . we determined the rate of growth , changes in ph of the media , and gas production of the four clostridia strains cultured in msm or scm . we also analyzed the extent of consumption of the carbon source and the metabolic products from the strains . ; this strain also generated the most gas , even after entering stationary phase , while all other strains stopped producing gas at the stationary phase . gc analysis of the headspace gas showed that the maximum percentage of hydrogen ( h2 ) produced by clostridium sp . however , the medium 's ph slightly increased from near neutral to around ph 7.8 , due to the concurrent consumption of citric acid ( figure 1 ) . however , growing the clostridia in media supplemented with a ph buffer to stabilize ph alleviated the drop in ph due to the production of organic acid during glucose fermentation ( figure 2 ) . when cultured in msm medium supplemented with 50 mm pipes ( ph 6.8 ) , ph of the medium reached around 5 to 6 by the late - log phase of growth for clostridium sp . furthermore , the ph - buffered medium affected the growth kinetics and final cell density of the strains . grew much slower initially when the medium was buffered to ph 6.8 , although growth later accelerated and it reached the stationary phase at the same time ( 40 hours ) and at the same optimal density ( od600 nm = 0.8 ) . grew much faster in the medium buffered to ph 6.2 , reaching the stationary phase 20 hours earlier at the same optimal density ( od600 nm = 0.8 ) ( figure 2 ) . , its growth was much lower at the first 15 hours ; thereafter , it reached the same level as that in unbuffered culture . addition of buffers did not result in an increase in final cell density ( figure 2 ) . the growth of c. pasteurianum increased in the ph 6.8 and 6.2 adjusted buffered medium ( figure 2 ) . the similar trends also occurred in the production of total gas by these strains ( figure 2 ) . glucose consumption and production of the organic acids were influenced by the initial ph of the medium ; buffered medium at ph 6.2 showed the rapid glucose consumption and production of acetic and butyric acids . concurrently , butyric acid production attained its peak at the same time point as that of 100% glucose consumption , and the highest concentration measured was 13.5 , 11.5 , and 10.5 mm in unbuffered , buffered medium at ph 6.2 and 6.8 , respectively . we demonstrated that gaseous hydrogen , in the presence of clostridia cells , could result in both u(vi ) and fe(iii ) reduction ( figure 4 ) . when hydrogen was provided as the headspace gas , either uranium(vi ) or iron(iii ) reduction occurred in the presence of whole cells of clostridium sp . this is in contrast to experiments which used nitrogen as the headspace gas . without whole cells , hydrogen alone did not result in either uranium(vi ) or iron(iii ) reduction , suggesting that hydrogenase indeed mediated both the uranium(vi ) and iron(iii ) reduction using hydrogen as an electron donor ( figures 4(a ) and 4(b ) ) . when concentration of cu(ii ) reached 20 mm , the u(vi ) reduction activity by bc1 was 100% inhibited ( figure 5 ) . in terms of inhibition effect on uranium reduction , no difference was observed between simultaneous and stepwise addition of cu(ii ) and u(vi ) into assay ( data not shown ) , suggesting that the inhibition effect of cu(ii ) is immediate and most likely occurs at enzymatic level . cells cultured in iron deficient media compromised its capability for uranium reduction ( figures 6(a ) and 6(b ) ) . compared with iron - rich medium which contained 10 m fe(ii ) , bc1 cells cultured in iron - depleted medium containing < 0.01 m , fe(ii ) performed much poorer in u(vi ) reduction to u(iv ) during the time period of assay . previously , we demonstrated that clostridium sp . later , we expanded this list to c. acetobutylicum and c. pasteurianum that were identified many years ago and have been widely used in basic and applied studies . for instance , atcc 824 , the type strain of species c. acetobutylicum , was isolated in 1924 from garden soil in connecticut and is one of the best - studied solventogenic clostridia used to develop an industrial starch - based acetone , butanol , and ethanol ( abe ) fermentation process . our study found that both c. acetobutylicum and c. pasteurianum can reduce uranium so bolstering our previous conclusion that this ability is a common phenomenon among clostridia bacteria . we demonstrated that not only all of the clostridia tested are able to reduce u(vi ) to u(iv ) , but also there are considerable differences in the extent of their ability to do so . the extent for uranium reduction varies among clostridia strains and that ph of medium strongly influences the dynamic process of uranium reduction . without a buffer supplement , our experiments showed that the ph of the cultures could drop to 3 to 4 when the late - log phase of growth was reached ; then , only the clostridium sp . in this study , we demonstrated that clostridium sp . nevertheless , the reduction rate of u(vi ) by this strain was much better at a near - neutral ph , and its highest rate mostly closely approached that of clostridium sp . thus , an optimal ph alone apparently is important to u(vi ) reduction by clostridia , even though it does not necessarily improve fermentation efficiency at the same time . we found that a suboptimal ph could compromise the ability of clostridia to reduce u(vi ) : we suggest that the underlying mechanism for this phenomenon is as follows . thus , we speculate that the physiological adaptations of clostridia to suboptimal ph also compete with u(vi ) reduction . under suboptimal conditions , the bacterial response to the stressful environments becomes their priority , and more energy than normal is consumed in activities , such as amino acid metabolism , transcriptional regulation and signal transduction , transport , maintaining cell - membrane structure , and protection against oxidative stress , as was suggested by a transcriptome analysis of shewanella oneidensis cells exposed to acidic and alkaline phs . meanwhile , under the same conditions , other activities become secondary ones , such as u(vi ) reduction , and little energy is allocated to them . the mechanisms of uranium(vi ) reduction by anaerobic respiratory bacteria , including desulfovibrio , geobacter , and shewanella , are being extensively investigated [ 5 , 6 ] . however , although uranium reduction by clostridia is established , the mechanisms underlying the reaction remain unclear . our previous results showed that it was an enzymatic process since it happened only in the presence of growing or resting cells ; neither the organic - acid metabolites generated nor the extracellular components of the culture , nor heat - killed cells could reduce uranium anaerobically . previous conjectures were that the reducing power generated from fermentation , such as that of glucose , caused uranium reduction . demonstrated that glucose amendments of the growth medium enriched the numbers of gram - positive spore - forming bacteria , and since some of the highest rates of u(vi ) reduction in situ occurred upon amendment , that fermentative processes were involved . indeed , improving fermentation conditions by supplementing the medium with a ph buffer increased the u(vi ) reduction rate of clostridia strains . apparently , the efficiency of fermentation is positively related to the u(vi ) reduction rate of individual strains . in this study , we demonstrate that the hydrogen metabolism could play an important role in both uranium(vi ) and iron(iii ) reduction by clostridium sp . when hydrogen gas ( h2 ) was provided in the headspace of the serum bottle , either uranium(vi ) or iron(iii ) reduction occurred in the presence of whole cells without carbon source . this is in contrast to the introduction of nitrogen gas ( n2 ) into the headspace . in the absence of whole cells , hydrogen alone could result neither uranium(vi ) nor iron(iii ) reduction , suggesting that a hydrogenase mediated both the uranium(vi ) and iron(iii ) reduction using hydrogen as the electron donor ( figure 4 ) . evidence supporting this hypothesis also came from cu(ii ) inhibition effects on uranium reduction of clostridia ( figure 5 ) . since cu(ii ) is a documented hydrogenase inhibitor , the inhibition of u(vi ) reduction is most likely through the inhibition of hydrogenase activity of clostridia cells . our experiment also showed that clostridium cells cultured in iron deficient media compromised its capability for uranium reduction ( figure 6 ) . still , our recent results showing that methyl viologen ( mv ) addition affects hydrogenase activity with a significant reduction in hydrogen production also agree with this hypothesis . taken together , one scenario explaining molecular mechanisms of uranium(vi ) reduction by clostridia is emerging and is described as below . ferredoxins or flavodoxins are essential components of the electron transport chains of clostridia . hydrogenase , a molecular hydrogen evolving enzyme , receives electrons from pyruvate oxidation through up to seven fd clusters to produce hydrogen ( h2 ) by the reaction as follows : 2h + 2e h2 . in some strains of clostridia , the presence of nitrogenase complicated hydrogen metabolism . in the presence of nitrogen and absence of ammonia , nitrogenases catalyze the production of hydrogen and ammonia at the expense of atp . the molecular basis for biological hydrogen production is dependent upon the presence of hydrogen - producing enzymes . generally , the cytoplasmic fe - hydrogenase ( hydrogenase-1 ) functions as the generator of hydrogen by removing excess reducing equivalents during fermentations of strict anaerobic bacteria , whereas the periplasmic fe - hydrogenase ( hydrogenase-2 ) functions in hydrogen oxidation . as shown in figure 7 , we postulate that both hydrogenases 1 and 2 are involved in uranium reduction of clostridia . while hydrogenase 1 functions as the generator of hydrogen by removing excess reducing equivalents during fermentations , hydrogenase 2 functions in hydrogen oxidation and more directly involved in uranium reduction . it is not clear how the hydrogenases of clostridia consume hydrogen and transfer electrons to iron(iii ) or uranium(vi ) . in addition to hydrogenase , it is not known if additional proteins are involved in this process . the presence of cytochrome in clostridia has been documented previously . from the annotated genome sequence of clostridium acetobutylicum ( atcc 824 ) , around we also postulate that iron depletion could result in the shutdown or downregulation of hydrogenase synthesis as well as other iron - containing proteins like ferredoxin . meanwhile , as a substitution , the expression of some noniron proteins such as flavodoxin could be upregulated . due to this shift in protein synthesis , clostridia compromise their metabolism of hydrogen production , as well as their capability for uranium reduction . note that our proposed model herein does not necessarily exclude possible other pathways and other components also used by clostridia for uranium reduction , such as spore - mediated metal reduction . with their widespread occurrence in soils , sediments , and low - level radioactive wastes , clostridia could play a significant role in the in situ reduction of uranium and other metals particularly at acidic ph and in nitrate - rich environments , as suggested by a number of studies [ 18 , 20 , 21 , 25 , 30 ] . however , compared with anaerobic respiratory bacteria including desulfovibrio , geobacter , and shewanella , molecular mechanisms underpinning uranium(vi ) reduction to uranium(iv ) are still not very clear . future study toward this direction should be encouraged and we expect more detailed mechanisms of uranium reduction by clostridia will be revealed in the future .

subjects were stratified by age group ( < 45 vs 45 yrs ) and were randomly assigned in a 1:1:1 ratio to receive bimatoprost 0.03% twice daily ( bid ) , bimatoprost 0.03% once daily ( qd ) , or vehicle bid for 7 months of treatment . randomization was managed through automated interactive voice / web response systems . in all 3 treatment groups , subjects were instructed to apply study medication twice daily , once in the morning ( am dose ) and once in the evening ( pm dose ) . in the bimatoprost qd group , the am dose consisted of vehicle with the pm dose consisting of bimatoprost 0.03% . in the bimatoprost bid group , both the am and pm doses consisted of bimatoprost 0.03% . in the vehicle group , both the am and pm doses consisted of vehicle . treatment group assignment was masked to both the subjects and the investigators throughout the study . the total duration of study participation from the screening visit was approximately 9 months , with a total of 11 visits : at screening ( day 21 to 7 ) , day 1 ( baseline ) , week 1 , months 1 , 2 , 3 , 4 , 5 , 6 , and 7 ( or early study discontinuation ) , and at month 8 posttreatment follow - up . eyebrow stencils were used to standardize both the application of the study medication and the area of grooming . subjects were instructed to groom their eyebrows outside the treatment area , as delineated by the stencil , with scissors or tweezers only ; grooming within the eyebrow treatment area was prohibited . the stencil was also used as a guide to determine the area of study medication application . eligible subjects were female or male , aged 18 years or older with eyebrow hypotrichosis , defined as either a grade 1 or 2 on the allergan global eyebrow assessment ( geba ) scale with photonumeric guide . the geba instrument is a validated 4-point scale used to grade fullness of the eyebrows ( 1 = very sparse , 2 = sparse , 3 = full , and 4 = very full ) . subjects were excluded if they had an uncontrolled systemic disease or any known disease , infection , or abnormality in the eyebrow area or hair shaft . subjects with systemic diseases , including all types of alopecia areata , which could prevent hair growth or produce abnormal growth were excluded , along with subjects with substantial permanent eyebrow loss due to overgrooming ; any skin pigmentation / depigmentation , tattoos , moles , scars , or other characteristics within the eyebrow area that could confound evaluation . subjects who used permanent injectable fillers in and around the eyebrow area within 12 months ; or systemic treatment within 12 months of screening with agents that may affect hair growth were excluded , as were subjects who had undergone any facial surgery ( e.g. , brow lift ) or eyebrow extension within 3 months of screening . subjects who used temporary or semipermanent eyebrow fillers or eyebrow tint , dye , or bleaching within 2 months of screening and female subjects who were pregnant , nursing , or planning for a pregnancy during the study were excluded . efficacy and safety were assessed posttreatment at week 1 , at months 1 through 7 ( or on early discontinuation ) , and at the month 8 follow - up visit . the primary efficacy endpoint was the investigator 's assessment of the subject 's overall eyebrow fullness at month 7 using the geba scale . the primary efficacy variable was defined as the proportion of subjects with at least a 1-grade increase ( improvement ) in the geba grade from baseline at month 7 . secondary efficacy endpoints at month 7 included subjects ' eyebrow fullness ( mm ) and darkness ( intensity units [ iu ] ; negative values are representative of eyebrow darkening ) as measured using digital monitoring system image analysis ( dmsia ) . subject satisfaction with eyebrow treatment at month 7 was measured by item 6 of the 9-item eyebrow satisfaction scale ( ess , follow - up version ) . assessments could range from 1 ( very satisfied ) to 5 ( very dissatisfied ) . safety measures included treatment - emergent adverse events ( teaes ) , vital sign measurements ( i.e. , blood pressure , pulse rate , and urine pregnancy test ) , and laboratory tests ( i.e. , thyroid - stimulating hormone and ferritin ) and hematology values . to have statistical power to evaluate the primary efficacy variable , a sample size of 348 was proposed , with a 1:1:1 randomization ratio to study groups . accounting for an attrition rate of 15% , 295 subjects were anticipated to complete the study . assuming a 25% vehicle response rate based on the primary efficacy endpoint and a 2-sided type 1 error of 0.05 , a sample size of 348 with a 1:1:1 randomization ratio for bimatoprost bid , bimatoprost qd , or vehicle was chosen to detect a difference of 20% , 22% , and 25% in favor of bimatoprost bid , with a statistical power of 86% , 92% , and 97% , respectively . efficacy analyses reported here were performed on the intent - to - treat population , defined as all randomized subjects . the geba and ess item 6 grades were analyzed using a pooled point estimate for the treatment difference , based on the mantel haenszel method , stratified by age group . the 95% cis for the differences were constructed based on the greenland robins formula . treatment - by - age interaction was tested using the breslow day test , with a significance level of 0.1 . the statistical analysis aimed to compare bimatoprost dose groups versus vehicle . bimatoprost bid or bimatoprost qd was considered to be superior to vehicle if a 2-sided mantel haenszel test showed p .05 . to control the type 1 error rate for the primary efficacy endpoint , a serial gatekeeping approach was used : the comparison between bimatoprost qd and vehicle was tested only if statistical analysis of bimatoprost bid versus vehicle was p .05 . to control the type 1 error rate for multiple secondary endpoints , a serial gatekeeping approach was used based on the importance of the secondary variables : eyebrow fullness , eyebrow darkness , and ess item 6 . in addition , a gatekeeping approach was used for the statistical analysis : bimatoprost bid versus vehicle was tested at the 2-sided = 0.05 level ; if p value was 0.05 , bimatoprost qd versus vehicle was tested at the 2-sided = 0.05 level . changes in eyebrow fullness and darkness were analyzed using dmsia at month 7 ; pairwise comparisons were made using the van elteren test , stratified by age group . safety analyses were performed on the safety population , defined as all subjects who received at least 1 dose of study treatment . this multicenter , double - masked , randomized , vehicle - controlled , 3-arm , parallel - group study was designed to evaluate the safety and efficacy of bimatoprost 0.03% compared with vehicle in subjects with eyebrow hypotrichosis ( clinicaltrials.gov identifier : nct01765764 ) . this study was approved by the institutional review board or independent ethics committee at each site and was conducted in compliance with good clinical practice guidelines . subjects were stratified by age group ( < 45 vs 45 yrs ) and were randomly assigned in a 1:1:1 ratio to receive bimatoprost 0.03% twice daily ( bid ) , bimatoprost 0.03% once daily ( qd ) , or vehicle bid for 7 months of treatment . randomization was managed through automated interactive voice / web response systems . in all 3 treatment groups , subjects were instructed to apply study medication twice daily , once in the morning ( am dose ) and once in the evening ( pm dose ) . in the bimatoprost qd group , the am dose consisted of vehicle with the pm dose consisting of bimatoprost 0.03% . in the bimatoprost bid group , both the am and pm doses consisted of bimatoprost 0.03% . in the vehicle group , both the am and pm doses consisted of vehicle . treatment group assignment was masked to both the subjects and the investigators throughout the study . the total duration of study participation from the screening visit was approximately 9 months , with a total of 11 visits : at screening ( day 21 to 7 ) , day 1 ( baseline ) , week 1 , months 1 , 2 , 3 , 4 , 5 , 6 , and 7 ( or early study discontinuation ) , and at month 8 posttreatment follow - up . eyebrow stencils were used to standardize both the application of the study medication and the area of grooming . subjects were instructed to groom their eyebrows outside the treatment area , as delineated by the stencil , with scissors or tweezers only ; grooming within the eyebrow treatment area was prohibited . the stencil was also used as a guide to determine the area of study medication application . eligible subjects were female or male , aged 18 years or older with eyebrow hypotrichosis , defined as either a grade 1 or 2 on the allergan global eyebrow assessment ( geba ) scale with photonumeric guide . the geba instrument is a validated 4-point scale used to grade fullness of the eyebrows ( 1 = very sparse , 2 = sparse , 3 = full , and 4 = very full ) . subjects were excluded if they had an uncontrolled systemic disease or any known disease , infection , or abnormality in the eyebrow area or hair shaft . subjects with systemic diseases , including all types of alopecia areata , which could prevent hair growth or produce abnormal growth were excluded , along with subjects with substantial permanent eyebrow loss due to overgrooming ; any skin pigmentation / depigmentation , tattoos , moles , scars , or other characteristics within the eyebrow area that could confound evaluation . subjects who used permanent injectable fillers in and around the eyebrow area within 12 months ; or systemic treatment within 12 months of screening with agents that may affect hair growth were excluded , as were subjects who had undergone any facial surgery ( e.g. , brow lift ) or eyebrow extension within 3 months of screening . subjects who used temporary or semipermanent eyebrow fillers or eyebrow tint , dye , or bleaching within 2 months of screening and female subjects who were pregnant , nursing , or planning for a pregnancy during the study were excluded . efficacy and safety were assessed posttreatment at week 1 , at months 1 through 7 ( or on early discontinuation ) , and at the month 8 follow - up visit . the primary efficacy endpoint was the investigator 's assessment of the subject 's overall eyebrow fullness at month 7 using the geba scale . the primary efficacy variable was defined as the proportion of subjects with at least a 1-grade increase ( improvement ) in the geba grade from baseline at month 7 . secondary efficacy endpoints at month 7 included subjects ' eyebrow fullness ( mm ) and darkness ( intensity units [ iu ] ; negative values are representative of eyebrow darkening ) as measured using digital monitoring system image analysis ( dmsia ) . subject satisfaction with eyebrow treatment at month 7 was measured by item 6 of the 9-item eyebrow satisfaction scale ( ess , follow - up version ) . assessments could range from 1 ( very satisfied ) to 5 ( very dissatisfied ) . safety measures included treatment - emergent adverse events ( teaes ) , vital sign measurements ( i.e. , blood pressure , pulse rate , and urine pregnancy test ) , and laboratory tests ( i.e. , thyroid - stimulating hormone and ferritin ) and hematology values . to have statistical power to evaluate the primary efficacy variable , a sample size of 348 was proposed , with a 1:1:1 randomization ratio to study groups . accounting for an attrition rate of 15% , 295 subjects were anticipated to complete the study . assuming a 25% vehicle response rate based on the primary efficacy endpoint and a 2-sided type 1 error of 0.05 , a sample size of 348 with a 1:1:1 randomization ratio for bimatoprost bid , bimatoprost qd , or vehicle was chosen to detect a difference of 20% , 22% , and 25% in favor of bimatoprost bid , with a statistical power of 86% , 92% , and 97% , respectively . efficacy analyses reported here were performed on the intent - to - treat population , defined as all randomized subjects . the geba and ess item 6 grades were analyzed using a pooled point estimate for the treatment difference , based on the mantel haenszel method , stratified by age group . the 95% cis for the differences were constructed based on the greenland robins formula . treatment - by - age interaction was tested using the breslow day test , with a significance level of 0.1 . the statistical analysis aimed to compare bimatoprost dose groups versus vehicle . bimatoprost bid or bimatoprost qd was considered to be superior to vehicle if a 2-sided mantel haenszel test showed p .05 . to control the type 1 error rate for the primary efficacy endpoint , a serial gatekeeping approach was used : the comparison between bimatoprost qd and vehicle was tested only if statistical analysis of bimatoprost bid versus vehicle was p .05 . to control the type 1 error rate for multiple secondary endpoints , a serial gatekeeping approach was used based on the importance of the secondary variables : eyebrow fullness , eyebrow darkness , and ess item 6 . in addition , a gatekeeping approach was used for the statistical analysis : bimatoprost bid versus vehicle was tested at the 2-sided = 0.05 level ; if p value was 0.05 , bimatoprost qd versus vehicle was tested at the 2-sided = 0.05 level . changes in eyebrow fullness and darkness were analyzed using dmsia at month 7 ; pairwise comparisons were made using the van elteren test , stratified by age group . safety analyses were performed on the safety population , defined as all subjects who received at least 1 dose of study treatment . a total of 357 subjects were enrolled in this study , 118 in each of the bimatoprost groups and 121 in the vehicle group ( figure 1 ) . overall , 339 of 357 ( 95.0% ) subjects completed the study : 115 of 118 ( 97.5% ) , 109 of 118 ( 92.4% ) , and 115 of 121 ( 95.0% ) in the bimatoprost bid , bimatoprost qd , and vehicle groups , respectively . eighteen ( 5.0% ) subjects discontinued the study ; 4 ( 1.1% ) discontinued because of aes , 5 ( 1.4% ) were lost to follow - up , 4 ( 1.1% ) discontinued because of personal reasons , and 5 ( 1.4% ) provided reason as other . the mean age of the subjects was 54.0 years ( range , 1982 years ) and most were female ( 94.7% ) and were white ( 76.8% ) . subjects had a similar distribution of geba scores across treatment groups at baseline . demographics and other baseline characteristics ( intent - to - treat population ) the proportion of subjects with at least a 1-grade increase ( improvement ) in the geba scale from baseline at month 7 was significantly higher in the bimatoprost bid group ( 83.9% ) and bimatoprost qd group ( 77.1% ) compared with the vehicle group ( 43.0% ) ( p < .001 , both comparisons ) ( figure 2 ) . note that the bimatoprost bid and qd groups showed similar results . after month 1 , significant improvements were observed in both bimatoprost treatment groups compared with the vehicle group during the treatment period and the posttreatment follow - up period ( p .035 , all comparisons ; figure 2 ) . photographic examples of geba changes over time are shown in figure 3 . percentage of subjects with at least a 1-grade improvement from baseline in geba by visit ( intent - to - treat population ) . one subject receiving bimatoprost bid ( a ) and one receiving qd ( b ) had geba grades of 2 ( sparse ) at baseline , 3 ( full ) at month 4 , and 4 ( very full ) at month 7 . the subject receiving vehicle ( c ) had a geba grade of 1 ( very sparse ) at baseline and 2 ( sparse ) at months 4 and 7 . at month 7 , the bimatoprost bid , bimatoprost qd , and vehicle groups experienced mean changes from baseline in eyebrow fullness of 34.51 , 30.96 , and 6.42 mm , respectively ( p < .001 for bimatoprost bid and bimatoprost qd vs vehicle ; figure 4a ) . as early as month 2 , significant increases were observed in both bimatoprost treatment groups compared with the vehicle group in eyebrow fullness ( p .001 , all comparisons ) . improvement from baseline in eyebrow fullness ( a ) and eyebrow darkness ( b ) by visit ( intent - to - treat population ) . bim , bimatoprost . at month 7 , the mean change from baseline in eyebrow darkness as measured by dmsia was significantly higher in the bimatoprost bid group ( 4.53 iu ) and bimatoprost qd group ( 3.76 iu ) compared with the vehicle group ( 0.11 iu ) ( p < .001 , both comparisons ; figure 4b ) . significant improvement was observed in both bimatoprost treatment groups compared with the vehicle group in eyebrow darkness ( p .001 , all comparisons ) as early as month 1 . the proportion of subjects who reported being very satisfied with how the treatment made their eyebrows look ( ess item 6 ) was 17.8% in the bimatoprost bid group , 16.9% in the bimatoprost qd group , and 11.6% in the vehicle group at month 7 . the proportion of subjects who reported being mostly satisfied with how the treatment made their eyebrows look was 52.5% in the bimatoprost bid group , 35.6% in the bimatoprost qd group , and 24.8% in the vehicle group . at month 2 and all subsequent time points , there was a significantly greater proportion of satisfied subjects ( very satisfied / mostly satisfied ) in both bimatoprost treatment groups compared with the vehicle group ( p .031 , all comparisons ; figure 5 ) . percentage of subjects who reported very satisfied / mostly satisfied on ess item 6 by visit ( intent - to - treat population ) . * p < .05 versus vehicle for composite very satisfied and mostly satisfied . overall , 38.1% of the bimatoprost bid subjects , 42.4% of the bimatoprost qd subjects , and 35.5% of subjects in the vehicle groups reported at least 1 teae during the study . the most frequent teae in all groups was upper respiratory tract infection ( 4.2% bimatoprost bid ; 4.2% bimatoprost qd , and 5.8% vehicle ) . treatment - emergent adverse events reported in at least 2% of subjects are shown in table 2 . skin - related teaes included application site pruritus , reported by subjects in all groups ( 0.8% bimatoprost bid , 2.5% bimatoprost qd , and 0.8% vehicle ) and actinic keratosis , reported in the bimatoprost qd and vehicle groups ( 0.8% bimatoprost qd ; 2.5% vehicle ) . ocular hypertension was noted in 1 subject ( 0.8% ) in the bimatoprost qd treatment group . overall , for bimatoprost bid , bimatoprost qd , and vehicle , respectively , teaes were mild in 18.6% , 22.9% , and 14.9% of subjects ; moderate in 12.7% , 16.9% , and 14.0% of subjects , and severe in 4.2% , 0.8% , and 5.0% of subjects . serious teaes were reported in 7 subjects ; 2 in the bimatoprost bid group ( basal cell carcinoma , breast cancer ) and 5 in the vehicle group ( sessile lesion of unknown behavior in hepatic flexure , infected toe , 2 cases of basal cell carcinoma , and cervical fusion separation ) ; no serious teaes were considered treatment related . treatment - emergent adverse events with incidence 2% in any treatment group during treatment period ( safety population ) four subjects discontinued because of teaes , 1 in the bimatoprost bid group because of breast cancer , 2 in the qd group because of treatment - related moderate dermatitis ( n = 1 ) and treatment - related moderate application site rash ( n = 1 ) , and 1 in the vehicle group because of treatment - related moderate application site pruritus . unintended hair growth in other facial areas ( i.e. , eyelashes ) was not observed , and no clinically meaningful changes in vital signs , laboratory findings , or hematology profiles were noted in any treatment group throughout the study . the proportion of subjects with at least a 1-grade increase ( improvement ) in the geba scale from baseline at month 7 was significantly higher in the bimatoprost bid group ( 83.9% ) and bimatoprost qd group ( 77.1% ) compared with the vehicle group ( 43.0% ) ( p < .001 , both comparisons ) ( figure 2 ) . after month 1 , significant improvements were observed in both bimatoprost treatment groups compared with the vehicle group during the treatment period and the posttreatment follow - up period ( p .035 , all comparisons ; figure 2 ) . percentage of subjects with at least a 1-grade improvement from baseline in geba by visit ( intent - to - treat population ) . one subject receiving bimatoprost bid ( a ) and one receiving qd ( b ) had geba grades of 2 ( sparse ) at baseline , 3 ( full ) at month 4 , and 4 ( very full ) at month 7 . the subject receiving vehicle ( c ) had a geba grade of 1 ( very sparse ) at baseline and 2 ( sparse ) at months 4 and 7 . at month 7 , the bimatoprost bid , bimatoprost qd , and vehicle groups experienced mean changes from baseline in eyebrow fullness of 34.51 , 30.96 , and 6.42 mm , respectively ( p < .001 for bimatoprost bid and bimatoprost as early as month 2 , significant increases were observed in both bimatoprost treatment groups compared with the vehicle group in eyebrow fullness ( p .001 , all comparisons ) . improvement from baseline in eyebrow fullness ( a ) and eyebrow darkness ( b ) by visit ( intent - to - treat population ) . bim , bimatoprost . at month 7 , the mean change from baseline in eyebrow darkness as measured by dmsia was significantly higher in the bimatoprost bid group ( 4.53 iu ) and bimatoprost qd group ( 3.76 iu ) compared with the vehicle group ( 0.11 iu ) ( p < .001 , both comparisons ; figure 4b ) . significant improvement was observed in both bimatoprost treatment groups compared with the vehicle group in eyebrow darkness ( p .001 , all comparisons ) as early as month 1 . the proportion of subjects who reported being very satisfied with how the treatment made their eyebrows look ( ess item 6 ) was 17.8% in the bimatoprost bid group , 16.9% in the bimatoprost qd group , and 11.6% in the vehicle group at month 7 . the proportion of subjects who reported being mostly satisfied with how the treatment made their eyebrows look was 52.5% in the bimatoprost bid group , 35.6% in the bimatoprost qd group , and 24.8% in the vehicle group . at month 2 and all subsequent time points , there was a significantly greater proportion of satisfied subjects ( very satisfied / mostly satisfied ) in both bimatoprost treatment groups compared with the vehicle group ( p .031 , all comparisons ; figure 5 ) . percentage of subjects who reported very satisfied / mostly satisfied on ess item 6 by visit ( intent - to - treat population ) . * p < .05 versus vehicle for composite very satisfied and mostly satisfied . p < .001 versus vehicle for composite very satisfied and mostly satisfied . overall , 38.1% of the bimatoprost bid subjects , 42.4% of the bimatoprost qd subjects , and 35.5% of subjects in the vehicle groups reported at least 1 teae during the study . the most frequent teae in all groups was upper respiratory tract infection ( 4.2% bimatoprost bid ; 4.2% bimatoprost qd , and 5.8% vehicle ) . treatment - emergent adverse events reported in at least 2% of subjects are shown in table 2 . skin - related teaes included application site pruritus , reported by subjects in all groups ( 0.8% bimatoprost bid , 2.5% bimatoprost qd , and 0.8% vehicle ) and actinic keratosis , reported in the bimatoprost qd and vehicle groups ( 0.8% bimatoprost qd ; 2.5% vehicle ) . ocular hypertension was noted in 1 subject ( 0.8% ) in the bimatoprost qd treatment group . overall , for bimatoprost bid , bimatoprost qd , and vehicle , respectively , teaes were mild in 18.6% , 22.9% , and 14.9% of subjects ; moderate in 12.7% , 16.9% , and 14.0% of subjects , and severe in 4.2% , 0.8% , and 5.0% of subjects . serious teaes were reported in 7 subjects ; 2 in the bimatoprost bid group ( basal cell carcinoma , breast cancer ) and 5 in the vehicle group ( sessile lesion of unknown behavior in hepatic flexure , infected toe , 2 cases of basal cell carcinoma , and cervical fusion separation ) ; no serious teaes were considered treatment related . treatment - emergent adverse events with incidence 2% in any treatment group during treatment period ( safety population ) four subjects discontinued because of teaes , 1 in the bimatoprost bid group because of breast cancer , 2 in the qd group because of treatment - related moderate dermatitis ( n = 1 ) and treatment - related moderate application site rash ( n = 1 ) , and 1 in the vehicle group because of treatment - related moderate application site pruritus . unintended hair growth in other facial areas ( i.e. , eyelashes ) was not observed , and no clinically meaningful changes in vital signs , laboratory findings , or hematology profiles were noted in any treatment group throughout the study . this first large , phase 3 , randomized , vehicle - controlled study in subjects with eyebrow hypotrichosis demonstrated that bimatoprost 0.03% applied once or twice daily to the eyebrows was effective and safe in subjects with eyebrow hypotrichosis . although statistical comparisons between once - daily and twice - daily application of bimatoprost 0.03% were not made , differences between the two treatment groups on the geba scale and dmsia do not suggest a substantially greater clinical benefit with twice - daily versus once - daily administration . improvements in eyebrow fullness in the bimatoprost groups were observed starting at month 2 on both the geba scale and dmsia , whereas improvements in eyebrow darkness on dmsia were seen as early as month 1 . improvements in eyebrow fullness were noted for subjects in the vehicle group up to month 4 . this may be attributed to natural regrowth of the eyebrows because subjects were instructed not to groom their eyebrows in the treatment area . improvements in the control setting have also been observed with other hair growth products , such as 2% minoxidil . in a randomized , double - masked , split - face study conducted in 39 men and women with eyebrow hypotrichosis receiving minoxidil 2% lotion or placebo , there was a slight increase in eyebrow enhancement in 51% of subjects receiving minoxidil and 23% of subjects receiving placebo . the improvements seen with bimatoprost treatment on eyebrow fullness and darkness confirm and add to earlier preliminary findings in case reports and exploratory studies of bimatoprost . substantial improvement in eyebrow growth was noted for all the reported cases . a single - center , randomized , double - masked , vehicle - controlled study evaluated bimatoprost 0.03% with daily application to the eyebrows for 9 months in 20 women with mild to moderate hypotrichosis . the bimatoprost group demonstrated improvement in eyebrow growth at all time points , with maximum changes seen at month 7 . subject satisfaction at month 9 with eyebrow fullness / thickness and darkness / color was higher in the bimatoprost group than that in the vehicle group . no aes were reported . in another study of 27 subjects with eyebrow hypotrichosis , bimatoprost increased eyebrow growth from baseline , as measured by hair diameter . although subjects in both bimatoprost groups in this study reported greater satisfaction with the effects of treatment than did subjects in the vehicle group , subjects in the bimatoprost bid group had the highest proportion of satisfied subjects at all time points . this observation could reflect the numerically higher treatment responses on the geba scale and dmsia in the bimatoprost bid group compared with the bimatoprost qd group . in this study , bimatoprost 0.03% applied to the eyebrows once or twice daily was safe and well tolerated in this population . overall , no new safety signals were encountered in this study compared with long - term use of bimatoprost for treatment of glaucoma and intraocular hypertension or for treatment of eyelash hypotrichosis . a total of 4 subjects were discontinued from the study because of aes , and the serious aes that occurred in 7 subjects were considered by the investigator to be not related to study treatment . the overall incidence and severity of teaes reported with bimatoprost in this study is comparable with that seen in studies of bimatoprost applied to the eyelashes to treat eyelash hypotrichosis . in those studies , teaes were typically mild to moderate , and the incidence of severe or serious aes and discontinuations due to teaes were relatively low . the most common teaes reported after eyelash application were ocular , such as conjunctival hyperemia , eye pruritus , pinguecula , eye irritation , dry eye , and erythema of the eyelid . the incidence of ocular teaes in this study was low compared with that in the eyelash studies ; this difference may be related to eyebrow application occurring further from the eyes than eyelash application . treatment - emergent adverse events such as skin hyperpigmentation , iris hyperpigmentation , or conjunctival hyperemia , reported at a low incidence in eyelash hypotrichosis studies , were not seen in this study . although this was required to standardize the areas of grooming and treatment across subjects and visits , it does not mirror use in the home environment . the results of this study demonstrate that bimatoprost 0.03% is a safe , well - tolerated , and effective treatment for hypotrichosis of the eyebrows in this population . improvements in eyebrow growth and subject satisfaction appeared within 1 to 2 months and were sustained throughout study treatment . the safety profile of bimatoprost in this study is in line with that shown in previous large , controlled studies of bimatoprost for treatment of eyelash hypotrichosis . based on these results , additional studies on the use of bimatoprost for the treatment of eyebrow hypotrichosis are warranted .

backgroundeyebrow loss may have substantial negative functional and social consequences.objectiveevaluate the safety and efficacy of bimatoprost 0.03% in subjects with eyebrow hypotrichosis.methodsthis multicenter , double - masked study randomized adult females or males with eyebrow hypotrichosis to receive bimatoprost 0.03% twice ( bid ) or once daily ( qd ) or vehicle bid for 7 months . primary endpoint was overall eyebrow fullness at month 7 . secondary endpoints included eyebrow fullness ( mm2 ) , darkness ( intensity units ) , and subject satisfaction with treatment . safety was also assessed.resultsat month 7 , the proportion of subjects with improvement was significantly higher in bimatoprost groups versus vehicle ( both , p < .001 ) . improvements occurred in both bimatoprost groups versus vehicle after month 1 and continued through follow - up ; eyebrow fullness and darkness improved as early as months 2 and 1 , respectively ( both , p < .001 ) . greater satisfaction was reported with bimatoprost versus vehicle at month 2 and all subsequent time points . overall , 38.1% , 42.4% , and 35.5% of subjects in the bimatoprost bid , qd , and vehicle groups , respectively , experienced 1 treatment - emergent adverse event ( teae ) . most frequent teaes were similar across groups . no skin or iris hyperpigmentation or conjunctival hyperemia occurred.conclusionbimatoprost 0.03% bid and qd is safe , well tolerated , and effective for eyebrow hypotrichosis .

METHODS Study Design Subjects Study Endpoints Statistical Analysis RESULTS Primary Endpoint: Global Eyebrow Assessment Additional Endpoints Safety Discussion

subjects were stratified by age group ( < 45 vs 45 yrs ) and were randomly assigned in a 1:1:1 ratio to receive bimatoprost 0.03% twice daily ( bid ) , bimatoprost 0.03% once daily ( qd ) , or vehicle bid for 7 months of treatment . in the bimatoprost qd group , the am dose consisted of vehicle with the pm dose consisting of bimatoprost 0.03% . in the bimatoprost bid group , both the am and pm doses consisted of bimatoprost 0.03% . the total duration of study participation from the screening visit was approximately 9 months , with a total of 11 visits : at screening ( day 21 to 7 ) , day 1 ( baseline ) , week 1 , months 1 , 2 , 3 , 4 , 5 , 6 , and 7 ( or early study discontinuation ) , and at month 8 posttreatment follow - up . efficacy and safety were assessed posttreatment at week 1 , at months 1 through 7 ( or on early discontinuation ) , and at the month 8 follow - up visit . the primary efficacy endpoint was the investigator 's assessment of the subject 's overall eyebrow fullness at month 7 using the geba scale . the primary efficacy variable was defined as the proportion of subjects with at least a 1-grade increase ( improvement ) in the geba grade from baseline at month 7 . secondary efficacy endpoints at month 7 included subjects ' eyebrow fullness ( mm ) and darkness ( intensity units [ iu ] ; negative values are representative of eyebrow darkening ) as measured using digital monitoring system image analysis ( dmsia ) . subject satisfaction with eyebrow treatment at month 7 was measured by item 6 of the 9-item eyebrow satisfaction scale ( ess , follow - up version ) . safety measures included treatment - emergent adverse events ( teaes ) , vital sign measurements ( i.e. assuming a 25% vehicle response rate based on the primary efficacy endpoint and a 2-sided type 1 error of 0.05 , a sample size of 348 with a 1:1:1 randomization ratio for bimatoprost bid , bimatoprost qd , or vehicle was chosen to detect a difference of 20% , 22% , and 25% in favor of bimatoprost bid , with a statistical power of 86% , 92% , and 97% , respectively . to control the type 1 error rate for the primary efficacy endpoint , a serial gatekeeping approach was used : the comparison between bimatoprost qd and vehicle was tested only if statistical analysis of bimatoprost bid versus vehicle was p .05 . changes in eyebrow fullness and darkness were analyzed using dmsia at month 7 ; pairwise comparisons were made using the van elteren test , stratified by age group . this multicenter , double - masked , randomized , vehicle - controlled , 3-arm , parallel - group study was designed to evaluate the safety and efficacy of bimatoprost 0.03% compared with vehicle in subjects with eyebrow hypotrichosis ( clinicaltrials.gov identifier : nct01765764 ) . subjects were stratified by age group ( < 45 vs 45 yrs ) and were randomly assigned in a 1:1:1 ratio to receive bimatoprost 0.03% twice daily ( bid ) , bimatoprost 0.03% once daily ( qd ) , or vehicle bid for 7 months of treatment . in the bimatoprost qd group , the am dose consisted of vehicle with the pm dose consisting of bimatoprost 0.03% . in the bimatoprost bid group , both the am and pm doses consisted of bimatoprost 0.03% . the total duration of study participation from the screening visit was approximately 9 months , with a total of 11 visits : at screening ( day 21 to 7 ) , day 1 ( baseline ) , week 1 , months 1 , 2 , 3 , 4 , 5 , 6 , and 7 ( or early study discontinuation ) , and at month 8 posttreatment follow - up . efficacy and safety were assessed posttreatment at week 1 , at months 1 through 7 ( or on early discontinuation ) , and at the month 8 follow - up visit . the primary efficacy endpoint was the investigator 's assessment of the subject 's overall eyebrow fullness at month 7 using the geba scale . the primary efficacy variable was defined as the proportion of subjects with at least a 1-grade increase ( improvement ) in the geba grade from baseline at month 7 . secondary efficacy endpoints at month 7 included subjects ' eyebrow fullness ( mm ) and darkness ( intensity units [ iu ] ; negative values are representative of eyebrow darkening ) as measured using digital monitoring system image analysis ( dmsia ) . subject satisfaction with eyebrow treatment at month 7 was measured by item 6 of the 9-item eyebrow satisfaction scale ( ess , follow - up version ) . safety measures included treatment - emergent adverse events ( teaes ) , vital sign measurements ( i.e. assuming a 25% vehicle response rate based on the primary efficacy endpoint and a 2-sided type 1 error of 0.05 , a sample size of 348 with a 1:1:1 randomization ratio for bimatoprost bid , bimatoprost qd , or vehicle was chosen to detect a difference of 20% , 22% , and 25% in favor of bimatoprost bid , with a statistical power of 86% , 92% , and 97% , respectively . to control the type 1 error rate for the primary efficacy endpoint , a serial gatekeeping approach was used : the comparison between bimatoprost qd and vehicle was tested only if statistical analysis of bimatoprost bid versus vehicle was p .05 . changes in eyebrow fullness and darkness were analyzed using dmsia at month 7 ; pairwise comparisons were made using the van elteren test , stratified by age group . overall , 339 of 357 ( 95.0% ) subjects completed the study : 115 of 118 ( 97.5% ) , 109 of 118 ( 92.4% ) , and 115 of 121 ( 95.0% ) in the bimatoprost bid , bimatoprost qd , and vehicle groups , respectively . eighteen ( 5.0% ) subjects discontinued the study ; 4 ( 1.1% ) discontinued because of aes , 5 ( 1.4% ) were lost to follow - up , 4 ( 1.1% ) discontinued because of personal reasons , and 5 ( 1.4% ) provided reason as other . demographics and other baseline characteristics ( intent - to - treat population ) the proportion of subjects with at least a 1-grade increase ( improvement ) in the geba scale from baseline at month 7 was significantly higher in the bimatoprost bid group ( 83.9% ) and bimatoprost qd group ( 77.1% ) compared with the vehicle group ( 43.0% ) ( p < .001 , both comparisons ) ( figure 2 ) . note that the bimatoprost bid and qd groups showed similar results . after month 1 , significant improvements were observed in both bimatoprost treatment groups compared with the vehicle group during the treatment period and the posttreatment follow - up period ( p .035 , all comparisons ; figure 2 ) . one subject receiving bimatoprost bid ( a ) and one receiving qd ( b ) had geba grades of 2 ( sparse ) at baseline , 3 ( full ) at month 4 , and 4 ( very full ) at month 7 . at month 7 , the bimatoprost bid , bimatoprost qd , and vehicle groups experienced mean changes from baseline in eyebrow fullness of 34.51 , 30.96 , and 6.42 mm , respectively ( p < .001 for bimatoprost bid and bimatoprost qd vs vehicle ; figure 4a ) . as early as month 2 , significant increases were observed in both bimatoprost treatment groups compared with the vehicle group in eyebrow fullness ( p .001 , all comparisons ) . improvement from baseline in eyebrow fullness ( a ) and eyebrow darkness ( b ) by visit ( intent - to - treat population ) . at month 7 , the mean change from baseline in eyebrow darkness as measured by dmsia was significantly higher in the bimatoprost bid group ( 4.53 iu ) and bimatoprost qd group ( 3.76 iu ) compared with the vehicle group ( 0.11 iu ) ( p < .001 , both comparisons ; figure 4b ) . significant improvement was observed in both bimatoprost treatment groups compared with the vehicle group in eyebrow darkness ( p .001 , all comparisons ) as early as month 1 . the proportion of subjects who reported being very satisfied with how the treatment made their eyebrows look ( ess item 6 ) was 17.8% in the bimatoprost bid group , 16.9% in the bimatoprost qd group , and 11.6% in the vehicle group at month 7 . the proportion of subjects who reported being mostly satisfied with how the treatment made their eyebrows look was 52.5% in the bimatoprost bid group , 35.6% in the bimatoprost qd group , and 24.8% in the vehicle group . at month 2 and all subsequent time points , there was a significantly greater proportion of satisfied subjects ( very satisfied / mostly satisfied ) in both bimatoprost treatment groups compared with the vehicle group ( p .031 , all comparisons ; figure 5 ) . overall , 38.1% of the bimatoprost bid subjects , 42.4% of the bimatoprost qd subjects , and 35.5% of subjects in the vehicle groups reported at least 1 teae during the study . the most frequent teae in all groups was upper respiratory tract infection ( 4.2% bimatoprost bid ; 4.2% bimatoprost qd , and 5.8% vehicle ) . treatment - emergent adverse events reported in at least 2% of subjects are shown in table 2 . skin - related teaes included application site pruritus , reported by subjects in all groups ( 0.8% bimatoprost bid , 2.5% bimatoprost qd , and 0.8% vehicle ) and actinic keratosis , reported in the bimatoprost qd and vehicle groups ( 0.8% bimatoprost qd ; 2.5% vehicle ) . overall , for bimatoprost bid , bimatoprost qd , and vehicle , respectively , teaes were mild in 18.6% , 22.9% , and 14.9% of subjects ; moderate in 12.7% , 16.9% , and 14.0% of subjects , and severe in 4.2% , 0.8% , and 5.0% of subjects . serious teaes were reported in 7 subjects ; 2 in the bimatoprost bid group ( basal cell carcinoma , breast cancer ) and 5 in the vehicle group ( sessile lesion of unknown behavior in hepatic flexure , infected toe , 2 cases of basal cell carcinoma , and cervical fusion separation ) ; no serious teaes were considered treatment related . treatment - emergent adverse events with incidence 2% in any treatment group during treatment period ( safety population ) four subjects discontinued because of teaes , 1 in the bimatoprost bid group because of breast cancer , 2 in the qd group because of treatment - related moderate dermatitis ( n = 1 ) and treatment - related moderate application site rash ( n = 1 ) , and 1 in the vehicle group because of treatment - related moderate application site pruritus . the proportion of subjects with at least a 1-grade increase ( improvement ) in the geba scale from baseline at month 7 was significantly higher in the bimatoprost bid group ( 83.9% ) and bimatoprost qd group ( 77.1% ) compared with the vehicle group ( 43.0% ) ( p < .001 , both comparisons ) ( figure 2 ) . after month 1 , significant improvements were observed in both bimatoprost treatment groups compared with the vehicle group during the treatment period and the posttreatment follow - up period ( p .035 , all comparisons ; figure 2 ) . one subject receiving bimatoprost bid ( a ) and one receiving qd ( b ) had geba grades of 2 ( sparse ) at baseline , 3 ( full ) at month 4 , and 4 ( very full ) at month 7 . at month 7 , the bimatoprost bid , bimatoprost qd , and vehicle groups experienced mean changes from baseline in eyebrow fullness of 34.51 , 30.96 , and 6.42 mm , respectively ( p < .001 for bimatoprost bid and bimatoprost as early as month 2 , significant increases were observed in both bimatoprost treatment groups compared with the vehicle group in eyebrow fullness ( p .001 , all comparisons ) . improvement from baseline in eyebrow fullness ( a ) and eyebrow darkness ( b ) by visit ( intent - to - treat population ) . at month 7 , the mean change from baseline in eyebrow darkness as measured by dmsia was significantly higher in the bimatoprost bid group ( 4.53 iu ) and bimatoprost qd group ( 3.76 iu ) compared with the vehicle group ( 0.11 iu ) ( p < .001 , both comparisons ; figure 4b ) . significant improvement was observed in both bimatoprost treatment groups compared with the vehicle group in eyebrow darkness ( p .001 , all comparisons ) as early as month 1 . the proportion of subjects who reported being very satisfied with how the treatment made their eyebrows look ( ess item 6 ) was 17.8% in the bimatoprost bid group , 16.9% in the bimatoprost qd group , and 11.6% in the vehicle group at month 7 . the proportion of subjects who reported being mostly satisfied with how the treatment made their eyebrows look was 52.5% in the bimatoprost bid group , 35.6% in the bimatoprost qd group , and 24.8% in the vehicle group . at month 2 and all subsequent time points , there was a significantly greater proportion of satisfied subjects ( very satisfied / mostly satisfied ) in both bimatoprost treatment groups compared with the vehicle group ( p .031 , all comparisons ; figure 5 ) . p < .001 versus vehicle for composite very satisfied and mostly satisfied . overall , 38.1% of the bimatoprost bid subjects , 42.4% of the bimatoprost qd subjects , and 35.5% of subjects in the vehicle groups reported at least 1 teae during the study . the most frequent teae in all groups was upper respiratory tract infection ( 4.2% bimatoprost bid ; 4.2% bimatoprost qd , and 5.8% vehicle ) . treatment - emergent adverse events reported in at least 2% of subjects are shown in table 2 . skin - related teaes included application site pruritus , reported by subjects in all groups ( 0.8% bimatoprost bid , 2.5% bimatoprost qd , and 0.8% vehicle ) and actinic keratosis , reported in the bimatoprost qd and vehicle groups ( 0.8% bimatoprost qd ; 2.5% vehicle ) . overall , for bimatoprost bid , bimatoprost qd , and vehicle , respectively , teaes were mild in 18.6% , 22.9% , and 14.9% of subjects ; moderate in 12.7% , 16.9% , and 14.0% of subjects , and severe in 4.2% , 0.8% , and 5.0% of subjects . serious teaes were reported in 7 subjects ; 2 in the bimatoprost bid group ( basal cell carcinoma , breast cancer ) and 5 in the vehicle group ( sessile lesion of unknown behavior in hepatic flexure , infected toe , 2 cases of basal cell carcinoma , and cervical fusion separation ) ; no serious teaes were considered treatment related . treatment - emergent adverse events with incidence 2% in any treatment group during treatment period ( safety population ) four subjects discontinued because of teaes , 1 in the bimatoprost bid group because of breast cancer , 2 in the qd group because of treatment - related moderate dermatitis ( n = 1 ) and treatment - related moderate application site rash ( n = 1 ) , and 1 in the vehicle group because of treatment - related moderate application site pruritus . this first large , phase 3 , randomized , vehicle - controlled study in subjects with eyebrow hypotrichosis demonstrated that bimatoprost 0.03% applied once or twice daily to the eyebrows was effective and safe in subjects with eyebrow hypotrichosis . improvements in eyebrow fullness in the bimatoprost groups were observed starting at month 2 on both the geba scale and dmsia , whereas improvements in eyebrow darkness on dmsia were seen as early as month 1 . improvements in eyebrow fullness were noted for subjects in the vehicle group up to month 4 . in a randomized , double - masked , split - face study conducted in 39 men and women with eyebrow hypotrichosis receiving minoxidil 2% lotion or placebo , there was a slight increase in eyebrow enhancement in 51% of subjects receiving minoxidil and 23% of subjects receiving placebo . the improvements seen with bimatoprost treatment on eyebrow fullness and darkness confirm and add to earlier preliminary findings in case reports and exploratory studies of bimatoprost . a single - center , randomized , double - masked , vehicle - controlled study evaluated bimatoprost 0.03% with daily application to the eyebrows for 9 months in 20 women with mild to moderate hypotrichosis . the bimatoprost group demonstrated improvement in eyebrow growth at all time points , with maximum changes seen at month 7 . subject satisfaction at month 9 with eyebrow fullness / thickness and darkness / color was higher in the bimatoprost group than that in the vehicle group . although subjects in both bimatoprost groups in this study reported greater satisfaction with the effects of treatment than did subjects in the vehicle group , subjects in the bimatoprost bid group had the highest proportion of satisfied subjects at all time points . in this study , bimatoprost 0.03% applied to the eyebrows once or twice daily was safe and well tolerated in this population . the overall incidence and severity of teaes reported with bimatoprost in this study is comparable with that seen in studies of bimatoprost applied to the eyelashes to treat eyelash hypotrichosis . treatment - emergent adverse events such as skin hyperpigmentation , iris hyperpigmentation , or conjunctival hyperemia , reported at a low incidence in eyelash hypotrichosis studies , were not seen in this study . the results of this study demonstrate that bimatoprost 0.03% is a safe , well - tolerated , and effective treatment for hypotrichosis of the eyebrows in this population .

a growing body of studies suggests that social capital has a significant influence on health and health behaviors.1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 the concept of social capital is used in two distinct approaches : the network - based approach and the social cohesion approach . most public health research adopts the latter social cohesion approach , which clarifies the contextual effect of community - level social capital as a group attribute or collective property . community - level social capital is important for older adults to maintain health and well - being , as they are likely to spend many hours in the community . the proportion of older people is currently 26.0% and is predicted to reach 30.3% by 2025 . to tackle issues associated with this situation , the japanese government started a novel public health agenda for the health of older adults called integrated community care for older adults . this agenda aims to build social capital at the community level , improve local healthcare governance , and enrich local resources / environments supporting older residents . therefore , interest in measuring and monitoring social capital at the community level has increased among central and local governments . to date , several scales have been developed to measure social capital , including scales that can be used in the workplace , in the school setting,12 , 13 and for caregivers of children requiring special care needs , as well as for trainees in clinical and translational science . however , to our knowledge , no community social capital scale is available that is useful for studies of older people in industrialized countries like japan . the generalizability of existing social capital scales might be limited , as most of them have been developed in only a few or single communities . information on the validity and reliability of those scales is widely lacking.2 , 16 available scales also fail to capture multiple dimensions of community - level social capital , such as cognitive and structural social capital.2 , 16 in this paper , using large - scale data from a survey of community - dwelling older adults , we developed and validated an instrument to measure community social capital in older community - dwelling populations . various definitions of community social capital have been offered.16 , 17 , 18 , 19 , 20 , 21 of these , influential definitions in the fields of epidemiology and public health include the definition by coleman : a variety of different entities having two characteristics in common : they all consist of some aspect of social structure , and they facilitate certain actions of individuals who are within the structure . putnam 's definition is also well known : features of social organization such as networks , norms , and social trust that facilitate coordination and cooperation for mutual benefit . in social epidemiology , kawachi and berkman introduced a more straightfoward definition that is useful in public health settings : resources that are accessed by individuals as a result of their membership of a network or a group . referring to these definitions , we have developed a health - related social capital scale at the community level because we assumed that our scale would be used to conduct community diagnosis ( i.e. , to evaluate the characteristics of the community and individual residents ) . evaluating the contextual effects of community characteristics on individual health is a key interest of studies and activities of public health . we analyzed cross - sectional data derived from the year 2013 wave of the japan gerontological evaluation study ( jages ) project . jages investigated people aged 65 years or older who did not have physical or cognitive disabilities , a state which was defined as not receiving public long - term care insurance benefits in 30 municipalities . the municipalities were not randomly selected but covered a wide range of characteristics in terms of regions and population sizes in japan . in 13 relatively small municipalities , self - administered questionnaires were mailed to all functionally independent older adults , and in 17 municipalities , questionnaires were mailed to randomly selected older adults based on the official residential registers ( response rate , 71.1% ) . the respondents were 129,739 residents nested in 832 communities that were primarily based on school districts , with some municipal exceptions . communities containing < 50 respondents were excluded to avoid non - precise values due to small samples . ultimately , we derived data from 702 communities comprising 123,760 individuals . the mean number of observations per community was 176 ( standard deviation [ sd ] , 226 ) . we aggregated individual responses into small areas ( i.e. , school districts ) to assess social capital at the community level . although social capital could be evaluated at various levels of aggregations , such as municipality , prefecture , and country levels , we selected the school district as the unit of community in this paper for the following reasons . first , in most regions , school district could represent a geographical scale in which older adults can travel easily by foot or bicycle , and many local activities by community organizations , such as senior citizens club and sports clubs , are performed within each school district . second , school district is valuable unit for considering local public health activities . using school districts as the sampling unit , we could evaluate regional variability in social capital within each municipality , which may help local public health practitioners in conducting their activities . third , it is the smallest area size in which we could maintain sufficient precision of the aggregated information , in terms of the number of samples within each community . referring to available concepts of social capital,16 , 17 , 18 , 19 , 20 , 21 we selected 53 indicators that were potentially associated with social capital ( see etable 1 for full lists of variables ) . for example , selected variables included the proportion of residents in each community who reported participating in community - based activities ( e.g. , volunteer groups ; sports groups or clubs ; hobby activity groups ; senior citizen clubs ; community associations ; study or cultural groups ; nursing care prevention activities ; activities to teach skills or pass on experiences to others ; local events , including festivals and dances ; activities to support older people requiring protection ; activities to support older people requiring nursing care ; activities to support parents raising children ; and local living arrangement improvement or beautification activities ) . items also assessed social integration ( e.g. , average number of friends and frequency of contact with them , the proportion of people who received or provided social support , and interactions with neighbors ) , trust , norms of reciprocity , and attachment . the concurrent validity of our social capital scale was evaluated using the health indicators of self - rated health ( srh ) and depressive symptoms . these are valid predictors of mortality regardless of other medical , behavioral , or psychosocial factors.25 , 26 we measured srh using the question how do you feel about your current health status : excellent , good , fair , or poor ? depressive symptoms were assessed using the 15-item geriatric depression scale ( gds ) that was developed for self - administration in the community using a simple binary ( yes / no ) format.27 , 28 scores 5 on the gds indicate mild to severe depression . first , we aggregated each selected variable into the community ( school district ) level because a community social capital scale should be created from the multiple indicators representing community - level characteristics . for example , when perceptions about trust are aggregated to the group level , it is no longer a measure of personal perceptions but a measure of the trustworthiness of people in the group . second , to extract the variables related to health outcomes , we calculated partial correlations between each candidate variable and the health indicators of srh and gds , after controlling for population density and the proportion of older individuals ( ecological analysis ) . candidate variables with moderate or strong correlations with either srh or gds were then extracted ( r > 0.150 ) . when several variables were conceptually similar , we adopted the variable with the closest relationship to be the health indicators . third , we conducted exploratory factor analysis and eliminated low - communality variables so that the remaining variables maximized internal consistency , as evaluated based on cronbach 's alpha test . fourth , we applied the maximum likelihood method with promax rotations for these factor analyses to account for the correlations among the factors identified . the utilization of multiple community indicators rather than a single indicator in creating a community social capital scale increases the reliability of the scale created . we did not attempt to improve the fit index of our confirmatory factor analysis model via basing the analysis on residual covariance matrices . the concurrent validity of our scale was determined using multilevel poisson regression predicting individual srh and gds . to model contextual effects of community social capital , we used multilevel analysis to account for the variability in health outcomes due to individual compositions ( i.e. , individual 's sociodemographic backgrounds and the responses to the questions used for making our community social capital scale ) . to model potentially different associations between community social capital and individual health across individual characteristics , we also applied a cross - level interaction term . we used stata 12.1 ( statacorp , college station , tx , usa ) and mlwin 2.32 ( centre for multilevel modelling , bristol university , bristol , uk ) for statistical analysis . jages participants were informed that participation in the present study was voluntary and that completing and returning the self - administered questionnaire via mail indicated their consent to participate in the study . ethics approval was obtained from the ethics committee at nihon fukushi university ( 13 - 14 ) . we analyzed cross - sectional data derived from the year 2013 wave of the japan gerontological evaluation study ( jages ) project . jages investigated people aged 65 years or older who did not have physical or cognitive disabilities , a state which was defined as not receiving public long - term care insurance benefits in 30 municipalities . the municipalities were not randomly selected but covered a wide range of characteristics in terms of regions and population sizes in japan . in 13 relatively small municipalities , self - administered questionnaires were mailed to all functionally independent older adults , and in 17 municipalities , questionnaires were mailed to randomly selected older adults based on the official residential registers ( response rate , 71.1% ) . the respondents were 129,739 residents nested in 832 communities that were primarily based on school districts , with some municipal exceptions . communities containing < 50 respondents were excluded to avoid non - precise values due to small samples . ultimately , we derived data from 702 communities comprising 123,760 individuals . the mean number of observations per community was 176 ( standard deviation [ sd ] , 226 ) . we aggregated individual responses into small areas ( i.e. , school districts ) to assess social capital at the community level . although social capital could be evaluated at various levels of aggregations , such as municipality , prefecture , and country levels , we selected the school district as the unit of community in this paper for the following reasons . first , in most regions , school district could represent a geographical scale in which older adults can travel easily by foot or bicycle , and many local activities by community organizations , such as senior citizens club and sports clubs , are performed within each school district . second , school district is valuable unit for considering local public health activities . using school districts as the sampling unit , we could evaluate regional variability in social capital within each municipality , which may help local public health practitioners in conducting their activities . third , it is the smallest area size in which we could maintain sufficient precision of the aggregated information , in terms of the number of samples within each community . referring to available concepts of social capital,16 , 17 , 18 , 19 , 20 , 21 we selected 53 indicators that were potentially associated with social capital ( see etable 1 for full lists of variables ) . for example , selected variables included the proportion of residents in each community who reported participating in community - based activities ( e.g. , volunteer groups ; sports groups or clubs ; hobby activity groups ; senior citizen clubs ; community associations ; study or cultural groups ; nursing care prevention activities ; activities to teach skills or pass on experiences to others ; local events , including festivals and dances ; activities to support older people requiring protection ; activities to support older people requiring nursing care ; activities to support parents raising children ; and local living arrangement improvement or beautification activities ) . items also assessed social integration ( e.g. , average number of friends and frequency of contact with them , the proportion of people who received or provided social support , and interactions with neighbors ) , trust , norms of reciprocity , and attachment . the concurrent validity of our social capital scale was evaluated using the health indicators of self - rated health ( srh ) and depressive symptoms . these are valid predictors of mortality regardless of other medical , behavioral , or psychosocial factors.25 , 26 we measured srh using the question how do you feel about your current health status : excellent , good , fair , or poor ? depressive symptoms were assessed using the 15-item geriatric depression scale ( gds ) that was developed for self - administration in the community using a simple binary ( yes / no ) format.27 , 28 scores 5 on the gds indicate mild to severe depression . first , we aggregated each selected variable into the community ( school district ) level because a community social capital scale should be created from the multiple indicators representing community - level characteristics . for example , when perceptions about trust are aggregated to the group level , it is no longer a measure of personal perceptions but a measure of the trustworthiness of people in the group . second , to extract the variables related to health outcomes , we calculated partial correlations between each candidate variable and the health indicators of srh and gds , after controlling for population density and the proportion of older individuals ( ecological analysis ) . candidate variables with moderate or strong correlations with either srh or gds were then extracted ( r > 0.150 ) . when several variables were conceptually similar , we adopted the variable with the closest relationship to be the health indicators . third , we conducted exploratory factor analysis and eliminated low - communality variables so that the remaining variables maximized internal consistency , as evaluated based on cronbach 's alpha test . fourth , we applied the maximum likelihood method with promax rotations for these factor analyses to account for the correlations among the factors identified . the utilization of multiple community indicators rather than a single indicator in creating a community social capital scale increases the reliability of the scale created . we did not attempt to improve the fit index of our confirmatory factor analysis model via basing the analysis on residual covariance matrices . the concurrent validity of our scale was determined using multilevel poisson regression predicting individual srh and gds . to model contextual effects of community social capital , we used multilevel analysis to account for the variability in health outcomes due to individual compositions ( i.e. , individual 's sociodemographic backgrounds and the responses to the questions used for making our community social capital scale ) . to model potentially different associations between community social capital and individual health across individual characteristics , we also applied a cross - level interaction term . we used stata 12.1 ( statacorp , college station , tx , usa ) and mlwin 2.32 ( centre for multilevel modelling , bristol university , bristol , uk ) for statistical analysis . first , we aggregated each selected variable into the community ( school district ) level because a community social capital scale should be created from the multiple indicators representing community - level characteristics . for example , when perceptions about trust are aggregated to the group level , it is no longer a measure of personal perceptions but a measure of the trustworthiness of people in the group . second , to extract the variables related to health outcomes , we calculated partial correlations between each candidate variable and the health indicators of srh and gds , after controlling for population density and the proportion of older individuals ( ecological analysis ) . candidate variables with moderate or strong correlations with either srh or gds were then extracted ( r > 0.150 ) . when several variables were conceptually similar , we adopted the variable with the closest relationship to be the health indicators . third , we conducted exploratory factor analysis and eliminated low - communality variables so that the remaining variables maximized internal consistency , as evaluated based on cronbach 's alpha test . fourth , we applied the maximum likelihood method with promax rotations for these factor analyses to account for the correlations among the factors identified . the utilization of multiple community indicators rather than a single indicator in creating a community social capital scale increases the reliability of the scale created . we did not attempt to improve the fit index of our confirmatory factor analysis model via basing the analysis on residual covariance matrices . the concurrent validity of our scale was determined using multilevel poisson regression predicting individual srh and gds . to model contextual effects of community social capital , we used multilevel analysis to account for the variability in health outcomes due to individual compositions ( i.e. , individual 's sociodemographic backgrounds and the responses to the questions used for making our community social capital scale ) . to model potentially different associations between community social capital and individual health across individual characteristics , we also applied a cross - level interaction term . we used stata 12.1 ( statacorp , college station , tx , usa ) and mlwin 2.32 ( centre for multilevel modelling , bristol university , bristol , uk ) for statistical analysis . jages participants were informed that participation in the present study was voluntary and that completing and returning the self - administered questionnaire via mail indicated their consent to participate in the study . ethics approval was obtained from the ethics committee at nihon fukushi university ( 13 - 14 ) . based on the results of the correlation analysis , we selected 14 of the 53 candidate variables that were strongly or moderately associated with health indicators ( table 1 ) . we excluded three variables to improve communalities and ultimately adopted 11 for inclusion in our health - related community social capital scale based on internal consistency ( = 0.752 ) ( table 2 ) . exploratory factor analysis ( table 3 ) suggested that three factors ( eigenvalues : 3.317 , 2.633 , and 1.424 ) composed of the 11 variables , with cumulative contribution of 67.0% . the first factor was mainly associated with the participation in volunteer groups , sports groups , hobby activities , study or cultural groups , and activities for teaching skills ( = 0.797 ) . the second factor that was strongly associated with trust , community trust and attachment ( = 0.853 ) , was named social cohesion . the third factor that was strongly associated with receiving and providing emotional support and receiving instrumental support ( = 0.732 ) was named reciprocity . social cohesion score significantly correlated with reciprocity score ( r = 0.436 , p confirmatory factor analysis showed that the root mean square error of approximation was 0.089 , the comparative fit index was 0.925 , the tucker lewis index was 0.899 , and the standardized root mean square residual was 0.058 , which were almost comparable to the criterion of the fit index . table 4 shows descriptive statistics for the variables in the multilevel poisson regression model . individual - level civic participation was calculated via summing the number of civic groups ( up to five ) in which respondents participated once or more per month . strongly / moderately agree and any one or more compared with all other responses . even after controlling for individual socio - demographic status ( i.e. , age , gender , marital status , education , and annual household income ) , all community - level social capital scores were significantly associated with depressive symptoms ( table 5 ) . the prevalence ratio ( pr ) was 0.94 ( 95% confidence interval [ ci ] , 0.920.95 ) per 1 sd increase in the score for civic participation . the prs for social cohesion and reciprocity were 0.97 ( 95% ci , 0.950.99 ) and 0.96 ( 95% ci , 0.950.98 ) , respectively . when the outcome was changed to self - rated health , the prs of social capital scores were similar to the associations with gds scores , although statistical significance was marginal for social cohesion and reciprocity . additional adjustments for individual - level responses to the questions used to form our scale for evaluating social capital did not affect the prs for civic participation ( pr for poor / fair srh , 0.96 ; 95% ci , 0.940.98 ; pr for gds , 0.95 ; 95% ci , 0.930.97 ; model 2 ) . reciprocity was also associated with individual gds ( pr 0.98 ; 95% ci , 0.961.00 ) . on the other hand , the same adjustment attenuated the association between community - level social cohesion and individual health indicators . most cross - level ( individual and community ) interactions were not statistically significant ( model 3 ) . applying an indicator method using a dummy variable as categorical data , which was done to account for non - normal distribution and missing data , did not alter the major results and trends ( etable 2 ) . in addition , correlation coefficients between each community - level social capital score and individual responses were not high ( 0.090.17 ) . we developed and validated an 11-item scale , which was comprised of sub - dimensions of civic participation , social cohesion , and reciprocity , to measure social capital at the community level . evaluations of communalities of factors and internal consistency , as well as confirmatory factor analysis , demonstrated that these 11 indicators formed a reliable scale . we also found evidence to support convergent validity : the indicators were correlated with health outcomes in expected directions . our instrument might be useful for gerontological studies and activities in japan , and , although further studies are needed , the scale may be useful in other countries with a similar context to that of japan . the distinction among civic participation , social cohesion , and reciprocity dimensions are fundamental in social capital theory.3 , 16 , 17 , 29 our factor analysis statistically identified these three components . according to islam et al , the structural dimension of social capital includes externally observable aspects of social organization and is characterized by behavioral manifestations of network connections or civic engagement . this concept was reflected in the variables included in our civic participation and reciprocity variables . that the factors reflecting subjective attitudes , such as trust , norms of reciprocity , and attachment within the community , were collectively named social cohesion was also theoretically reasonable . the reciprocity component of our social capital scale may capture the dimension of community social capital that promotes the exchange of individual social supports within the community . our results , which showed strong correlation between reciprocity and social cohesion , are also consistent with the theoretical framework . we did not find remarkable inflations of confidence intervals or strong correlation between community - level social capital scores and individual responses after adjusting for individual measures . social capital studies in public health have so far yielded mixed findings , potentially as a result of inconsistencies in the manner in which investigators have operationalized and measured the concept of social capital , often by resorting to proxy variables available through secondary data sets . previous studies have used indicators , such as community voting rates in elections or the local crime rate , as proxies for social capital . although these variables could be viewed as either antecedents or consequences of social capital , they do not directly capture its core concepts . in addition , differences between study findings might relate to variations in culture , region , units of analysis , and age cohorts . although communities in japan might have closer social ties than those in the united states , generalized trust , which is the central concept of social cohesion or cognitive social capital , is low in cohesive societies because human relations in such a society are based on mutual assurance rather than trust . this has resulted in an unfavorable effect of trust on health in some japanese studies.31 , 32 nonetheless , studies to date are difficult to compare due to their use of conventionally created alternative scales based on an assortment of concepts . hence , we believe that our novel scale for measuring community social capital could provide a useful option for such studies . using a common scale would contribute to the discussion about between - study differences of the true interests of investigators . our findings suggest that community - level civic participation is more closely correlated with health outcomes than social cohesion . this might be because the indicators of civic participation applied in our study were more objective than those used to determine social cohesion . our analysis also showed a reverse predictive value of community - level social cohesion for health indicators . portes 's concepts of the dark side of social capital may help understanding the reasons . portes has pointed out four potentially harmful characteristics of group - level social capital : the exclusion of outsiders , excess claims on group members , restrictions on individual freedoms , and downward - leveling norms . a japanese empirical study demonstrated that stronger social cohesion was associated with depressive symptoms in residents whose hometown of origin differed from the communities where they currently resided . alternatively , the weaker predictive value of community - level social cohesion might be explained via measurement bias due to the potential non - participation of those who trust others less . compared with information about individual memberships in community groups , individual perceptions regarding trust or reciprocity could be more easily influenced by various temporary conditions . this might also reflect some degree of reverse causation , in which healthier people are more likely to participate . although individual - level perceptions of social capital were more strongly associated with individual health compared with community - level social capital , this is not unexpected , since more proximal exposure to the individual is likely to correlate with individual - level health outcomes . the survey was originally designed to measure social capital , which allowed the use of various conceptually appropriate candidate variables . the large sample size in terms of numbers of individual participants and communities in particular is an important strength , as our analysis has sufficient power to create a community - level scale . we created a social capital scale specifically for the elderly population , which is the first of its kind . the cross - sectional design might have included reverse causality that potentially biased the results of our evaluation of concurrent validity . moreover , it would be better to conduct further validations . for example , criterion validity could be evaluated using more objectively measured community - level variables , such as the proportion of participation in each organization and the voting rate . although the response rate to our survey was relatively high ( 71.1% ) , selection bias can not be fully excluded . generalizability might be limited because our dataset was not a nationally representative sample and was created for older adults . caution is needed when applying our community social capital scale to data obtained from alternative contexts , such as younger populations or using data created via alternative survey methods . however , the geographic and cultural variations of the municipalities included in our sample were high , and the municipalities included metropolitan and rural areas . moreover , we used school districts as community units , but we do not know whether our scale would be similarly valid when evaluating a community defined using an alternative area unit . ideally , a social capital index that can be applicable at any level of aggregation should be created . however , we found that the three factors based on our alternative factor analyses using individual - level data also showed the same items in each factor , suggesting the potential generalizability of our social capital scores to alternative units of aggregation . the survey was originally designed to measure social capital , which allowed the use of various conceptually appropriate candidate variables . the large sample size in terms of numbers of individual participants and communities in particular is an important strength , as our analysis has sufficient power to create a community - level scale . we created a social capital scale specifically for the elderly population , which is the first of its kind . the cross - sectional design might have included reverse causality that potentially biased the results of our evaluation of concurrent validity . for example , criterion validity could be evaluated using more objectively measured community - level variables , such as the proportion of participation in each organization and the voting rate . although the response rate to our survey was relatively high ( 71.1% ) , selection bias can not be fully excluded . generalizability might be limited because our dataset was not a nationally representative sample and was created for older adults . caution is needed when applying our community social capital scale to data obtained from alternative contexts , such as younger populations or using data created via alternative survey methods . however , the geographic and cultural variations of the municipalities included in our sample were high , and the municipalities included metropolitan and rural areas . moreover , we used school districts as community units , but we do not know whether our scale would be similarly valid when evaluating a community defined using an alternative area unit . ideally , a social capital index that can be applicable at any level of aggregation should be created . however , we found that the three factors based on our alternative factor analyses using individual - level data also showed the same items in each factor , suggesting the potential generalizability of our social capital scores to alternative units of aggregation . we developed a health - related community social capital scale , which was composed of 11 items assessing civic participation , social cohesion , and reciprocity . this new standard social capital measure could shed light on public health and gerontological issues , as well as other matters associated with community social capital . conceived and designed the analysis : ms , nk , ja . performed the survey : kk , the jages group . contributed to the writing of the manuscript : ms , nk , ja , ik , kk , to .

backgroundwe developed and validated an instrument to measure community - level social capital based on data derived from older community dwellers in japan.methodswe used cross - sectional data from the japan gerontological evaluation study , a nationwide survey involving 123,760 functionally independent older people nested within 702 communities ( i.e. , school districts ) . we conducted exploratory and confirmatory factor analyses on survey items to determine the items in a multi - dimensional scale to measure community social capital . internal consistency was checked with cronbach 's alpha . convergent construct validity was assessed via correlating the scale with health outcomes.resultsfrom 53 candidate variables , 11 community - level variables were extracted : participation in volunteer groups , sports groups , hobby activities , study or cultural groups , and activities for teaching specific skills ; trust , norms of reciprocity , and attachment to one 's community ; received emotional support ; provided emotional support ; and received instrumental support . using factor analysis , these variables were determined to belong to three sub - scales : civic participation ( eigenvalue = 3.317 , = 0.797 ) , social cohesion ( eigenvalue = 2.633 , = 0.853 ) , and reciprocity ( eigenvalue = 1.424 , = 0.732 ) . confirmatory factor analysis indicated the goodness of fit of this model . multilevel poisson regression analysis revealed that civic participation score was robustly associated with individual subjective health ( self - rated health : prevalence ratio [ pr ] 0.96 ; 95% confidence interval [ ci ] , 0.940.98 ; geriatric depression scale [ gds ] : pr 0.95 ; 95% ci , 0.930.97 ) . reciprocity score was also associated with individual gds ( pr 0.98 ; 95% ci , 0.961.00 ) . social cohesion score was not consistently associated with individual health indicators.conclusionsour scale for measuring social capital at the community level might be useful for future studies of older community dwellers .

Introduction Methods Data Selection of candidate variables for social capital scale Subjective health indicators Statistical analysis Selection of variables for social capital scale Evaluating concurrent validity Ethical considerations Results Discussion Strengths and limitations Conclusion Author's contributions Conflicts of interest Supplementary data

a growing body of studies suggests that social capital has a significant influence on health and health behaviors.1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 the concept of social capital is used in two distinct approaches : the network - based approach and the social cohesion approach . most public health research adopts the latter social cohesion approach , which clarifies the contextual effect of community - level social capital as a group attribute or collective property . community - level social capital is important for older adults to maintain health and well - being , as they are likely to spend many hours in the community . to tackle issues associated with this situation , the japanese government started a novel public health agenda for the health of older adults called integrated community care for older adults . this agenda aims to build social capital at the community level , improve local healthcare governance , and enrich local resources / environments supporting older residents . therefore , interest in measuring and monitoring social capital at the community level has increased among central and local governments . however , to our knowledge , no community social capital scale is available that is useful for studies of older people in industrialized countries like japan . the generalizability of existing social capital scales might be limited , as most of them have been developed in only a few or single communities . information on the validity and reliability of those scales is widely lacking.2 , 16 available scales also fail to capture multiple dimensions of community - level social capital , such as cognitive and structural social capital.2 , 16 in this paper , using large - scale data from a survey of community - dwelling older adults , we developed and validated an instrument to measure community social capital in older community - dwelling populations . various definitions of community social capital have been offered.16 , 17 , 18 , 19 , 20 , 21 of these , influential definitions in the fields of epidemiology and public health include the definition by coleman : a variety of different entities having two characteristics in common : they all consist of some aspect of social structure , and they facilitate certain actions of individuals who are within the structure . putnam 's definition is also well known : features of social organization such as networks , norms , and social trust that facilitate coordination and cooperation for mutual benefit . referring to these definitions , we have developed a health - related social capital scale at the community level because we assumed that our scale would be used to conduct community diagnosis ( i.e. evaluating the contextual effects of community characteristics on individual health is a key interest of studies and activities of public health . we analyzed cross - sectional data derived from the year 2013 wave of the japan gerontological evaluation study ( jages ) project . in 13 relatively small municipalities , self - administered questionnaires were mailed to all functionally independent older adults , and in 17 municipalities , questionnaires were mailed to randomly selected older adults based on the official residential registers ( response rate , 71.1% ) . the respondents were 129,739 residents nested in 832 communities that were primarily based on school districts , with some municipal exceptions . , school districts ) to assess social capital at the community level . although social capital could be evaluated at various levels of aggregations , such as municipality , prefecture , and country levels , we selected the school district as the unit of community in this paper for the following reasons . first , in most regions , school district could represent a geographical scale in which older adults can travel easily by foot or bicycle , and many local activities by community organizations , such as senior citizens club and sports clubs , are performed within each school district . using school districts as the sampling unit , we could evaluate regional variability in social capital within each municipality , which may help local public health practitioners in conducting their activities . referring to available concepts of social capital,16 , 17 , 18 , 19 , 20 , 21 we selected 53 indicators that were potentially associated with social capital ( see etable 1 for full lists of variables ) . , volunteer groups ; sports groups or clubs ; hobby activity groups ; senior citizen clubs ; community associations ; study or cultural groups ; nursing care prevention activities ; activities to teach skills or pass on experiences to others ; local events , including festivals and dances ; activities to support older people requiring protection ; activities to support older people requiring nursing care ; activities to support parents raising children ; and local living arrangement improvement or beautification activities ) . , average number of friends and frequency of contact with them , the proportion of people who received or provided social support , and interactions with neighbors ) , trust , norms of reciprocity , and attachment . the concurrent validity of our social capital scale was evaluated using the health indicators of self - rated health ( srh ) and depressive symptoms . depressive symptoms were assessed using the 15-item geriatric depression scale ( gds ) that was developed for self - administration in the community using a simple binary ( yes / no ) format.27 , 28 scores 5 on the gds indicate mild to severe depression . first , we aggregated each selected variable into the community ( school district ) level because a community social capital scale should be created from the multiple indicators representing community - level characteristics . second , to extract the variables related to health outcomes , we calculated partial correlations between each candidate variable and the health indicators of srh and gds , after controlling for population density and the proportion of older individuals ( ecological analysis ) . when several variables were conceptually similar , we adopted the variable with the closest relationship to be the health indicators . third , we conducted exploratory factor analysis and eliminated low - communality variables so that the remaining variables maximized internal consistency , as evaluated based on cronbach 's alpha test . the utilization of multiple community indicators rather than a single indicator in creating a community social capital scale increases the reliability of the scale created . to model contextual effects of community social capital , we used multilevel analysis to account for the variability in health outcomes due to individual compositions ( i.e. , individual 's sociodemographic backgrounds and the responses to the questions used for making our community social capital scale ) . to model potentially different associations between community social capital and individual health across individual characteristics , we also applied a cross - level interaction term . we analyzed cross - sectional data derived from the year 2013 wave of the japan gerontological evaluation study ( jages ) project . in 13 relatively small municipalities , self - administered questionnaires were mailed to all functionally independent older adults , and in 17 municipalities , questionnaires were mailed to randomly selected older adults based on the official residential registers ( response rate , 71.1% ) . the respondents were 129,739 residents nested in 832 communities that were primarily based on school districts , with some municipal exceptions . ultimately , we derived data from 702 communities comprising 123,760 individuals . we aggregated individual responses into small areas ( i.e. , school districts ) to assess social capital at the community level . first , in most regions , school district could represent a geographical scale in which older adults can travel easily by foot or bicycle , and many local activities by community organizations , such as senior citizens club and sports clubs , are performed within each school district . referring to available concepts of social capital,16 , 17 , 18 , 19 , 20 , 21 we selected 53 indicators that were potentially associated with social capital ( see etable 1 for full lists of variables ) . , volunteer groups ; sports groups or clubs ; hobby activity groups ; senior citizen clubs ; community associations ; study or cultural groups ; nursing care prevention activities ; activities to teach skills or pass on experiences to others ; local events , including festivals and dances ; activities to support older people requiring protection ; activities to support older people requiring nursing care ; activities to support parents raising children ; and local living arrangement improvement or beautification activities ) . , average number of friends and frequency of contact with them , the proportion of people who received or provided social support , and interactions with neighbors ) , trust , norms of reciprocity , and attachment . the concurrent validity of our social capital scale was evaluated using the health indicators of self - rated health ( srh ) and depressive symptoms . depressive symptoms were assessed using the 15-item geriatric depression scale ( gds ) that was developed for self - administration in the community using a simple binary ( yes / no ) format.27 , 28 scores 5 on the gds indicate mild to severe depression . first , we aggregated each selected variable into the community ( school district ) level because a community social capital scale should be created from the multiple indicators representing community - level characteristics . second , to extract the variables related to health outcomes , we calculated partial correlations between each candidate variable and the health indicators of srh and gds , after controlling for population density and the proportion of older individuals ( ecological analysis ) . when several variables were conceptually similar , we adopted the variable with the closest relationship to be the health indicators . third , we conducted exploratory factor analysis and eliminated low - communality variables so that the remaining variables maximized internal consistency , as evaluated based on cronbach 's alpha test . the utilization of multiple community indicators rather than a single indicator in creating a community social capital scale increases the reliability of the scale created . we did not attempt to improve the fit index of our confirmatory factor analysis model via basing the analysis on residual covariance matrices . the concurrent validity of our scale was determined using multilevel poisson regression predicting individual srh and gds . to model contextual effects of community social capital , we used multilevel analysis to account for the variability in health outcomes due to individual compositions ( i.e. , individual 's sociodemographic backgrounds and the responses to the questions used for making our community social capital scale ) . to model potentially different associations between community social capital and individual health across individual characteristics , we also applied a cross - level interaction term . first , we aggregated each selected variable into the community ( school district ) level because a community social capital scale should be created from the multiple indicators representing community - level characteristics . second , to extract the variables related to health outcomes , we calculated partial correlations between each candidate variable and the health indicators of srh and gds , after controlling for population density and the proportion of older individuals ( ecological analysis ) . when several variables were conceptually similar , we adopted the variable with the closest relationship to be the health indicators . third , we conducted exploratory factor analysis and eliminated low - communality variables so that the remaining variables maximized internal consistency , as evaluated based on cronbach 's alpha test . the utilization of multiple community indicators rather than a single indicator in creating a community social capital scale increases the reliability of the scale created . we did not attempt to improve the fit index of our confirmatory factor analysis model via basing the analysis on residual covariance matrices . the concurrent validity of our scale was determined using multilevel poisson regression predicting individual srh and gds . to model contextual effects of community social capital , we used multilevel analysis to account for the variability in health outcomes due to individual compositions ( i.e. to model potentially different associations between community social capital and individual health across individual characteristics , we also applied a cross - level interaction term . jages participants were informed that participation in the present study was voluntary and that completing and returning the self - administered questionnaire via mail indicated their consent to participate in the study . based on the results of the correlation analysis , we selected 14 of the 53 candidate variables that were strongly or moderately associated with health indicators ( table 1 ) . we excluded three variables to improve communalities and ultimately adopted 11 for inclusion in our health - related community social capital scale based on internal consistency ( = 0.752 ) ( table 2 ) . exploratory factor analysis ( table 3 ) suggested that three factors ( eigenvalues : 3.317 , 2.633 , and 1.424 ) composed of the 11 variables , with cumulative contribution of 67.0% . the first factor was mainly associated with the participation in volunteer groups , sports groups , hobby activities , study or cultural groups , and activities for teaching skills ( = 0.797 ) . the second factor that was strongly associated with trust , community trust and attachment ( = 0.853 ) , was named social cohesion . the third factor that was strongly associated with receiving and providing emotional support and receiving instrumental support ( = 0.732 ) was named reciprocity . social cohesion score significantly correlated with reciprocity score ( r = 0.436 , p confirmatory factor analysis showed that the root mean square error of approximation was 0.089 , the comparative fit index was 0.925 , the tucker lewis index was 0.899 , and the standardized root mean square residual was 0.058 , which were almost comparable to the criterion of the fit index . table 4 shows descriptive statistics for the variables in the multilevel poisson regression model . individual - level civic participation was calculated via summing the number of civic groups ( up to five ) in which respondents participated once or more per month . even after controlling for individual socio - demographic status ( i.e. , age , gender , marital status , education , and annual household income ) , all community - level social capital scores were significantly associated with depressive symptoms ( table 5 ) . the prevalence ratio ( pr ) was 0.94 ( 95% confidence interval [ ci ] , 0.920.95 ) per 1 sd increase in the score for civic participation . the prs for social cohesion and reciprocity were 0.97 ( 95% ci , 0.950.99 ) and 0.96 ( 95% ci , 0.950.98 ) , respectively . when the outcome was changed to self - rated health , the prs of social capital scores were similar to the associations with gds scores , although statistical significance was marginal for social cohesion and reciprocity . additional adjustments for individual - level responses to the questions used to form our scale for evaluating social capital did not affect the prs for civic participation ( pr for poor / fair srh , 0.96 ; 95% ci , 0.940.98 ; pr for gds , 0.95 ; 95% ci , 0.930.97 ; model 2 ) . reciprocity was also associated with individual gds ( pr 0.98 ; 95% ci , 0.961.00 ) . on the other hand , the same adjustment attenuated the association between community - level social cohesion and individual health indicators . in addition , correlation coefficients between each community - level social capital score and individual responses were not high ( 0.090.17 ) . we developed and validated an 11-item scale , which was comprised of sub - dimensions of civic participation , social cohesion , and reciprocity , to measure social capital at the community level . evaluations of communalities of factors and internal consistency , as well as confirmatory factor analysis , demonstrated that these 11 indicators formed a reliable scale . we also found evidence to support convergent validity : the indicators were correlated with health outcomes in expected directions . our instrument might be useful for gerontological studies and activities in japan , and , although further studies are needed , the scale may be useful in other countries with a similar context to that of japan . the distinction among civic participation , social cohesion , and reciprocity dimensions are fundamental in social capital theory.3 , 16 , 17 , 29 our factor analysis statistically identified these three components . this concept was reflected in the variables included in our civic participation and reciprocity variables . that the factors reflecting subjective attitudes , such as trust , norms of reciprocity , and attachment within the community , were collectively named social cohesion was also theoretically reasonable . the reciprocity component of our social capital scale may capture the dimension of community social capital that promotes the exchange of individual social supports within the community . we did not find remarkable inflations of confidence intervals or strong correlation between community - level social capital scores and individual responses after adjusting for individual measures . in addition , differences between study findings might relate to variations in culture , region , units of analysis , and age cohorts . although communities in japan might have closer social ties than those in the united states , generalized trust , which is the central concept of social cohesion or cognitive social capital , is low in cohesive societies because human relations in such a society are based on mutual assurance rather than trust . hence , we believe that our novel scale for measuring community social capital could provide a useful option for such studies . our findings suggest that community - level civic participation is more closely correlated with health outcomes than social cohesion . this might be because the indicators of civic participation applied in our study were more objective than those used to determine social cohesion . our analysis also showed a reverse predictive value of community - level social cohesion for health indicators . portes has pointed out four potentially harmful characteristics of group - level social capital : the exclusion of outsiders , excess claims on group members , restrictions on individual freedoms , and downward - leveling norms . a japanese empirical study demonstrated that stronger social cohesion was associated with depressive symptoms in residents whose hometown of origin differed from the communities where they currently resided . alternatively , the weaker predictive value of community - level social cohesion might be explained via measurement bias due to the potential non - participation of those who trust others less . although individual - level perceptions of social capital were more strongly associated with individual health compared with community - level social capital , this is not unexpected , since more proximal exposure to the individual is likely to correlate with individual - level health outcomes . the survey was originally designed to measure social capital , which allowed the use of various conceptually appropriate candidate variables . the large sample size in terms of numbers of individual participants and communities in particular is an important strength , as our analysis has sufficient power to create a community - level scale . the cross - sectional design might have included reverse causality that potentially biased the results of our evaluation of concurrent validity . for example , criterion validity could be evaluated using more objectively measured community - level variables , such as the proportion of participation in each organization and the voting rate . generalizability might be limited because our dataset was not a nationally representative sample and was created for older adults . caution is needed when applying our community social capital scale to data obtained from alternative contexts , such as younger populations or using data created via alternative survey methods . ideally , a social capital index that can be applicable at any level of aggregation should be created . however , we found that the three factors based on our alternative factor analyses using individual - level data also showed the same items in each factor , suggesting the potential generalizability of our social capital scores to alternative units of aggregation . the survey was originally designed to measure social capital , which allowed the use of various conceptually appropriate candidate variables . the large sample size in terms of numbers of individual participants and communities in particular is an important strength , as our analysis has sufficient power to create a community - level scale . the cross - sectional design might have included reverse causality that potentially biased the results of our evaluation of concurrent validity . for example , criterion validity could be evaluated using more objectively measured community - level variables , such as the proportion of participation in each organization and the voting rate . generalizability might be limited because our dataset was not a nationally representative sample and was created for older adults . caution is needed when applying our community social capital scale to data obtained from alternative contexts , such as younger populations or using data created via alternative survey methods . ideally , a social capital index that can be applicable at any level of aggregation should be created . however , we found that the three factors based on our alternative factor analyses using individual - level data also showed the same items in each factor , suggesting the potential generalizability of our social capital scores to alternative units of aggregation . we developed a health - related community social capital scale , which was composed of 11 items assessing civic participation , social cohesion , and reciprocity . this new standard social capital measure could shed light on public health and gerontological issues , as well as other matters associated with community social capital .

human immunodeficiency virus ( hiv ) is the second leading cause of death in the world[13 ] , the first in the caribbean ( among 15 - 49 year olds ) and the second in jamaica . the risk of contracting hiv is considered higher in low - income countries , and lowers in industrialized countries . factors that contribute to such discrepancies in sexual and reproductive risk all over the world are weak and uneven distribution of health services , the concentration of poverty among certain population groups and geographic areas , gender inequalities and harmful social practices . this represents almost 3 billion people in the world , and almost half of all new hiv infections . in jamaica the public health dilemma of hiv among youths has led to commitments made by various nations ( 189 states ) of the world at the united nations general assembly special session ( ungass ) on hiv and aids in new york in 2001 . such commitment was made via the signing of a declaration of commitment which encapsulates promises to acknowledge the role and contributions of young people in addressing all aspects of hiv and aids , recognizing the full involvement and participation of the youth , in designing , planning , implementing and evaluating programs relating to responses to the epidemic ; reducing the prevalence of hiv among youths within the range of 15 - 24 years of age by 25% by 2010 , ensuring that 90% of youths have access to information and services that would reduce their vulnerability to hiv infection , ensuring access to information through primary and secondary school curricula on matters of safe and secure environment , strengthening sexual and reproductive health programs , and so on . under the caricom - pancap , the strategic objectives for national hiv responses are to prevent the sexual transmission of hiv , to decrease the vulnerability to sexual transmission of hiv ; to establish comprehensive , gender - sensitive , and targeted prevention programs for children ( 9 - 14 years old ) and the youth ( 15 - 24 years old ) , to achieve universal access to targeted prevention interventions among the most at - risk populations ( such as msm , sw , drug users , prisoners , and migrant populations ) , to provide services for the prevention of mother - to - child transmission of hiv to all pregnant women and their families ; to strengthen prevention efforts among plhiv as part of comprehensive care ; and to reduce vulnerability to hiv through early identification and treatment of other sexually transmitted infections ( stis ) . the achievement of these objectives can also be hindered by policies ( for example , legislation against men having sex with men ( msm ) , the capacity to address legal constraints that hinder access to services , the lack of integration of hiv policies and programs into national development plans and programs , the lack of political support and incongruities between policies and legislations . within the context of the high hiv incidence and prevalence rates among people in developing nations , and in particular the caribbean , and more so among young people , the attitude toward consistent condom usage is problematic and needs to be examined in developing countries . hence , we wanted to elucidate information as to whether there are differences in the knowledge , attitude and practices of adults in their reproductive years regarding their reproductive health issues , compared with those who have hiv in a caribbean society , as well as to model factors which account for their willingness to do an hiv test in the future . no study emerged in the caribbean that has comprehensively examined adults in their reproductive ages ( 15 - 49 years ) on their sexual and reproductive health attitudes , knowledge and practices , and compares the result with those who are hiv - infected youth , as well as factors which explain people 's willingness to do an hiv test in the future . the present study examines core issues of sexual and reproductive health among youths , particularly with respect to those who are hiv - infected in jamaica in order to provide a comprehensive understanding of people 's perceptions , which will be used to fashion public health intervention programs . the study population comprised people aged 15 - 49 years who resided in jamaica at the time of the survey in 2004 ( may - august ) . the population data for this research were collected by hope enterprises limited on behalf of the jamaican ministry of health . each of the 14 parishes in jamaica was stratified into constituencies , with each constituency stratified into three areas - rural areas , parish capitals ( urban areas ) and main towns ( semi - urban areas ) . the areas which comprised a constituency were then stratified into primary sampling units ( psus ) or enumeration districts ( eds ) . a random sample of each psu 23 eds in urban areas , 25eds in semi - urban areas , and 24 eds in rural areas . twenty - five households were systematically chosen from each ed , and cluster sampling was carried out , where all the people living in the household of the designated areas were interviewed for the survey . the questions were demographic characteristics , sexual history ( including number and type of partners , and having sexual relations with commercial sex workers ) , condom usage , stis and health issues , knowledge of hiv / aids ( including have you ever had an hiv test ? , did you go back for the results yourself or were you contacted by a health worker ? , and would you be willing to do an hiv test [ in the future ] ? ) . the interviewers were trained for a 5-day period , of which 2 days were devoted to field practices . interviewers were assigned to a team composed of two females , two males and a supervisor . interviewees were informed of confidentiality and their right to stop the interview at any time , if they should so desire . no names , addresses or other personal information were collected from respondents in order to ensure anonymity and confidentiality . the instrument used in the survey utilized indicator measures and definitions consistent with unaids and the usaid priority prevention indicator . data were entered , stored and retrieved using spss for windows , version 16.0 spss inc ; chicago , il , usa ) . statistical analyses used were an independent sample t - test , anova , and pearson 's product moment correlation . multivariate logistic regressions were fitted using one outcome measure : self - reported , confirmed positive hiv test results . where collinearity existed ( r > 0.7 ) , variables were entered independently into the model to determine those that should be retained during the final model construction . a p - value < 0.05 ( two - tailed ) was used to establish statistical significance . for this study , the analytic model used is one that can accommodate multiple independent variables on a single binary dependent variable ( positive hiv test result , which was confirmed by an agent of the state ) . using logistic regression , this paper tested variables identified in the literature as being associated with having a positive hiv test result ( equation ) : hivti = f(ai , xi , edi , ei , msi , ci , sii , ni , asi , li , cui , ki , fi , pi , ti , stii , ri , qi) eqn where hivti denotes currently having a positive hiv test result for individual i , ai is age of individual i , edi represents educational level of individual i , ui , means employment status of individual i , ssi is social class of individual i , ari indicates area of residence of individual i , pi denotes currently having sexual relations with a commercial sex worker for individual i , msi is marital status of individual i , ci means length of time dwelling in community for individual i , sli is age of first sexual intercourse of individual i , si represents type of sexual practice of individual i , ni is number of sexual partners of individual i , ri denotes actively practicing religion of individual i , ki is having had an sti of individual i , wi represents crowding in household of individual i , qi denotes frequency of condom usage of the individual i , and the parameter i is the model 's error term . using logistic regression to test the hypothesis ( equation ) , we now know that marital status , employment status , age of respondents , and other variables are associated with those who are currently hiv positive individuals , and can write equation . hivti = f(ai , msi , ui , pi , i) .. eqn crowding is the total number of persons who dwell in a room ( excluding kitchen , bathroom and verandah ) . these methods include tubal ligation , vasectomy , implant ( norplant ) , injection , emergency contraceptive protection , pill , condom , foaming tablets , creams , jellies , diaphragm , abstinence , withdrawal , the rhythm method , calendar or billings . non - steady sexual partner denotes sexual relations that are casual , with someone with whom the individual is not having a common law sexual union , a visiting relationship or to whom the individual is not legally married . education is taken from the question , how many years did you attend school? this is coded as junior high or below ( 0 9 years ) , secondary ( 10 - 12 years ) and tertiary ( 13 + years ) . shared facility is taken from are these [ sanitary conveniences ] shared with another household ? did you go back for the results yourself or were you contacted by a health worker ? if the individual indicated that he / she was contacted by a health worker , this was used to indicate a positive hiv result . old hiv infected people were measured using self - reported information on the do you know the result of the test [ hiv ] , and whether this was positive or negative ( 1= know positive status , 0 = otherwise ) . knowledge in this study was measured using the following issues : have you heard of hiv ? , have you heard of a disease called aids ? , do you think that a healthy looking person can be infected with hiv , the virus that causes aids ? attitude for this research was measured using the following issues : if a member of your family became sick with hiv , the virus that causes aids , would you be willing to care for him or her in your household ? , would you be willing to do an hiv test [ in the future ] ? practice was evaluated from the following questions : have you ever had sexual intercourse ? by sexual intercourse i mean vaginal sex ( penis in vagina ) or anal sex ( penis in bottom ) . at what age , frequency of condom usage with recent , next recent or next most recent partners ( in the last 12 months ) , what did you do to avoid getting pregnant ? the last time you had sex , did you or this partner do anything to delay or avoid getting pregnant ? , and have you ever had sex with someone whom you paid for sex , that is , a commercial partner? the study population comprised people aged 15 - 49 years who resided in jamaica at the time of the survey in 2004 ( may - august ) . the population data for this research were collected by hope enterprises limited on behalf of the jamaican ministry of health . each of the 14 parishes in jamaica was stratified into constituencies , with each constituency stratified into three areas - rural areas , parish capitals ( urban areas ) and main towns ( semi - urban areas ) . the areas which comprised a constituency were then stratified into primary sampling units ( psus ) or enumeration districts ( eds ) . a random sample of each psu 23 eds in urban areas , 25eds in semi - urban areas , and 24 eds in rural areas . twenty - five households were systematically chosen from each ed , and cluster sampling was carried out , where all the people living in the household of the designated areas were interviewed for the survey . the questions were demographic characteristics , sexual history ( including number and type of partners , and having sexual relations with commercial sex workers ) , condom usage , stis and health issues , knowledge of hiv / aids ( including have you ever had an hiv test ? , did you go back for the results yourself or were you contacted by a health worker ? , and would you be willing to do an hiv test [ in the future ] ? ) . the interviewers were trained for a 5-day period , of which 2 days were devoted to field practices . interviewers were assigned to a team composed of two females , two males and a supervisor . interviewees were informed of confidentiality and their right to stop the interview at any time , if they should so desire . no names , addresses or other personal information were collected from respondents in order to ensure anonymity and confidentiality . the instrument used in the survey utilized indicator measures and definitions consistent with unaids and the usaid priority prevention indicator . data were entered , stored and retrieved using spss for windows , version 16.0 spss inc ; chicago , il , usa ) . statistical analyses used were an independent sample t - test , anova , and pearson 's product moment correlation . multivariate logistic regressions were fitted using one outcome measure : self - reported , confirmed positive hiv test results . where collinearity existed ( r > 0.7 ) , variables were entered independently into the model to determine those that should be retained during the final model construction . a p - value < 0.05 ( two - tailed ) was used to establish statistical significance . for this study , the analytic model used is one that can accommodate multiple independent variables on a single binary dependent variable ( positive hiv test result , which was confirmed by an agent of the state ) . using logistic regression , this paper tested variables identified in the literature as being associated with having a positive hiv test result ( equation ) : hivti = f(ai , xi , edi , ei , msi , ci , sii , ni , asi , li , cui , ki , fi , pi , ti , stii , ri , qi) eqn where hivti denotes currently having a positive hiv test result for individual i , ai is age of individual i , edi represents educational level of individual i , ui , means employment status of individual i , ssi is social class of individual i , ari indicates area of residence of individual i , pi denotes currently having sexual relations with a commercial sex worker for individual i , msi is marital status of individual i , ci means length of time dwelling in community for individual i , sli is age of first sexual intercourse of individual i , si represents type of sexual practice of individual i , ni is number of sexual partners of individual i , ri denotes actively practicing religion of individual i , ki is having had an sti of individual i , wi represents crowding in household of individual i , qi denotes frequency of condom usage of the individual i , and the parameter i is the model 's error term . using logistic regression to test the hypothesis ( equation ) , we now know that marital status , employment status , age of respondents , and other variables are associated with those who are currently hiv positive individuals , and can write equation . hivti = f(ai , msi , ui , pi , i) .. eqn crowding is the total number of persons who dwell in a room ( excluding kitchen , bathroom and verandah ) . these methods include tubal ligation , vasectomy , implant ( norplant ) , injection , emergency contraceptive protection , pill , condom , foaming tablets , creams , jellies , diaphragm , abstinence , withdrawal , the rhythm method , calendar or billings . non - steady sexual partner denotes sexual relations that are casual , with someone with whom the individual is not having a common law sexual union , a visiting relationship or to whom the individual is not legally married . education is taken from the question , how many years did you attend school? this is coded as junior high or below ( 0 9 years ) , secondary ( 10 - 12 years ) and tertiary ( 13 + years ) . shared facility is taken from are these [ sanitary conveniences ] shared with another household ? did you go back for the results yourself or were you contacted by a health worker ? if the individual indicated that he / she was contacted by a health worker , this was used to indicate a positive hiv result . old hiv infected people were measured using self - reported information on the do you know the result of the test [ hiv ] , and whether this was positive or negative ( 1= know positive status , 0 = otherwise ) . knowledge in this study was measured using the following issues : have you heard of hiv ? , have you heard of a disease called aids ? , do you think that a healthy looking person can be infected with hiv , the virus that causes aids ? attitude for this research was measured using the following issues : if a member of your family became sick with hiv , the virus that causes aids , would you be willing to care for him or her in your household ? , would you be willing to do an hiv test [ in the future ] ? practice was evaluated from the following questions : have you ever had sexual intercourse ? by sexual intercourse i mean vaginal sex ( penis in vagina ) or anal sex ( penis in bottom ) . at what age , frequency of condom usage with recent , next recent or next most recent partners ( in the last 12 months ) , what did you do to avoid getting pregnant ? the last time you had sex , did you or this partner do anything to delay or avoid getting pregnant ? , and have you ever had sex with someone whom you paid for sex , that is , a commercial partner? the study population comprised people aged 15 - 49 years who resided in jamaica at the time of the survey in 2004 ( may - august ) . the population data for this research were collected by hope enterprises limited on behalf of the jamaican ministry of health . each of the 14 parishes in jamaica was stratified into constituencies , with each constituency stratified into three areas - rural areas , parish capitals ( urban areas ) and main towns ( semi - urban areas ) . the areas which comprised a constituency were then stratified into primary sampling units ( psus ) or enumeration districts ( eds ) . a random sample of each psu 23 eds in urban areas , 25eds in semi - urban areas , and 24 eds in rural areas . twenty - five households were systematically chosen from each ed , and cluster sampling was carried out , where all the people living in the household of the designated areas were interviewed for the survey . the questions were demographic characteristics , sexual history ( including number and type of partners , and having sexual relations with commercial sex workers ) , condom usage , stis and health issues , knowledge of hiv / aids ( including have you ever had an hiv test ? , did you go back for the results yourself or were you contacted by a health worker ? , and would you be willing to do an hiv test [ in the future ] ? ) . the interviewers were trained for a 5-day period , of which 2 days were devoted to field practices . interviewers were assigned to a team composed of two females , two males and a supervisor . interviewees were informed of confidentiality and their right to stop the interview at any time , if they should so desire . no names , addresses or other personal information were collected from respondents in order to ensure anonymity and confidentiality . the instrument used in the survey utilized indicator measures and definitions consistent with unaids and the usaid priority prevention indicator . data were entered , stored and retrieved using spss for windows , version 16.0 spss inc ; chicago , il , usa ) . statistical analyses used were an independent sample t - test , anova , and pearson 's product moment correlation . multivariate logistic regressions were fitted using one outcome measure : self - reported , confirmed positive hiv test results . where collinearity existed ( r > 0.7 ) , variables were entered independently into the model to determine those that should be retained during the final model construction . a p - value < 0.05 ( two - tailed ) was used to establish statistical significance . for this study , the analytic model used is one that can accommodate multiple independent variables on a single binary dependent variable ( positive hiv test result , which was confirmed by an agent of the state ) . using logistic regression , this paper tested variables identified in the literature as being associated with having a positive hiv test result ( equation ) : hivti = f(ai , xi , edi , ei , msi , ci , sii , ni , asi , li , cui , ki , fi , pi , ti , stii , ri , qi) eqn where hivti denotes currently having a positive hiv test result for individual i , ai is age of individual i , edi represents educational level of individual i , ui , means employment status of individual i , ssi is social class of individual i , ari indicates area of residence of individual i , pi denotes currently having sexual relations with a commercial sex worker for individual i , msi is marital status of individual i , ci means length of time dwelling in community for individual i , sli is age of first sexual intercourse of individual i , si represents type of sexual practice of individual i , ni is number of sexual partners of individual i , ri denotes actively practicing religion of individual i , ki is having had an sti of individual i , wi represents crowding in household of individual i , qi denotes frequency of condom usage of the individual i , and the parameter i is the model 's error term . using logistic regression to test the hypothesis ( equation ) , we now know that marital status , employment status , age of respondents , and other variables are associated with those who are currently hiv positive individuals , and can write equation . crowding is the total number of persons who dwell in a room ( excluding kitchen , bathroom and verandah ) . these methods include tubal ligation , vasectomy , implant ( norplant ) , injection , emergency contraceptive protection , pill , condom , foaming tablets , creams , jellies , diaphragm , abstinence , withdrawal , the rhythm method , calendar or billings . non - steady sexual partner denotes sexual relations that are casual , with someone with whom the individual is not having a common law sexual union , a visiting relationship or to whom the individual is not legally married . education is taken from the question , how many years did you attend school? this is coded as junior high or below ( 0 9 years ) , secondary ( 10 - 12 years ) and tertiary ( 13 + years ) . shared facility is taken from are these [ sanitary conveniences ] shared with another household ? the options are shared , not shared or not stated . did you go back for the results yourself or were you contacted by a health worker ? if the individual indicated that he / she was contacted by a health worker , this was used to indicate a positive hiv result . old hiv infected people were measured using self - reported information on the do you know the result of the test [ hiv ] , and whether this was positive or negative ( 1= know positive status , 0 = otherwise ) . knowledge in this study was measured using the following issues : have you heard of hiv ? , have you heard of a disease called aids ? , do you think that a healthy looking person can be infected with hiv , the virus that causes aids ? attitude for this research was measured using the following issues : if a member of your family became sick with hiv , the virus that causes aids , would you be willing to care for him or her in your household ? , have you ever had an hiv test ? , would you be willing to do an hiv test [ in the future ] ? practice was evaluated from the following questions : have you ever had sexual intercourse ? by sexual intercourse i mean vaginal sex ( penis in vagina ) or anal sex ( penis in bottom ) . at what age , frequency of condom usage with recent , next recent or next most recent partners ( in the last 12 months ) , what did you do to avoid getting pregnant ? , the last time you had sex , did you or this partner do anything to delay or avoid getting pregnant ? , and have you ever had sex with someone whom you paid for sex , that is , a commercial partner? no significant statistical association existed between the gender of a respondent and his / her positive hiv test result ( = 0.900 , p = 0.343 ) . there was a statistical difference between the mean age at first sexual relations for males ( 14.1 years ( sd = 3.2 years ) and of females ( 16.7 years ( sd = 2.7 ) ) t - test = 16.416 , p < 0.0001 . the mean age of females in the sample was 28.7 years ( sd = 11.3 years ) compared to that for males ( 27.8 years , sd = 10.9 years ) t - test = 1.656 , p = 0.098 . the mean age of first sexual relations for the sample was 15.4 years ( sd = 3.2 years ) , and a significant statistical difference was found for the mean age of first sexual intercourse among the union statuses ( f - statistic = 31.96 , p < 0.0001 ) : the mean age of first sexual relations for married people was 16.2 years ( sd = 3.1 years ) ; partner who stays overnight ( 15.2 years ( sd=3.0 years ) ) , sees partner occasionally ( 14.2 years ( sd = 3.0 years ) ) , and single ( 15.6 years ( sd = 3.3 years ) ) . furthermore , the mean age of first sexual relations was greater for those who are actively practicing religion ( 15.9 years ( sd = 3.4 years ) ) compared to those who are not actively practicing religion ( 14.8 years ( sd = 2.9 years ) ) t - test = -6.768 , p < 0.0001 . likewise , there was a significant statistical difference for the mean age of first sexual relations and typology of sexual acts performed the mean age at first sexual relations for those who have had anal sex was 14.0 years ( sd = 0.0 ) compared to those who practice vaginal sex ( 15.4 years , sd = 3.2 ) or those who did both ( vaginal and anal sex ) , 12.9 years , sd = 4.17 years . a statistical correlation existed between the age at first sexual relations and number of sexual partners in the last 12 months ( r = - 0.246 , p < 0.0001 ) . when the sample was asked do you think this partner [ current ] has other partner(s ) ? 35% indicated yes ; do you sometimes feel embarrassed to buy a condom ? only 12% remarked yes , and to what extent do you usually have a condom on you ? every time , 20.4% ; most times , 14.2% ; sometimes , 13.2% ; rarely , 13.6% and never , 38.7% . when asked can you always find your favorite brand [ condom ] when you need one in a hurry ? if that brand [ condom ] is not available would you take another brand or you would rather do without ? , only 1.6% indicated that they would rather go without using a condom , and 22.5% stated that their partner would be upset if he / she found that they had a condom ready available . the sociodemographic characteristics of those with positive hiv test results were examined in table 1 . of those who have had sexual relations with a sex worker , 75% indicated that they had used a condom . approximately 16% of those with hiv had contracted an sti infection in the past , all of them knew that they had the hiv virus , and 61% were actively practicing religion . twenty - nine percent of the hiv - infected individuals had given birth in the last 2 years or were at least 6 months pregnant . twenty - seven percent of the sample indicated that they always used a condom ; most times , 13.4% ; occasionally , 25.4% ; and never , 34.3% , with their most recent partner . when respondents were asked why they did not used a condom on the last sexual relations , most of them indicated that they knew the person well ( 44.7% ) , 2.6% said they both had hiv , 2.6% indicated that a condom was not available , 5.3% mentioned that the other partner objected to its usage , 7.9% mentioned that they used other contraceptive methods , 5.3% said that they did not need to , 7.9% said no special reason and 13.2% indicated that they had not thought of it . table 2 presents information on knowledge of the sample and those who are infected with hiv . among those who are infected with the hiv virus , almost 73% indicated that they had at most a slight chance of contracting the virus compared to 86.6% of the sample . only 8.1% of the infected respondents stated that there was a good probability of their contracting the virus compared to 6.1% of the sample . knowledge , attitude and practices of sample and of hiv infected sample seventy - eight percent of the sample indicated a willingness to do a hiv test , and when asked the majority did not want to know their status , 59.9% ; no need to know ( because not sexually active ) , 15.3% , and 9.9% mentioned that they know that they do not have the virus . figure 1 shows that more people who are hiv positive indicated that they had never used a condom with their recent partner as well as the next most recent partner . frequency of condom usage with recent , next recent and next most recent partner for sample and hiv infected sample . table 3 shows the variables which explain those who are willing to do an hiv test in the future . using logistic regression analyses , four variables emerged as statistically significant factors of jamaicans willingness to do an hiv test in the future . the model had statistically significant predictive power ( model = 31.86 , p = 0.032 ; hosmer and lemeshow goodness of fit = 5.17 , p = 0.74 ) , and correctly classified 87.0% of the sample . approximately 33.2 million people in the world are living with hiv or aids , of which 30.8 million are adults and 15.5 million are women , while 2.0 million are children . in the caribbean , hiv prevalence represents 1% in 2007 , with 15,000 - 17,000 newly infected cases and approximately 11,000 deaths , with a higher infection rate among men than women ( ratio = 2:1 ) . however , young women are more likely to become infected with the virus because the tissue lining of the genital tract is not fully developed ; hence their thinner mucus membranes are less protective than older women . in other words , the transmission of hiv from male to female is two to ten times more likely than female to male . as a result , the people of thailand and uganda , for example , blame women for the transmission of the virus ; while in east africa , the word sti is referred to as a disease of women . in the case of jamaica , approximately 1.3% of the adult population is infected , with two - thirds not knowing their status . of the number of infected adults , 4,447 ( out of the 27,000 infected cases ) started antiretroviral treatment thus representing 69% coverage of the estimated 7,000 persons who require antiretroviral therapy . this is tantamount to bringing jamaica closer to providing universal coverage with respect to hiv treatment . adolescent females ( age 10 - 19 ) have a two and a half times higher risk of hiv infection than boys of the same age group . this is owing to social factors such as young girls having sexual intercourse with older men . in terms of sexual orientation , the hiv / aids endemic has plagued humanity for more than 20 years ; and infection rates continue to grow . persistent behavioral , social and cultural factors continue to fuel the hiv epidemic ; coupled with the fear that friendship with an hiv positive person would cause self - stigmatization . owing to the stigma and discrimination , people living with hiv ( plhiv ) tend not to disclose their hiv status , other factors include fear of rejection , side effects of hiv drugs , uncertain life span , disclosure of transmission and the impact of loss . the authors also noted that without support it becomes extremely difficult for adolescents and youth , the most vulnerable group ( 15 - 24 year olds ) , to adhere to treatment . research has shown that more than 25% individuals are not cognizant of the status of their sexual partner and that 40% do not use condoms . with regard to jamaica , approximately eight out of every hundred persons ( 7.9% of the population ) engages in risky sexual activityresearch has shown significant relationships between age , relationship status and condom use . in addition , condom usage was not prevalent among main partners , especially where multiple partnerships existed ( 75.1% males aged 25 - 49 ; 70.1% females aged 15 - 24 ) , thus resulting in approximately 25 - 30% of individuals who expose themselves as well as their partners to hiv and other stis . sti case rates ( per 100,000 persons ) reported a steady increase over the period 2006 ( 637.77 ) , 2007 ( 787.17 ) and 2008 ( 850.43 ) for infections such as pelvic inflammatory disease , herpes , chancroid , bacterial vaginosis , trichomoniasis , candidiasis , opthalmia neonatorum and congenital syphilis . where cost of and access to condoms poses a challenge , youths become more vulnerable to hiv . this results in 90% of girls ( 10 - 15 year olds ) refraining from using male condoms , while both cost and concerns regarding the female condom become a barrier . in some instances , access to contraceptives ( especially male condoms ) is more favorable to males than females , as the latter encounter barriers such as being shunned or chastised . in terms of sexual orientation and status , approximately 10% of hiv cases in the caribbean represent men having sex with men ( msm ) ; while commercial sex workers ( csw ) vary from 9% to 31% . while there are persons who are ignorant of their hiv status because of non - testing , there still remain those who lack knowledge about hiv . the literature pointed out that where there is a lack of knowledge , combined with early sexual activity , these factors put youths at risk of not only unintended pregnancy , but stis and hiv this resulted in 86% of 1,000 participants who were surveyed not considering themselves personally responsible for being pregnant and/or contracting stis and hiv . despite widespread information on hiv , for instance , in 24 sub - saharan countries approximately two - thirds of young women lack adequate knowledge of hiv transmission ; also fewer than one in five people at risk for infection globally have access to basic prevention services . sexual relation is mainly the medium through which most people contract hiv / aids , indicating that the lack of knowledge ( or low ) is affecting the risky sexual behavior . it is imperative to note that a lack of knowledge regarding hiv is fuelled in part by poverty , which makes it difficult for persons to learn about hiv or to purchase condoms or antiretroviral drugs . however , the adoption of voluntary counseling and testing ( vct ) is seen as a way to remedy a lack of knowledge regarding the infection and thus facilitates safer behavior . nonetheless , although there has been evidence of success regarding vct , the issue of confidentiality is often expressed . adults and young people , who refrain from vct , claim that they fear being identified at a testing site , with the possibility of having a health care provider who knows them and may share their information with someone else . it is also recognized that many young people , especially adolescents , do not have independent access to hiv prevention services , despite the fact that the age of sexual debut is earlier than the age of legal majority . another way of curtailing the spread of hiv among adolescents and reducing new infection was the recommendation for routine , opt - out hiv screening without separate written consent or prevention counseling for persons within the age range of 13 to 65 years . jamaica 's prevention and service strategies for young females ( 15 - 24 years of age ) encompasses components such as legislation , policy , programs , service availability , participation and rights . the social reality is that the age at first sexual intercourse of jamaicans was 15.4 years and 15.6 years for those with hiv , which indicates that intervention measures must be instituted with urgency to address this public health concern . among the realities that emerged from this study is the inconsistency with which jamaicans used a condom , and this was also the case with hiv patients . a study by wilks et al found that 24.4% of jamaicans ( ages 15 - 74 years ) had more than one partner , 48.4% had sex once per week , while those with secondary education were more likely to have more partners compared to those with at most primary level education , while those with tertiary education were the least likely to have multiple partners . it is also evident that parental consent is deemed to be the greatest legal barrier to minors / adolescents being able to access hiv testing on their own . in cases where parental consent is not required under state law or policy , an increased number of minors visit test sites and receive antibody tests . the literature recommended therefore that the desire of minors to receive hiv testing without parental consent should be treated as a right of the said minors . in summary , hiv is not a homosexual virus but a heterosexual phenomenon . while more jamaicans between the ages of 15 - 49 years who were diagnosed with hiv were in visiting relationships , marginally less of them were married or in common - law unions .

background : south and southeast asia represent the largest number of new hiv infections , while sub - saharan africa represents the highest rate of new infections , followed by latin america and the caribbean . yet no study that has emerged in the caribbean has comprehensively examined young people 's sexual and reproductive health attitudes , knowledge and practices , comparing the result with those who are hiv infected.aim:the present study examines core issues of sexual and reproductive health among youths , particularly with respect to hiv.material and methods : the sample was 1,800 respondents ages 15 - 49 years . multivariate logistic regressions were fitted using one outcome measure : self - reported confirmed positive hiv test results.results:almost 34% of the sample had been tested for hiv , and 16.9% had done this in the past 12 months . only 0.2% of the sample knew that they were hiv positive and 4% had positive hiv test results when they did the test . of those with a positive hiv test result , 58.1% were females . approximately 16% of those with hiv have had an sti infection in the past , and 61% were actively practicing religion . the mean age of first sexual relations for the sample was 15.4 years ( sd = 3.2 years ) , and 15.6 years for those infected with hiv . four variables emerged as statistically significant factors of jamaicans willingness to do an hiv test in the future.conclusion:the findings of this research are far - reaching and can be used to guide public health policy formulation .

Introduction Materials and Methods None Sample Data sources Statistical analyses Analytic Model Measurement Results Discussion Conclusion

human immunodeficiency virus ( hiv ) is the second leading cause of death in the world[13 ] , the first in the caribbean ( among 15 - 49 year olds ) and the second in jamaica . this represents almost 3 billion people in the world , and almost half of all new hiv infections . in jamaica the public health dilemma of hiv among youths has led to commitments made by various nations ( 189 states ) of the world at the united nations general assembly special session ( ungass ) on hiv and aids in new york in 2001 . such commitment was made via the signing of a declaration of commitment which encapsulates promises to acknowledge the role and contributions of young people in addressing all aspects of hiv and aids , recognizing the full involvement and participation of the youth , in designing , planning , implementing and evaluating programs relating to responses to the epidemic ; reducing the prevalence of hiv among youths within the range of 15 - 24 years of age by 25% by 2010 , ensuring that 90% of youths have access to information and services that would reduce their vulnerability to hiv infection , ensuring access to information through primary and secondary school curricula on matters of safe and secure environment , strengthening sexual and reproductive health programs , and so on . under the caricom - pancap , the strategic objectives for national hiv responses are to prevent the sexual transmission of hiv , to decrease the vulnerability to sexual transmission of hiv ; to establish comprehensive , gender - sensitive , and targeted prevention programs for children ( 9 - 14 years old ) and the youth ( 15 - 24 years old ) , to achieve universal access to targeted prevention interventions among the most at - risk populations ( such as msm , sw , drug users , prisoners , and migrant populations ) , to provide services for the prevention of mother - to - child transmission of hiv to all pregnant women and their families ; to strengthen prevention efforts among plhiv as part of comprehensive care ; and to reduce vulnerability to hiv through early identification and treatment of other sexually transmitted infections ( stis ) . within the context of the high hiv incidence and prevalence rates among people in developing nations , and in particular the caribbean , and more so among young people , the attitude toward consistent condom usage is problematic and needs to be examined in developing countries . hence , we wanted to elucidate information as to whether there are differences in the knowledge , attitude and practices of adults in their reproductive years regarding their reproductive health issues , compared with those who have hiv in a caribbean society , as well as to model factors which account for their willingness to do an hiv test in the future . no study emerged in the caribbean that has comprehensively examined adults in their reproductive ages ( 15 - 49 years ) on their sexual and reproductive health attitudes , knowledge and practices , and compares the result with those who are hiv - infected youth , as well as factors which explain people 's willingness to do an hiv test in the future . the present study examines core issues of sexual and reproductive health among youths , particularly with respect to those who are hiv - infected in jamaica in order to provide a comprehensive understanding of people 's perceptions , which will be used to fashion public health intervention programs . the study population comprised people aged 15 - 49 years who resided in jamaica at the time of the survey in 2004 ( may - august ) . twenty - five households were systematically chosen from each ed , and cluster sampling was carried out , where all the people living in the household of the designated areas were interviewed for the survey . the questions were demographic characteristics , sexual history ( including number and type of partners , and having sexual relations with commercial sex workers ) , condom usage , stis and health issues , knowledge of hiv / aids ( including have you ever had an hiv test ? , and would you be willing to do an hiv test [ in the future ] ? ) multivariate logistic regressions were fitted using one outcome measure : self - reported , confirmed positive hiv test results . for this study , the analytic model used is one that can accommodate multiple independent variables on a single binary dependent variable ( positive hiv test result , which was confirmed by an agent of the state ) . using logistic regression , this paper tested variables identified in the literature as being associated with having a positive hiv test result ( equation ) : hivti = f(ai , xi , edi , ei , msi , ci , sii , ni , asi , li , cui , ki , fi , pi , ti , stii , ri , qi) eqn where hivti denotes currently having a positive hiv test result for individual i , ai is age of individual i , edi represents educational level of individual i , ui , means employment status of individual i , ssi is social class of individual i , ari indicates area of residence of individual i , pi denotes currently having sexual relations with a commercial sex worker for individual i , msi is marital status of individual i , ci means length of time dwelling in community for individual i , sli is age of first sexual intercourse of individual i , si represents type of sexual practice of individual i , ni is number of sexual partners of individual i , ri denotes actively practicing religion of individual i , ki is having had an sti of individual i , wi represents crowding in household of individual i , qi denotes frequency of condom usage of the individual i , and the parameter i is the model 's error term . using logistic regression to test the hypothesis ( equation ) , we now know that marital status , employment status , age of respondents , and other variables are associated with those who are currently hiv positive individuals , and can write equation . old hiv infected people were measured using self - reported information on the do you know the result of the test [ hiv ] , and whether this was positive or negative ( 1= know positive status , 0 = otherwise ) . , do you think that a healthy looking person can be infected with hiv , the virus that causes aids ? , would you be willing to do an hiv test [ in the future ] ? at what age , frequency of condom usage with recent , next recent or next most recent partners ( in the last 12 months ) , what did you do to avoid getting pregnant ? the study population comprised people aged 15 - 49 years who resided in jamaica at the time of the survey in 2004 ( may - august ) . twenty - five households were systematically chosen from each ed , and cluster sampling was carried out , where all the people living in the household of the designated areas were interviewed for the survey . the questions were demographic characteristics , sexual history ( including number and type of partners , and having sexual relations with commercial sex workers ) , condom usage , stis and health issues , knowledge of hiv / aids ( including have you ever had an hiv test ? , and would you be willing to do an hiv test [ in the future ] ? ) multivariate logistic regressions were fitted using one outcome measure : self - reported , confirmed positive hiv test results . for this study , the analytic model used is one that can accommodate multiple independent variables on a single binary dependent variable ( positive hiv test result , which was confirmed by an agent of the state ) . using logistic regression , this paper tested variables identified in the literature as being associated with having a positive hiv test result ( equation ) : hivti = f(ai , xi , edi , ei , msi , ci , sii , ni , asi , li , cui , ki , fi , pi , ti , stii , ri , qi) eqn where hivti denotes currently having a positive hiv test result for individual i , ai is age of individual i , edi represents educational level of individual i , ui , means employment status of individual i , ssi is social class of individual i , ari indicates area of residence of individual i , pi denotes currently having sexual relations with a commercial sex worker for individual i , msi is marital status of individual i , ci means length of time dwelling in community for individual i , sli is age of first sexual intercourse of individual i , si represents type of sexual practice of individual i , ni is number of sexual partners of individual i , ri denotes actively practicing religion of individual i , ki is having had an sti of individual i , wi represents crowding in household of individual i , qi denotes frequency of condom usage of the individual i , and the parameter i is the model 's error term . using logistic regression to test the hypothesis ( equation ) , we now know that marital status , employment status , age of respondents , and other variables are associated with those who are currently hiv positive individuals , and can write equation . if the individual indicated that he / she was contacted by a health worker , this was used to indicate a positive hiv result . old hiv infected people were measured using self - reported information on the do you know the result of the test [ hiv ] , and whether this was positive or negative ( 1= know positive status , 0 = otherwise ) . , do you think that a healthy looking person can be infected with hiv , the virus that causes aids ? , would you be willing to do an hiv test [ in the future ] ? at what age , frequency of condom usage with recent , next recent or next most recent partners ( in the last 12 months ) , what did you do to avoid getting pregnant ? the study population comprised people aged 15 - 49 years who resided in jamaica at the time of the survey in 2004 ( may - august ) . twenty - five households were systematically chosen from each ed , and cluster sampling was carried out , where all the people living in the household of the designated areas were interviewed for the survey . the questions were demographic characteristics , sexual history ( including number and type of partners , and having sexual relations with commercial sex workers ) , condom usage , stis and health issues , knowledge of hiv / aids ( including have you ever had an hiv test ? , and would you be willing to do an hiv test [ in the future ] ? ) multivariate logistic regressions were fitted using one outcome measure : self - reported , confirmed positive hiv test results . for this study , the analytic model used is one that can accommodate multiple independent variables on a single binary dependent variable ( positive hiv test result , which was confirmed by an agent of the state ) . using logistic regression , this paper tested variables identified in the literature as being associated with having a positive hiv test result ( equation ) : hivti = f(ai , xi , edi , ei , msi , ci , sii , ni , asi , li , cui , ki , fi , pi , ti , stii , ri , qi) eqn where hivti denotes currently having a positive hiv test result for individual i , ai is age of individual i , edi represents educational level of individual i , ui , means employment status of individual i , ssi is social class of individual i , ari indicates area of residence of individual i , pi denotes currently having sexual relations with a commercial sex worker for individual i , msi is marital status of individual i , ci means length of time dwelling in community for individual i , sli is age of first sexual intercourse of individual i , si represents type of sexual practice of individual i , ni is number of sexual partners of individual i , ri denotes actively practicing religion of individual i , ki is having had an sti of individual i , wi represents crowding in household of individual i , qi denotes frequency of condom usage of the individual i , and the parameter i is the model 's error term . using logistic regression to test the hypothesis ( equation ) , we now know that marital status , employment status , age of respondents , and other variables are associated with those who are currently hiv positive individuals , and can write equation . if the individual indicated that he / she was contacted by a health worker , this was used to indicate a positive hiv result . old hiv infected people were measured using self - reported information on the do you know the result of the test [ hiv ] , and whether this was positive or negative ( 1= know positive status , 0 = otherwise ) . , do you think that a healthy looking person can be infected with hiv , the virus that causes aids ? attitude for this research was measured using the following issues : if a member of your family became sick with hiv , the virus that causes aids , would you be willing to care for him or her in your household ? , have you ever had an hiv test ? , would you be willing to do an hiv test [ in the future ] ? at what age , frequency of condom usage with recent , next recent or next most recent partners ( in the last 12 months ) , what did you do to avoid getting pregnant ? there was a statistical difference between the mean age at first sexual relations for males ( 14.1 years ( sd = 3.2 years ) and of females ( 16.7 years ( sd = 2.7 ) ) t - test = 16.416 , p < 0.0001 . the mean age of females in the sample was 28.7 years ( sd = 11.3 years ) compared to that for males ( 27.8 years , sd = 10.9 years ) t - test = 1.656 , p = 0.098 . the mean age of first sexual relations for the sample was 15.4 years ( sd = 3.2 years ) , and a significant statistical difference was found for the mean age of first sexual intercourse among the union statuses ( f - statistic = 31.96 , p < 0.0001 ) : the mean age of first sexual relations for married people was 16.2 years ( sd = 3.1 years ) ; partner who stays overnight ( 15.2 years ( sd=3.0 years ) ) , sees partner occasionally ( 14.2 years ( sd = 3.0 years ) ) , and single ( 15.6 years ( sd = 3.3 years ) ) . furthermore , the mean age of first sexual relations was greater for those who are actively practicing religion ( 15.9 years ( sd = 3.4 years ) ) compared to those who are not actively practicing religion ( 14.8 years ( sd = 2.9 years ) ) t - test = -6.768 , p < 0.0001 . likewise , there was a significant statistical difference for the mean age of first sexual relations and typology of sexual acts performed the mean age at first sexual relations for those who have had anal sex was 14.0 years ( sd = 0.0 ) compared to those who practice vaginal sex ( 15.4 years , sd = 3.2 ) or those who did both ( vaginal and anal sex ) , 12.9 years , sd = 4.17 years . a statistical correlation existed between the age at first sexual relations and number of sexual partners in the last 12 months ( r = - 0.246 , p < 0.0001 ) . the sociodemographic characteristics of those with positive hiv test results were examined in table 1 . of those who have had sexual relations with a sex worker , 75% indicated that they had used a condom . approximately 16% of those with hiv had contracted an sti infection in the past , all of them knew that they had the hiv virus , and 61% were actively practicing religion . twenty - nine percent of the hiv - infected individuals had given birth in the last 2 years or were at least 6 months pregnant . when respondents were asked why they did not used a condom on the last sexual relations , most of them indicated that they knew the person well ( 44.7% ) , 2.6% said they both had hiv , 2.6% indicated that a condom was not available , 5.3% mentioned that the other partner objected to its usage , 7.9% mentioned that they used other contraceptive methods , 5.3% said that they did not need to , 7.9% said no special reason and 13.2% indicated that they had not thought of it . table 2 presents information on knowledge of the sample and those who are infected with hiv . among those who are infected with the hiv virus , almost 73% indicated that they had at most a slight chance of contracting the virus compared to 86.6% of the sample . knowledge , attitude and practices of sample and of hiv infected sample seventy - eight percent of the sample indicated a willingness to do a hiv test , and when asked the majority did not want to know their status , 59.9% ; no need to know ( because not sexually active ) , 15.3% , and 9.9% mentioned that they know that they do not have the virus . figure 1 shows that more people who are hiv positive indicated that they had never used a condom with their recent partner as well as the next most recent partner . table 3 shows the variables which explain those who are willing to do an hiv test in the future . using logistic regression analyses , four variables emerged as statistically significant factors of jamaicans willingness to do an hiv test in the future . the model had statistically significant predictive power ( model = 31.86 , p = 0.032 ; hosmer and lemeshow goodness of fit = 5.17 , p = 0.74 ) , and correctly classified 87.0% of the sample . approximately 33.2 million people in the world are living with hiv or aids , of which 30.8 million are adults and 15.5 million are women , while 2.0 million are children . as a result , the people of thailand and uganda , for example , blame women for the transmission of the virus ; while in east africa , the word sti is referred to as a disease of women . this is tantamount to bringing jamaica closer to providing universal coverage with respect to hiv treatment . persistent behavioral , social and cultural factors continue to fuel the hiv epidemic ; coupled with the fear that friendship with an hiv positive person would cause self - stigmatization . in addition , condom usage was not prevalent among main partners , especially where multiple partnerships existed ( 75.1% males aged 25 - 49 ; 70.1% females aged 15 - 24 ) , thus resulting in approximately 25 - 30% of individuals who expose themselves as well as their partners to hiv and other stis . despite widespread information on hiv , for instance , in 24 sub - saharan countries approximately two - thirds of young women lack adequate knowledge of hiv transmission ; also fewer than one in five people at risk for infection globally have access to basic prevention services . it is also recognized that many young people , especially adolescents , do not have independent access to hiv prevention services , despite the fact that the age of sexual debut is earlier than the age of legal majority . the social reality is that the age at first sexual intercourse of jamaicans was 15.4 years and 15.6 years for those with hiv , which indicates that intervention measures must be instituted with urgency to address this public health concern . a study by wilks et al found that 24.4% of jamaicans ( ages 15 - 74 years ) had more than one partner , 48.4% had sex once per week , while those with secondary education were more likely to have more partners compared to those with at most primary level education , while those with tertiary education were the least likely to have multiple partners . while more jamaicans between the ages of 15 - 49 years who were diagnosed with hiv were in visiting relationships , marginally less of them were married or in common - law unions .

the mental health care bill 2013 was introduced to the rajya sabha , india 's upper house of parliament , on 19 august 2013 . the legislation aimed to provide for mental health care and services for persons with mental illness and to protect , promote , and fulfill the rights of such persons during delivery of mental health care and services and for matters connected therewith or incidental thereto ( preamble ) . the twin emphasis on providing care and promoting rights is critically important in india , as it is elsewhere . this legislative initiative is therefore an exceptionally important one with real potential to improve the position of the mentally ill and enhance their experiences of good mental health , social justice , and liberty . the current paper examines this development in the broader global context of human rights , mental health , and mental illness . more specifically , the first part of the paper looks at the background to current human rights standards relating to mental illness , and the second part focuses on the convention on the rights of persons with disabilities ( crpd ) , which was adapted by the united nations ( un ) in 2006 and ratified by india in 2007 . the 2013 bill sought explicitly to harmonize indian legislation with the crpd , so this part of the paper focuses on two key issues in this context : involuntary care and mental capacity . finally , some conclusions are presented , emphasizing the need to increase the level of social justice experienced by the mentally ill and their families , and to take a broad view of rights , spanning not just mental health services but the experiences of the mentally ill across broader society in india , the rest of asia and elsewhere around the globe . in light of the unprecedented humanitarian atrocities of the second world war , the un was established in october 1945 to promote international peace and security and reduce the possibility of further wars . one of the primary aims of the new organization was to articulate an intellectual and legal framework that would support the observance of human rights among member states and promote a culture of human rights throughout the world . to promote these goals , the universal declaration of human rights ( udhr ) was adopted by the un general assembly at palais de chaillot in paris on 10 december 1948 . the udhr was presented as a nonbinding statement of rights , the first stage in a process which continued with the drafting of the international covenant on civil and political rights and the international covenant on economic , social and cultural rights , adapted by the un general assembly in 1966 . the udhr comprises thirty articles , preceded by a short preamble which recognizes that the inherent dignity ( and ) the equal and inalienable rights of all members of the human family is the foundation of freedom , justice and peace in the world , and that it is essential , if man is not to be compelled to have recourse , as a last resort , to rebellion against tyranny and oppression , that human rights should be protected by the rule of law ( preamble ) . the first article of the udhr states that all human beings are born free and equal in dignity and rights . they are endowed with reason and conscience and should act toward one another in a spirit of brotherhood article 2 emphasizes the universal nature of rights : everyone is entitled to all the rights and freedoms set forth in this declaration , without distinction of any kind , such as race , color , sex , language , religion , political or other opinion , national or social origin , property , birth or other status . everyone is entitled to all the rights and freedoms set forth in this declaration , without distinction of any kind , such as race , color , sex , language , religion , political or other opinion , national or social origin , property , birth or other status . this emphasis on universality is both useful and necessary , not least because previous declarations of rights had commonly been interpreted in such a way as to exclude certain groups . while mental illness was not mentioned explicitly in the list of factors which were not to form the basis of discrimination , it undoubtedly belongs under the term other status . in 1991 , the un made this more explicit in its principles for the protection of persons with mental illness and the improvement of mental health care : every person with a mental illness shall have the right to exercise all civil , political , economic , social and cultural rights as recognized in the universal declaration of human rights , the international covenant on economic , social and cultural rights , the international covenant on civil and political rights , and in other relevant instruments , such as the declaration on the rights of disabled persons and the body of principles for the protection of all persons under any form of detention or imprisonment ( principle 1 ) . the remainder of the udhr went on to articulate a range of rights fundamentally rooted in the principle of liberty , including the right to life , liberty , and security of person the explicit articulation of this right , especially in the context of universal rights , is particularly relevant to the mentally ill , not least because of their increased risk of lengthy involuntary detention in various institutions . again , the need to respect the right to liberty , along with the other rights outlined in the udhr , was strongly re - emphasized in 1991 in the un 's principles for the protection of persons with mental illness and the improvement of mental health care . historically , however , people with mental disorder have often experienced high levels of abuse or neglect of human rights , including the rights to liberty and treatment . the first comprehensive statement of the rights of persons with mental illness was un 's principles for the protection of persons with mental illness and the improvement of mental health care in 1991 . key principles include as follows : all people are entitled to receive the best mental health care available and be treated with humanity and respectthere should be no discrimination on the grounds of mental illness . all people with mental illness have the same rights to medical and social care as otherseveryone with mental illnesses has the right to live , work , and receive treatment in the community , as far as possiblemental health care should be based on internationally accepted ethical standardseach patient 's treatment plan should be reviewed regularly with the patientthere shall be no misuse of mental health skills and knowledgemedication should meet the health needs of the patient and shall not be administered for the convenience of others or as a punishmentfor voluntary patients , no treatment should be administered without informed consent , subject to some exceptions ( e.g. , patients with personal representatives empowered by law to provide consent)for involuntary patients , every effort should be made to inform the patient about treatmentphysical restraint or involuntary seclusion should be used only in accordance with official guidelinesrecords must be kept of all treatmentsmental health facilities must be appropriately structured and resourcedan impartial review body should , in consultation with mental health practitioners , review the cases of involuntary patients . all people are entitled to receive the best mental health care available and be treated with humanity and respect there should be no discrimination on the grounds of mental illness . all people with mental illness have the same rights to medical and social care as others everyone with mental illnesses has the right to live , work , and receive treatment in the community , as far as possible mental health care should be based on internationally accepted ethical standards each patient 's treatment plan should be reviewed regularly with the patient there shall be no misuse of mental health skills and knowledge medication should meet the health needs of the patient and shall not be administered for the convenience of others or as a punishment for voluntary patients , no treatment should be administered without informed consent , subject to some exceptions ( e.g. , patients with personal representatives empowered by law to provide consent ) for involuntary patients , every effort should be made to inform the patient about treatment physical restraint or involuntary seclusion should be used only in accordance with official guidelines records must be kept of all treatments mental health facilities must be appropriately structured and resourced an impartial review body should , in consultation with mental health practitioners , review the cases of involuntary patients . the 1991 statement of principles was important not only for its specific provisions , but also its acknowledgement of a particular need to protect the rights of persons with mental disorder , especially persons with enduring mental disorders whose rights have been significantly ignored in the past . against this background , the world health organization ( who ) went on to articulate ten basic principles of mental health care law in 1996 , further emphasizing many of the 1991 principles , and distilling them into ten key principles : all persons should benefit from the best possible measures to promote mental well - being and prevent mental disordersall persons in need should have access to basic mental health caremental health assessments should be performed in accordance with internationally accepted medical principles and instrumentsall persons with mental disorders should be provided with health care which is the least restrictive possibleconsent is needed before any type of interference with a person can occurif a patient experiences difficulties appreciating the implications of a decision , although not unable to decide , the patient shall benefit from the assistance of an appropriate third party of his or her choicethere should be a review procedure for any decision made by official , surrogate or representative decision - makers and health care providersfor decisions affecting integrity or liberty , with a long - lasting impact , there should be automatic periodical review mechanismsall decision - makers acting in official or surrogate capacity should be qualified to do soall decisions should be made in keeping with the body of law in force in the jurisdiction involved and not on any other basis , or an arbitrary basis . all persons should benefit from the best possible measures to promote mental well - being and prevent mental disorders all persons in need should have access to basic mental health care mental health assessments should be performed in accordance with internationally accepted medical principles and instruments all persons with mental disorders should be provided with health care which is the least restrictive possible consent is needed before any type of interference with a person can occur if a patient experiences difficulties appreciating the implications of a decision , although not unable to decide , the patient shall benefit from the assistance of an appropriate third party of his or her choice there should be a review procedure for any decision made by official , surrogate or representative decision - makers and health care providers for decisions affecting integrity or liberty , with a long - lasting impact , there should be automatic periodical review mechanisms all decision - makers acting in official or surrogate capacity should be qualified to do so all decisions should be made in keeping with the body of law in force in the jurisdiction involved and not on any other basis , or an arbitrary basis . specific aspects of the application of these principles were developed further in 2005 in the who resource book on mental health , human rights , and legislation which presents a detailed statement of human rights issues which , according to the who , need to be addressed at national level . more specifically , the resource book includes a detailed checklist on mental health legislation based , in large part , on previous un and who publications . the checklist is a companion to the who resource book on mental health , human rights , and legislation and its objectives are to : ( a ) assist countries in reviewing the adequacy and comprehensiveness of existing mental health legislation ; and ( b ) help countries in the process of drafting new law . this checklist can help countries assess whether key components are included in legislation or policy , and ensure that the broad recommendations contained in the resource book are carefully examined and considered . the checklist , although lengthy , detailed and explicitly informed by the udhr , is not a set of absolute rules , and is not legally binding . there are no sanctions for states which fail to accord with its standards and unlike the un international covenant on civil and political rights , the un human rights committee does not review member states compliance with it . the who checklist is , rather , designed to work by influencing member states as they redraft and implement national mental health laws and policies . given the checklist 's close links with the udhr and who documents outlining the rights of the mentally ill , the authors make the assumption that the checklist standards will be accepted by the international community and deemed worth reflecting in national mental health law and policies . the who also explicitly states that some countries may address some or all of these mental health issues in general legislation ( e.g. , equality legislation ) , other forms of ( not legally binding ) regulation , or mental health policy , rather than specific mental health legislation . the history of psychiatry , however , supports the unique importance of dedicated mental health legislation , rather than general law or nonbinding regulation , for protecting the rights of the mentally ill : while there were substantial advances in the articulation of human rights standards for the general population throughout the early twentieth century , the plight of the mentally ill remained bleak until much later in most jurisdictions , suggesting a need for specific and dedicated measures to protect their rights . the who implicitly acknowledges the centrality of law in this process when it presents its final checklist in the resource book as a checklist on mental health legislation ( italics added ) . the resource book is especially useful owing to its emphasis on a broad concept of human rights , encompassing not only just issues relating to the right to liberty but also social rights , which are commonly neglected among the mentally ill . it is this broader concept of human rights to which i will return in the conclusions to this paper . however , first , it is necessary to consider in some depth the crpd , which is undoubtedly the most significant and challenging development in this field in recent decades , and holds out real hope for significant progress on human rights in the decades ahead . the crpd commits ratifying countries to promote , protect , and ensure the full and equal enjoyment of all human rights and fundamental freedoms by all persons with disabilities , and to promote respect for their inherent dignity ( article 1 ) . it specifies that persons with disabilities include those who have long - term physical , mental , intellectual , or sensory impairments which in interaction with various barriers may hinder their full and effective participation in society on an equal basis with others . in the context of psychiatry , it seems clear that this definition does not include all people with mental illness , because many mental illnesses are not long - term . the crpd does not , however , present its definition of persons with disabilities as a comprehensive one but specifies that the term persons with disabilities includes people with long - term impairments ; others , presumably , also fit this definition . as a result , it is likely that some people with mental illness meet the definition at least some of the time ( e.g. , a person with chronic schizophrenia or an intellectual disability ) but others do not ( e.g. , a person with time - limited adjustment disorder ) . in india , india has signed and ratified the said convention on the 1 day of october , 2007 and it is necessary to align and harmonize the existing laws with the said convention . thus , the 2013 bill was explicitly intended to bring india into compliance with the requirements of the crpd . the spirit and principles of the 2013 bill were certainly in keeping with the crpd , and the measures outlined would go a long way toward promoting community - based treatment , ensuring access to care , increasing patients involvement in key care decisions , and strengthening governance in the mental health system . these would be important and historic steps in improving the position of the mentally ill , promoting their rights , and increasing their experiences of mental health care and social justice . it is not , entirely clear , however , if the 2013 bill would be compliant with the crpd in certain other respects , especially in relation to involuntary care , termed supported admission in the bill . according to the 2013 bill , supported admission could occur if , following independent examination , it appeared that the person has a mental illness of such severity that the person ( i ) has recently threatened or attempted or is threatening or attempting to cause bodily harm to himself ; or ( ii ) has recently behaved or is behaving violently toward another person or has caused or is causing another person to fear bodily harm from him ; or ( iii ) has recently shown or is showing an inability to care for himself to a degree that places the individual at risk of harm to himself ( section 98[a ] ) . in addition , the bill would require that : the psychiatrist or the mental health professionals or the medical practitioner , as the case may be , certify , after taking into account an advance directive , if any , that admission to the mental health establishment is the least restrictive care option possible in the circumstances ( section 98[b ] ) ; andthe person is ineligible to receive care and treatment as an independent patient because the person is unable to make mental health care and treatment decisions independently and needs very high support from his nominated representative in making decisions the psychiatrist or the mental health professionals or the medical practitioner , as the case may be , certify , after taking into account an advance directive , if any , that admission to the mental health establishment is the least restrictive care option possible in the circumstances ( section 98[b ] ) ; and the person is ineligible to receive care and treatment as an independent patient because the person is unable to make mental health care and treatment decisions independently and needs very high support from his nominated representative in making decisions there are two issues here : first , the crpd does not appear to permit involuntary care based on mental illness ; second , with regard to the reference to , the crpd might not permit any distinction between people on the grounds of mental capacity either ( let alone using mental capacity as a basis for deciding upon involuntary care , as in the 2013 bill ) . these two issues merit consideration . first is the issue of involuntary care . the crpd states that the existence of a disability shall in no case justify a deprivation of liberty ( article 14[b ] ) . if certain people with mental illness as defined in the 2013 bill ( e.g. , some people with chronic schizophrenia ) fit the un definition of persons with disabilities , then the 2013 bill would be inconsistent with the crpd in this respect , given the clear links it draws between mental illness , risk and involuntary admission ( see above ) . this is also the case for mental health legislation in england , wales , scotland , northern ireland , ireland and most other jurisdictions , all of which violate this article of the crpd . in 2009 , the un high commissioner for human rights ( 2009 ) underlined this issue by objecting explicitly to any link between preventive detention and risk to self or others stemming from mental illness : legislation authorising the institutionalisation of persons with disabilities on the grounds of their disability without their free and informed consent must be abolished . this must include the repeal of provisions authorising institutionalisation of persons with disabilities for their care and treatment without their free and informed consent , as well as provisions authorising the preventive detention of persons with disabilities on grounds such as the likelihood of them posing a danger to themselves or others , in all cases in which such grounds of care , treatment and public security are linked in legislation to an apparent or diagnosed mental illness legislation authorising the institutionalisation of persons with disabilities on the grounds of their disability without their free and informed consent must be abolished . this must include the repeal of provisions authorising institutionalisation of persons with disabilities for their care and treatment without their free and informed consent , as well as provisions authorising the preventive detention of persons with disabilities on grounds such as the likelihood of them posing a danger to themselves or others , in all cases in which such grounds of care , treatment and public security are linked in legislation to an apparent or diagnosed mental illness ( paragraph 49 ) . in 2011 , the un committee on the rights of persons with disabilities ( 2011 ) , reporting on tunisia , underscored this point : with reference to article 14 of the convention ( liberty and security of the person ) , the committee is concerned that having a disability , including an intellectual or psychosocial disability , can constitute a basis for the deprivation of liberty under current legislation ( paragraph 24)the committee recommends that the state party repeal legislative provisions which allow for the deprivation of liberty on the basis of disability , including a psychosocial or intellectual disability ( paragraph 25)the committee is concerned about the lack of clarity concerning the scope of legislation to protect persons with disabilities from being subjected to treatment without their free and informed consent , including forced treatment in mental health services ( paragraph 28)the committee recommends that the state party incorporate into the law the abolition of surgery and treatment without the full and informed consent of the patient , and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention ( paragraph 29 ) . with reference to article 14 of the convention ( liberty and security of the person ) , the committee is concerned that having a disability , including an intellectual or psychosocial disability , can constitute a basis for the deprivation of liberty under current legislation ( paragraph 24 ) the committee recommends that the state party repeal legislative provisions which allow for the deprivation of liberty on the basis of disability , including a psychosocial or intellectual disability ( paragraph 25 ) the committee is concerned about the lack of clarity concerning the scope of legislation to protect persons with disabilities from being subjected to treatment without their free and informed consent , including forced treatment in mental health services ( paragraph 28 ) the committee recommends that the state party incorporate into the law the abolition of surgery and treatment without the full and informed consent of the patient , and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention the committee ( 2011 ) also made similar points in relation to spain : the committee recommends that the state party : review its laws that allow for the deprivation of liberty on the basis of disability , including mental , psychosocial or intellectual disabilities ; repeal provisions that authorize involuntary internment linked to an apparent or diagnosed disability ; and adopt measures to ensure that health - care services , including all mental - health - care services , are based on the informed consent of the person concerned ( paragraph 36)the committee urges the state party to abolish the administration of medical treatment , in particular sterilization , without the full and informed consent of the patient ; and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention ( paragraph 38 ) . the committee recommends that the state party : review its laws that allow for the deprivation of liberty on the basis of disability , including mental , psychosocial or intellectual disabilities ; repeal provisions that authorize involuntary internment linked to an apparent or diagnosed disability ; and adopt measures to ensure that health - care services , including all mental - health - care services , are based on the informed consent of the person concerned ( paragraph 36 ) the committee urges the state party to abolish the administration of medical treatment , in particular sterilization , without the full and informed consent of the patient ; and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention ( paragraph 38 ) . the committee 's apparent opposition to involuntary care on the basis of mental illness or disability stands in remarkable contrast to the history of mental health services in india and most other countries , where involuntary care has always been based on the presence of mental illness and associated risk . it is not , however , entirely clear what proportion of persons with mental illness are persons with disabilities under the crpd , and not all psychiatric admission can be considered institutionalisation . in addition , denial of care ( especially to the most distressed ) on the basis of the crpd would be grossly inconsistent with the fundamental aims and purpose of the crpd : people with disabilities are entitled to all the levels and modalities of care that are available to everyone , without distinction of any description . on this basis , abolishing involuntary care in order ostensibly to comply with the crpd would be a historically radical , counter - productive and it would seem , unlikely step . the second area of apparent inconsistency between india 's mental health care bill 2013 and the crpd concerns the 2013 bill 's use of mental capacity when making decisions regarding involuntary care . this featured in the section 98 criteria for supported admission , which would require that the person is unable to make mental health care and treatment decisions independently and needs very high support from his nominated representative in making decisions ( section 98(1)(c ) ) . article 12 of the crpd states that persons with disabilities have the right to recognition everywhere as persons before the law and enjoy legal capacity on an equal basis with others in all aspects of life . legal capacity is a person 's authority or right under law to be recognized as an actor in law , as opposed to mental capacity , which is the cognitive ability to make decisions . article 12 requires ratifying states to take appropriate measures to provide access by persons with disabilities to the support they may require in exercising their legal capacity : states parties shall ensure that all measures that relate to the exercise of legal capacity provide for appropriate and effective safeguards to prevent abuse in accordance with international human rights law . such safeguards shall ensure that measures relating to the exercise of legal capacity respect the rights , will and preferences of the person , are free of conflict of interest and undue influence , are proportional and tailored to the person 's circumstances , apply for the shortest time possible and are subject to regular review by a competent , independent and impartial authority or judicial body . the safeguards shall be proportional to the degree to which such measures affect the person 's rights and interests states parties shall ensure that all measures that relate to the exercise of legal capacity provide for appropriate and effective safeguards to prevent abuse in accordance with international human rights law . such safeguards shall ensure that measures relating to the exercise of legal capacity respect the rights , will and preferences of the person , are free of conflict of interest and undue influence , are proportional and tailored to the person 's circumstances , apply for the shortest time possible and are subject to regular review by a competent , independent and impartial authority or judicial body . the safeguards shall be proportional to the degree to which such measures affect the person 's rights and interests general comment on article 12 , however , the committee on the rights of persons with disabilities , appointed by the un under the crpd , explicitly rejects the use of mental capacity in any form to determine what supports might be needed for the exercise of legal capacity , arguing that mental capacity is not , as is commonly presented , an objective , scientific and naturally occurring phenomenon . mental capacity is contingent on social and political contexts as are the disciplines , professions , and practices which play a dominant role in assessing mental capacity the committee contends that the functional approach to assessing mental capacity is often based on whether a person can understand the nature and consequences of a decision and/or whether he or she can use or weigh the relevant information , and argues that this approach is flawed for two key reasons : ( a ) it is discriminatorily applied to people with disabilities ; and ( b ) it presumes to be able to accurately assess the inner - workings of the human mind and when the person does not pass the assessment , it then denies him or her a core human right - the right to equal recognition before the law . in all of those approaches , a person 's disability and/or decision - making skills are taken as legitimate grounds for denying his or her legal capacity and lowering his or her status as a person before the law . ( a ) it is discriminatorily applied to people with disabilities ; and ( b ) it presumes to be able to accurately assess the inner - workings of the human mind and when the person does not pass the assessment , it then denies him or her a core human right - the right to equal recognition before the law . in all of those approaches , a person 's disability and/or decision - making skills are taken as legitimate grounds for denying his or her legal capacity and lowering his or her status as a person before the law . the committee concludes that the provision of support to exercise legal capacity should not hinge on mental capacity assessments ; new , nondiscriminatory indicators of support needs are required in the provision of support to exercise legal capacity ( paragraph 29[i ] ) . the committee also explicitly rejects the idea of substitute decision - making of any description : states parties obligation to replace substitute decision - making regimes by supported decision - making requires both the abolition of substitute decision - making regimes and the development of supported decision - making alternatives . the development of supported decision - making systems in parallel with the maintenance of substitute decision - making regimes is not sufficient to comply with article 12 of the convention states parties obligation to replace substitute decision - making regimes by supported decision - making requires both the abolition of substitute decision - making regimes and the development of supported decision - making alternatives . the development of supported decision - making systems in parallel with the maintenance of substitute decision - making regimes is not sufficient to comply with article 12 of the convention the fact that india 's 2013 bill used the concept of mental capacity as one of the criteria for supported admission appears to place it in discord with the committee 's interpretation of article 12 . general comment has , however , been critiqued strongly , chiefly because it dismisses not only the very concept of mental capacity but also substitute decision - making of any sort and diversion of the mentally ill from prison on the basis of mental incapacity , among other things . these interpretations of the crpd diverge significantly from the text of the convention itself and diverge very substantially from clinical and social realities . this is , presumably , attributable to narrow consultation with service - users and the paucity of clinical personnel on the committee . will create the impression that the crpd is simply impossible to implement and therefore irrelevant . this would be a real pity : the crpd , including article 12 , is a vital articulation of the rights of people with disabilities , including some people with mental illness . legislation along the lines of india 's 2013 bill offers much that is positive and progressive in terms of overall standards of care , revised processes for involuntary admission , and enhanced governance throughout the mental health system . in this way , this kind of legislation , although imperfect , promotes the principles enshrined in the crpd , true to the goals of such legislation and as clearly outlined in the preamble to the 2013 bill . the crpd states that the existence of a disability shall in no case justify a deprivation of liberty ( article 14[b ] ) . if certain people with mental illness as defined in the 2013 bill ( e.g. , some people with chronic schizophrenia ) fit the un definition of persons with disabilities , then the 2013 bill would be inconsistent with the crpd in this respect , given the clear links it draws between mental illness , risk and involuntary admission ( see above ) . this is also the case for mental health legislation in england , wales , scotland , northern ireland , ireland and most other jurisdictions , all of which violate this article of the crpd . in 2009 , the un high commissioner for human rights ( 2009 ) underlined this issue by objecting explicitly to any link between preventive detention and risk to self or others stemming from mental illness : legislation authorising the institutionalisation of persons with disabilities on the grounds of their disability without their free and informed consent must be abolished . this must include the repeal of provisions authorising institutionalisation of persons with disabilities for their care and treatment without their free and informed consent , as well as provisions authorising the preventive detention of persons with disabilities on grounds such as the likelihood of them posing a danger to themselves or others , in all cases in which such grounds of care , treatment and public security are linked in legislation to an apparent or diagnosed mental illness legislation authorising the institutionalisation of persons with disabilities on the grounds of their disability without their free and informed consent must be abolished . this must include the repeal of provisions authorising institutionalisation of persons with disabilities for their care and treatment without their free and informed consent , as well as provisions authorising the preventive detention of persons with disabilities on grounds such as the likelihood of them posing a danger to themselves or others , in all cases in which such grounds of care , treatment and public security are linked in legislation to an apparent or diagnosed mental illness ( paragraph 49 ) . in 2011 , the un committee on the rights of persons with disabilities ( 2011 ) , reporting on tunisia , underscored this point : with reference to article 14 of the convention ( liberty and security of the person ) , the committee is concerned that having a disability , including an intellectual or psychosocial disability , can constitute a basis for the deprivation of liberty under current legislation ( paragraph 24)the committee recommends that the state party repeal legislative provisions which allow for the deprivation of liberty on the basis of disability , including a psychosocial or intellectual disability ( paragraph 25)the committee is concerned about the lack of clarity concerning the scope of legislation to protect persons with disabilities from being subjected to treatment without their free and informed consent , including forced treatment in mental health services ( paragraph 28)the committee recommends that the state party incorporate into the law the abolition of surgery and treatment without the full and informed consent of the patient , and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention ( paragraph 29 ) . with reference to article 14 of the convention ( liberty and security of the person ) , the committee is concerned that having a disability , including an intellectual or psychosocial disability , can constitute a basis for the deprivation of liberty under current legislation ( paragraph 24 ) the committee recommends that the state party repeal legislative provisions which allow for the deprivation of liberty on the basis of disability , including a psychosocial or intellectual disability ( paragraph 25 ) the committee is concerned about the lack of clarity concerning the scope of legislation to protect persons with disabilities from being subjected to treatment without their free and informed consent , including forced treatment in mental health services ( paragraph 28 ) the committee recommends that the state party incorporate into the law the abolition of surgery and treatment without the full and informed consent of the patient , and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention ( paragraph 29 ) . the committee ( 2011 ) also made similar points in relation to spain : the committee recommends that the state party : review its laws that allow for the deprivation of liberty on the basis of disability , including mental , psychosocial or intellectual disabilities ; repeal provisions that authorize involuntary internment linked to an apparent or diagnosed disability ; and adopt measures to ensure that health - care services , including all mental - health - care services , are based on the informed consent of the person concerned ( paragraph 36)the committee urges the state party to abolish the administration of medical treatment , in particular sterilization , without the full and informed consent of the patient ; and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention ( paragraph 38 ) . the committee recommends that the state party : review its laws that allow for the deprivation of liberty on the basis of disability , including mental , psychosocial or intellectual disabilities ; repeal provisions that authorize involuntary internment linked to an apparent or diagnosed disability ; and adopt measures to ensure that health - care services , including all mental - health - care services , are based on the informed consent of the person concerned ( paragraph 36 ) the committee urges the state party to abolish the administration of medical treatment , in particular sterilization , without the full and informed consent of the patient ; and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention the committee 's apparent opposition to involuntary care on the basis of mental illness or disability stands in remarkable contrast to the history of mental health services in india and most other countries , where involuntary care has always been based on the presence of mental illness and associated risk . it is not , however , entirely clear what proportion of persons with mental illness are persons with disabilities under the crpd , and not all psychiatric admission can be considered institutionalisation . in addition , denial of care ( especially to the most distressed ) on the basis of the crpd would be grossly inconsistent with the fundamental aims and purpose of the crpd : people with disabilities are entitled to all the levels and modalities of care that are available to everyone , without distinction of any description . on this basis , abolishing involuntary care in order ostensibly to comply with the crpd would be a historically radical , counter - productive and it would seem , unlikely step . the second area of apparent inconsistency between india 's mental health care bill 2013 and the crpd concerns the 2013 bill 's use of mental capacity when making decisions regarding involuntary care . this featured in the section 98 criteria for supported admission , which would require that the person is unable to make mental health care and treatment decisions independently and needs very high support from his nominated representative in making decisions article 12 of the crpd states that persons with disabilities have the right to recognition everywhere as persons before the law and enjoy legal capacity on an equal basis with others in all aspects of life . legal capacity is a person 's authority or right under law to be recognized as an actor in law , as opposed to mental capacity , which is the cognitive ability to make decisions . article 12 requires ratifying states to take appropriate measures to provide access by persons with disabilities to the support they may require in exercising their legal capacity : states parties shall ensure that all measures that relate to the exercise of legal capacity provide for appropriate and effective safeguards to prevent abuse in accordance with international human rights law . such safeguards shall ensure that measures relating to the exercise of legal capacity respect the rights , will and preferences of the person , are free of conflict of interest and undue influence , are proportional and tailored to the person 's circumstances , apply for the shortest time possible and are subject to regular review by a competent , independent and impartial authority or judicial body . the safeguards shall be proportional to the degree to which such measures affect the person 's rights and interests states parties shall ensure that all measures that relate to the exercise of legal capacity provide for appropriate and effective safeguards to prevent abuse in accordance with international human rights law . such safeguards shall ensure that measures relating to the exercise of legal capacity respect the rights , will and preferences of the person , are free of conflict of interest and undue influence , are proportional and tailored to the person 's circumstances , apply for the shortest time possible and are subject to regular review by a competent , independent and impartial authority or judicial body . the safeguards shall be proportional to the degree to which such measures affect the person 's rights and interests general comment on article 12 , however , the committee on the rights of persons with disabilities , appointed by the un under the crpd , explicitly rejects the use of mental capacity in any form to determine what supports might be needed for the exercise of legal capacity , arguing that mental capacity is not , as is commonly presented , an objective , scientific and naturally occurring phenomenon . mental capacity is contingent on social and political contexts as are the disciplines , professions , and practices which play a dominant role in assessing mental capacity the committee contends that the functional approach to assessing mental capacity is often based on whether a person can understand the nature and consequences of a decision and/or whether he or she can use or weigh the relevant information , and argues that this approach is flawed for two key reasons : ( a ) it is discriminatorily applied to people with disabilities ; and ( b ) it presumes to be able to accurately assess the inner - workings of the human mind and when the person does not pass the assessment , it then denies him or her a core human right - the right to equal recognition before the law . in all of those approaches , a person 's disability and/or decision - making skills are taken as legitimate grounds for denying his or her legal capacity and lowering his or her status as a person before the law . ( a ) it is discriminatorily applied to people with disabilities ; and ( b ) it presumes to be able to accurately assess the inner - workings of the human mind and when the person does not pass the assessment , it then denies him or her a core human right - the right to equal recognition before the law . in all of those approaches , a person 's disability and/or decision - making skills are taken as legitimate grounds for denying his or her legal capacity and lowering his or her status as a person before the law . the committee concludes that the provision of support to exercise legal capacity should not hinge on mental capacity assessments ; new , nondiscriminatory indicators of support needs are required in the provision of support to exercise legal capacity the committee also explicitly rejects the idea of substitute decision - making of any description : states parties obligation to replace substitute decision - making regimes by supported decision - making requires both the abolition of substitute decision - making regimes and the development of supported decision - making alternatives . the development of supported decision - making systems in parallel with the maintenance of substitute decision - making regimes is not sufficient to comply with article 12 of the convention states parties obligation to replace substitute decision - making regimes by supported decision - making requires both the abolition of substitute decision - making regimes and the development of supported decision - making alternatives . the development of supported decision - making systems in parallel with the maintenance of substitute decision - making regimes is not sufficient to comply with article 12 of the convention the fact that india 's 2013 bill used the concept of mental capacity as one of the criteria for supported admission appears to place it in discord with the committee 's interpretation of article 12 . general comment has , however , been critiqued strongly , chiefly because it dismisses not only the very concept of mental capacity but also substitute decision - making of any sort and diversion of the mentally ill from prison on the basis of mental incapacity , among other things . these interpretations of the crpd diverge significantly from the text of the convention itself and diverge very substantially from clinical and social realities . this is , presumably , attributable to narrow consultation with service - users and the paucity of clinical personnel on the committee . will create the impression that the crpd is simply impossible to implement and therefore irrelevant . this would be a real pity : the crpd , including article 12 , is a vital articulation of the rights of people with disabilities , including some people with mental illness . legislation along the lines of india 's 2013 bill offers much that is positive and progressive in terms of overall standards of care , revised processes for involuntary admission , and enhanced governance throughout the mental health system . in this way , this kind of legislation , although imperfect , promotes the principles enshrined in the crpd , true to the goals of such legislation and as clearly outlined in the preamble to the 2013 bill . following some 134 official amendments , mental health care bill 2013 was passed by the rajya sabha in august 2016 . reforms of mental health law , such as this , present real opportunities to improve the experiences and lives of the mentally ill and their families , notwithstanding the various issues relating to the crpd ( discussed above ) . it is likely that future years will see these interpretative issues with the crpd explored further , producing ever greater recognition of the rights of the mentally ill based on the text of the convention itself . perhaps the most compelling element of the crpd in this context is the admirable breadth of its ambition . the convention seeks nothing less than to promote , protect and ensure the full and equal enjoyment of all human rights and fundamental freedoms by all persons with disabilities , and to promote respect for their inherent dignity ( article 1 ) . this is precisely as it should be . while the indian legislation is undoubtedly challenging for services , and there are preexisting issues with compliance with preexisting standards ( e.g. , in relation to prisoners ) , there can be no doubt that change is proceeding in the correct direction . it is important that such initiatives focus not only on the right to liberty but also on rights to treatment , social care , social inclusion and political empowerment of the mentally ill . as amartya sen points out , what matters most is whether or not measures designed to promote justice actually realize this goal in day - to - day life , and not just in theory . that is , we need to look at the real - life outcomes of measures intended to protect rights , as opposed to simply verifying that current legislation and other arrangements appear likely to promote human rights . that , to date , the mentally ill around the world have increased rates of imprisonment , homelessness , social exclusion , untreated illness , and various other denials of rights . the indian legislative initiative , in the context of the crpd , offers an important framework within which to start to address this situation . it is important that the relevant principles and values are applied not only in the development of legislation , but also in its application , and in mental health and social services , courtrooms , prisons , and other locations across india , the rest of asia and elsewhere around the world . globally , the rights of the mentally ill have been neglected for far , far too long .

the mental health care bill 2013 was introduced to the rajya sabha ( india 's upper house of parliament ) in august 2013 and following 134 official amendments , passed in august 2016 . properly implemented , mental health legislation such as this plays a key role in protecting the rights of the mentally ill , ensuring access to care , and promoting social justice for the mentally ill , their families and carers . in this context , the 2006 united nations convention on the rights of persons with disabilities ( crpd ) presents a real opportunity to improve the position of people with disabilities and those disabled by long - term mental illness . the crpd also presents many challenges to mental health legislators and service - providers , especially in relation to involuntary care , mental capacity , and substitute decision - making . nevertheless , the crpd has still generated strong incentive for reform and is an opportunity that should not be missed . legislation along the lines of india 's 2013 bill offers much that is positive and progressive in terms of standards of care , revised processes for involuntary admission , and enhanced governance throughout mental health services . in this way , this kind of legislation , although imperfect in certain respects , promotes the principles of the crpd ( as outlined in the preamble to india 's 2013 bill ) . it is important that such initiatives focus not only on the right to liberty but also on rights to treatment , social care , social inclusion , and political empowerment of the mentally ill . globally , the mentally ill have been neglected for far , far too long . it is time to fix this .

INTRODUCTION MENTAL HEALTH, MENTAL ILLNESS, AND HUMAN RIGHTS: WHAT ARE THE STANDARDS? CONVENTION ON THE RIGHTS OF PERSONS WITH DISABILITIES Involuntary care and the Convention on the Rights of Persons with Disabilities Mental capacity and the Convention on the Rights of Persons with Disabilities CONCLUSIONS: FIGHTING FOR JUSTICE FOR THE MENTALLY ILL Financial support and sponsorship Conflicts of interest

the mental health care bill 2013 was introduced to the rajya sabha , india 's upper house of parliament , on 19 august 2013 . this legislative initiative is therefore an exceptionally important one with real potential to improve the position of the mentally ill and enhance their experiences of good mental health , social justice , and liberty . more specifically , the first part of the paper looks at the background to current human rights standards relating to mental illness , and the second part focuses on the convention on the rights of persons with disabilities ( crpd ) , which was adapted by the united nations ( un ) in 2006 and ratified by india in 2007 . in 1991 , the un made this more explicit in its principles for the protection of persons with mental illness and the improvement of mental health care : every person with a mental illness shall have the right to exercise all civil , political , economic , social and cultural rights as recognized in the universal declaration of human rights , the international covenant on economic , social and cultural rights , the international covenant on civil and political rights , and in other relevant instruments , such as the declaration on the rights of disabled persons and the body of principles for the protection of all persons under any form of detention or imprisonment ( principle 1 ) . the remainder of the udhr went on to articulate a range of rights fundamentally rooted in the principle of liberty , including the right to life , liberty , and security of person the explicit articulation of this right , especially in the context of universal rights , is particularly relevant to the mentally ill , not least because of their increased risk of lengthy involuntary detention in various institutions . again , the need to respect the right to liberty , along with the other rights outlined in the udhr , was strongly re - emphasized in 1991 in the un 's principles for the protection of persons with mental illness and the improvement of mental health care . the first comprehensive statement of the rights of persons with mental illness was un 's principles for the protection of persons with mental illness and the improvement of mental health care in 1991 . all people with mental illness have the same rights to medical and social care as otherseveryone with mental illnesses has the right to live , work , and receive treatment in the community , as far as possiblemental health care should be based on internationally accepted ethical standardseach patient 's treatment plan should be reviewed regularly with the patientthere shall be no misuse of mental health skills and knowledgemedication should meet the health needs of the patient and shall not be administered for the convenience of others or as a punishmentfor voluntary patients , no treatment should be administered without informed consent , subject to some exceptions ( e.g. all people with mental illness have the same rights to medical and social care as others everyone with mental illnesses has the right to live , work , and receive treatment in the community , as far as possible mental health care should be based on internationally accepted ethical standards each patient 's treatment plan should be reviewed regularly with the patient there shall be no misuse of mental health skills and knowledge medication should meet the health needs of the patient and shall not be administered for the convenience of others or as a punishment for voluntary patients , no treatment should be administered without informed consent , subject to some exceptions ( e.g. the 1991 statement of principles was important not only for its specific provisions , but also its acknowledgement of a particular need to protect the rights of persons with mental disorder , especially persons with enduring mental disorders whose rights have been significantly ignored in the past . against this background , the world health organization ( who ) went on to articulate ten basic principles of mental health care law in 1996 , further emphasizing many of the 1991 principles , and distilling them into ten key principles : all persons should benefit from the best possible measures to promote mental well - being and prevent mental disordersall persons in need should have access to basic mental health caremental health assessments should be performed in accordance with internationally accepted medical principles and instrumentsall persons with mental disorders should be provided with health care which is the least restrictive possibleconsent is needed before any type of interference with a person can occurif a patient experiences difficulties appreciating the implications of a decision , although not unable to decide , the patient shall benefit from the assistance of an appropriate third party of his or her choicethere should be a review procedure for any decision made by official , surrogate or representative decision - makers and health care providersfor decisions affecting integrity or liberty , with a long - lasting impact , there should be automatic periodical review mechanismsall decision - makers acting in official or surrogate capacity should be qualified to do soall decisions should be made in keeping with the body of law in force in the jurisdiction involved and not on any other basis , or an arbitrary basis . all persons should benefit from the best possible measures to promote mental well - being and prevent mental disorders all persons in need should have access to basic mental health care mental health assessments should be performed in accordance with internationally accepted medical principles and instruments all persons with mental disorders should be provided with health care which is the least restrictive possible consent is needed before any type of interference with a person can occur if a patient experiences difficulties appreciating the implications of a decision , although not unable to decide , the patient shall benefit from the assistance of an appropriate third party of his or her choice there should be a review procedure for any decision made by official , surrogate or representative decision - makers and health care providers for decisions affecting integrity or liberty , with a long - lasting impact , there should be automatic periodical review mechanisms all decision - makers acting in official or surrogate capacity should be qualified to do so all decisions should be made in keeping with the body of law in force in the jurisdiction involved and not on any other basis , or an arbitrary basis . the history of psychiatry , however , supports the unique importance of dedicated mental health legislation , rather than general law or nonbinding regulation , for protecting the rights of the mentally ill : while there were substantial advances in the articulation of human rights standards for the general population throughout the early twentieth century , the plight of the mentally ill remained bleak until much later in most jurisdictions , suggesting a need for specific and dedicated measures to protect their rights . the spirit and principles of the 2013 bill were certainly in keeping with the crpd , and the measures outlined would go a long way toward promoting community - based treatment , ensuring access to care , increasing patients involvement in key care decisions , and strengthening governance in the mental health system . these would be important and historic steps in improving the position of the mentally ill , promoting their rights , and increasing their experiences of mental health care and social justice . it is not , entirely clear , however , if the 2013 bill would be compliant with the crpd in certain other respects , especially in relation to involuntary care , termed supported admission in the bill . in addition , the bill would require that : the psychiatrist or the mental health professionals or the medical practitioner , as the case may be , certify , after taking into account an advance directive , if any , that admission to the mental health establishment is the least restrictive care option possible in the circumstances ( section 98[b ] ) ; andthe person is ineligible to receive care and treatment as an independent patient because the person is unable to make mental health care and treatment decisions independently and needs very high support from his nominated representative in making decisions the psychiatrist or the mental health professionals or the medical practitioner , as the case may be , certify , after taking into account an advance directive , if any , that admission to the mental health establishment is the least restrictive care option possible in the circumstances ( section 98[b ] ) ; and the person is ineligible to receive care and treatment as an independent patient because the person is unable to make mental health care and treatment decisions independently and needs very high support from his nominated representative in making decisions there are two issues here : first , the crpd does not appear to permit involuntary care based on mental illness ; second , with regard to the reference to , the crpd might not permit any distinction between people on the grounds of mental capacity either ( let alone using mental capacity as a basis for deciding upon involuntary care , as in the 2013 bill ) . , some people with chronic schizophrenia ) fit the un definition of persons with disabilities , then the 2013 bill would be inconsistent with the crpd in this respect , given the clear links it draws between mental illness , risk and involuntary admission ( see above ) . in 2011 , the un committee on the rights of persons with disabilities ( 2011 ) , reporting on tunisia , underscored this point : with reference to article 14 of the convention ( liberty and security of the person ) , the committee is concerned that having a disability , including an intellectual or psychosocial disability , can constitute a basis for the deprivation of liberty under current legislation ( paragraph 24)the committee recommends that the state party repeal legislative provisions which allow for the deprivation of liberty on the basis of disability , including a psychosocial or intellectual disability ( paragraph 25)the committee is concerned about the lack of clarity concerning the scope of legislation to protect persons with disabilities from being subjected to treatment without their free and informed consent , including forced treatment in mental health services ( paragraph 28)the committee recommends that the state party incorporate into the law the abolition of surgery and treatment without the full and informed consent of the patient , and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention ( paragraph 29 ) . with reference to article 14 of the convention ( liberty and security of the person ) , the committee is concerned that having a disability , including an intellectual or psychosocial disability , can constitute a basis for the deprivation of liberty under current legislation ( paragraph 24 ) the committee recommends that the state party repeal legislative provisions which allow for the deprivation of liberty on the basis of disability , including a psychosocial or intellectual disability ( paragraph 25 ) the committee is concerned about the lack of clarity concerning the scope of legislation to protect persons with disabilities from being subjected to treatment without their free and informed consent , including forced treatment in mental health services ( paragraph 28 ) the committee recommends that the state party incorporate into the law the abolition of surgery and treatment without the full and informed consent of the patient , and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention the committee ( 2011 ) also made similar points in relation to spain : the committee recommends that the state party : review its laws that allow for the deprivation of liberty on the basis of disability , including mental , psychosocial or intellectual disabilities ; repeal provisions that authorize involuntary internment linked to an apparent or diagnosed disability ; and adopt measures to ensure that health - care services , including all mental - health - care services , are based on the informed consent of the person concerned ( paragraph 36)the committee urges the state party to abolish the administration of medical treatment , in particular sterilization , without the full and informed consent of the patient ; and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention ( paragraph 38 ) . the second area of apparent inconsistency between india 's mental health care bill 2013 and the crpd concerns the 2013 bill 's use of mental capacity when making decisions regarding involuntary care . the safeguards shall be proportional to the degree to which such measures affect the person 's rights and interests general comment on article 12 , however , the committee on the rights of persons with disabilities , appointed by the un under the crpd , explicitly rejects the use of mental capacity in any form to determine what supports might be needed for the exercise of legal capacity , arguing that mental capacity is not , as is commonly presented , an objective , scientific and naturally occurring phenomenon . mental capacity is contingent on social and political contexts as are the disciplines , professions , and practices which play a dominant role in assessing mental capacity the committee contends that the functional approach to assessing mental capacity is often based on whether a person can understand the nature and consequences of a decision and/or whether he or she can use or weigh the relevant information , and argues that this approach is flawed for two key reasons : ( a ) it is discriminatorily applied to people with disabilities ; and ( b ) it presumes to be able to accurately assess the inner - workings of the human mind and when the person does not pass the assessment , it then denies him or her a core human right - the right to equal recognition before the law . general comment has , however , been critiqued strongly , chiefly because it dismisses not only the very concept of mental capacity but also substitute decision - making of any sort and diversion of the mentally ill from prison on the basis of mental incapacity , among other things . legislation along the lines of india 's 2013 bill offers much that is positive and progressive in terms of overall standards of care , revised processes for involuntary admission , and enhanced governance throughout the mental health system . in this way , this kind of legislation , although imperfect , promotes the principles enshrined in the crpd , true to the goals of such legislation and as clearly outlined in the preamble to the 2013 bill . , some people with chronic schizophrenia ) fit the un definition of persons with disabilities , then the 2013 bill would be inconsistent with the crpd in this respect , given the clear links it draws between mental illness , risk and involuntary admission ( see above ) . in 2011 , the un committee on the rights of persons with disabilities ( 2011 ) , reporting on tunisia , underscored this point : with reference to article 14 of the convention ( liberty and security of the person ) , the committee is concerned that having a disability , including an intellectual or psychosocial disability , can constitute a basis for the deprivation of liberty under current legislation ( paragraph 24)the committee recommends that the state party repeal legislative provisions which allow for the deprivation of liberty on the basis of disability , including a psychosocial or intellectual disability ( paragraph 25)the committee is concerned about the lack of clarity concerning the scope of legislation to protect persons with disabilities from being subjected to treatment without their free and informed consent , including forced treatment in mental health services ( paragraph 28)the committee recommends that the state party incorporate into the law the abolition of surgery and treatment without the full and informed consent of the patient , and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention ( paragraph 29 ) . with reference to article 14 of the convention ( liberty and security of the person ) , the committee is concerned that having a disability , including an intellectual or psychosocial disability , can constitute a basis for the deprivation of liberty under current legislation ( paragraph 24 ) the committee recommends that the state party repeal legislative provisions which allow for the deprivation of liberty on the basis of disability , including a psychosocial or intellectual disability ( paragraph 25 ) the committee is concerned about the lack of clarity concerning the scope of legislation to protect persons with disabilities from being subjected to treatment without their free and informed consent , including forced treatment in mental health services ( paragraph 28 ) the committee recommends that the state party incorporate into the law the abolition of surgery and treatment without the full and informed consent of the patient , and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention ( paragraph 29 ) . the committee recommends that the state party : review its laws that allow for the deprivation of liberty on the basis of disability , including mental , psychosocial or intellectual disabilities ; repeal provisions that authorize involuntary internment linked to an apparent or diagnosed disability ; and adopt measures to ensure that health - care services , including all mental - health - care services , are based on the informed consent of the person concerned ( paragraph 36 ) the committee urges the state party to abolish the administration of medical treatment , in particular sterilization , without the full and informed consent of the patient ; and ensure that national law especially respects women 's rights under ( articles ) 23 ( respect for home and the family ) and 25 ( health ) of the convention the committee 's apparent opposition to involuntary care on the basis of mental illness or disability stands in remarkable contrast to the history of mental health services in india and most other countries , where involuntary care has always been based on the presence of mental illness and associated risk . the second area of apparent inconsistency between india 's mental health care bill 2013 and the crpd concerns the 2013 bill 's use of mental capacity when making decisions regarding involuntary care . this featured in the section 98 criteria for supported admission , which would require that the person is unable to make mental health care and treatment decisions independently and needs very high support from his nominated representative in making decisions article 12 of the crpd states that persons with disabilities have the right to recognition everywhere as persons before the law and enjoy legal capacity on an equal basis with others in all aspects of life . the safeguards shall be proportional to the degree to which such measures affect the person 's rights and interests general comment on article 12 , however , the committee on the rights of persons with disabilities , appointed by the un under the crpd , explicitly rejects the use of mental capacity in any form to determine what supports might be needed for the exercise of legal capacity , arguing that mental capacity is not , as is commonly presented , an objective , scientific and naturally occurring phenomenon . mental capacity is contingent on social and political contexts as are the disciplines , professions , and practices which play a dominant role in assessing mental capacity the committee contends that the functional approach to assessing mental capacity is often based on whether a person can understand the nature and consequences of a decision and/or whether he or she can use or weigh the relevant information , and argues that this approach is flawed for two key reasons : ( a ) it is discriminatorily applied to people with disabilities ; and ( b ) it presumes to be able to accurately assess the inner - workings of the human mind and when the person does not pass the assessment , it then denies him or her a core human right - the right to equal recognition before the law . general comment has , however , been critiqued strongly , chiefly because it dismisses not only the very concept of mental capacity but also substitute decision - making of any sort and diversion of the mentally ill from prison on the basis of mental incapacity , among other things . legislation along the lines of india 's 2013 bill offers much that is positive and progressive in terms of overall standards of care , revised processes for involuntary admission , and enhanced governance throughout the mental health system . in this way , this kind of legislation , although imperfect , promotes the principles enshrined in the crpd , true to the goals of such legislation and as clearly outlined in the preamble to the 2013 bill . following some 134 official amendments , mental health care bill 2013 was passed by the rajya sabha in august 2016 . reforms of mental health law , such as this , present real opportunities to improve the experiences and lives of the mentally ill and their families , notwithstanding the various issues relating to the crpd ( discussed above ) . it is important that such initiatives focus not only on the right to liberty but also on rights to treatment , social care , social inclusion and political empowerment of the mentally ill . it is important that the relevant principles and values are applied not only in the development of legislation , but also in its application , and in mental health and social services , courtrooms , prisons , and other locations across india , the rest of asia and elsewhere around the world . globally , the rights of the mentally ill have been neglected for far , far too long .

evidence - based medicine requires integrating individual clinician expertise with the best available external clinical evidence and patient choice.1 evidence - based medicine began with the development and application of categorical rules to identify individuals and populations at risk and target them with effective treatment and prevention . over time , it has evolved to incorporate increasingly detailed criteria to enhance accurate risk identification and treatment targeting . for example , the atp-3 dyslipidemia guideline uses comorbidities , risk factors , and coronary heart disease risk to sort individuals into risk categories.2 individualized clinical guidelines , taking into account individual risk factors and expected benefit from treatment , represent an even more precise application of evidence - based medicine.3 4 from the inception of modern evidence - based clinical guidelines , questions have consistently arisen about how best to disseminate and implement them.5 6 individualized guidelines heighten these concerns because of the requirement for detailed information about the person to whom they will be applied , for example , individual risk of morbidity or mortality and expected benefits of treatment . integration of guidelines in electronic health records ( ehr ) enables the ready availability of information necessary for personalized recommendations,7 but little is known about the use of individualized guidelines in routine clinical practice . demonstrating the potential benefits of individualized guidelines for a specific clinical problem is one challenge that must be met if they are to be used more broadly.7 a second challenge is clinician adoption . this report examines the feasibility of using individualized clinical guidelines in primary care and provides limited - scale observational data on medication use and cardiovascular risk . indigo individualized clinical guidelines are based on the archimedes model , a full - scale , rigorously validated biomathematical simulation model of human physiology , diseases , behavior , interventions , and healthcare systems.817 indigo uses regression equations to predict the likelihood of heart attack or stroke based on individual risk factors : cardiovascular disease history , comorbidities , medication history , biomarkers ( low - density lipoprotein ( ldl ) cholesterol , triglycerides , blood pressure , and body mass index ( bmi ) ) , and lifestyle behaviors . interventions modeled in indigo include smoking cessation , weight loss , and preventive medications : simvastatin , lisinopril , atenolol , hydrochlorthiazide , amlodipine , and lisinopril / hydrochlorthiazide . using patient - level clinical information in an ehr , indigo produces individualized benefit scores predicting risk reduction from maintaining each intervention over 5 years . the score is a weighted combination of the decreased risk of adverse outcomes ; weights approximate the expected 5-year reduction in quality of life that would result from each outcome . indigo also produces a total benefit score for each individual , the sum of benefit scores for all interventions . finally , indigo predicts individualized 5-year risk of heart attack or stroke under three scenarios : ( 1 ) maintaining current interventions for 5 years ; ( 2 ) discontinuing all current interventions ; or ( 3 ) maintaining all identified interventions for 5 years . complete details of the predictive modeling have been published.18 automated data interchange between the ehr and the biomathematical model updates benefit scores daily , which are displayed in an interactive graphical user interface ( gui ) . indigo 's unique strength is identifying two types of individuals : those for whom traditional population - based guidelines indicate treatment but who would benefit too little to justify the side effects or risks , and those missed by population - based guidelines but who would derive considerable benefit . a simulation applying indigo to a secondary dataset found that individualized guidelines could lead to improved quality of care and lower costs for patients with hypertension , compared to traditional guidelines.3 we report here on the initial implementation of indigo individualized guidelines in primary care , in which we assessed the feasibility of use in routine clinical practice , integration into shared decision - making , patients and physicians experience with their use , and effects on medication use and cardiovascular risk in a small population over a short observation period . we considered assessment of these factors a first step that could lead to a full - scale trial or to further refinement in practice . our quality improvement project used a matched control observational design in two clinics employing 10 primary care providers ( pcp ) in kaiser permanente 's hawaii region ( kphi ) . kphi serves 224 000 members with a hospital on oahu and 15 clinics across three islands . a comprehensive integrated ehr , kp health connect , contains all patient information.19 20 indigo individualized guidelines were implemented by automated daily extraction of relevant clinical information for all kphi adult members from kp health connect . after applying indigo calculations , risk and benefit scores were embedded in the panel support tool , a population management module of kp health connect , where pcp accessed them.2124 to encourage acceptance by practicing physicians and organizational leadership , algorithms were set such that all interventions that would be recommended by existing guidelines would also be recommended by indigo . the gui displayed each member 's current 5-year risk of heart attack or stroke and the comparable predicted risk after adopting and maintaining all identified interventions ( figure 1 ) . risk - reduction information was shown for each intervention ; selecting interventions as adopted dynamically changed the 5-year predicted risk display . gfr , glomerular filtration rate ; hba1c , hemoglobin a 1c ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein . patients were considered eligible for indigo use if they had a scheduled primary care visit and their indigo total benefit score was in the top third of the kphi population . pcp then selected individual patients for indigo use based on the patient 's chief complaint and availability of pcp time , and the extent to which pcp believed patients would be likely to accept treatment recommendations . physicians selected patients with primary care visits between november 2008 and april 2009 ; for the purposes of this project , patients were followed for 36 months . selected patients viewed a video explaining individualized guidelines , their benefits and limitations , and the gui . pcp and patients subsequently used indigo in 510-min shared decision - making sessions ; a printed summary displayed interventions chosen by the member and the predicted 5-year risk with no interventions , with selected interventions , and with all identified interventions . physicians made treatment recommendations based on existing guidelines , indigo benefit scores , and clinical judgment . for example , indigo modeling was based on specified medication doses , but physicians tailored doses to individual patient needs . the intervention described in this report thus includes three components : individualized guidelines , a gui to communicate risk - reduction options to patients and their physicians , and a brief face - to - face discussion of the risk - reduction options . we compared patients using indigo at the pilot clinics with those receiving usual care at other clinics . we identified the matched comparison cohort using nearest - neighbor propensity score matching with replacement . we estimated a logistic regression model with indigo use as the dependent variable and six matching variables : age ; gender ; pre - indigo history of cardiovascular disease , diabetes , or hypertension ; number of primary care visits during the 12 months before indigo use ; an indicator for the presence or absence of at least one health behavior benefit recommendation ; and an indicator for the presence or absence of at least one medication recommendation . the primary outcome measures were prescribing and dispensing of medications that have been shown to reduce the risk of heart attack or stroke : statins or other lipid - lowering medications , diuretics , calcium antagonists , -blockers , and ace inhibitors or angiotensin receptor blockers ( arb ) . because indigo often recommends multiple medications , we grouped them by likely indication to simplify the analysis and gauge any general effect on prescribing and dispensing rates . statins and other lipid - lowering medications were grouped , and we refer to them collectively as statins. medications and medication combinations intended primarily for hypertension control diuretics , calcium antagonists , and lisinopril / hydrochlorothiazide were collectively tracked as antihypertensives. we also included in this category recommendations for any of these medications in combination with a -blocker , ace inhibitor , or arb , which also have antihypertensive effect . we counted separately recommendations for -blockers alone and ace inhibitors or arb alone , because these may be indicated after a myocardial infarction and for chronic kidney disease , respectively . because -blockers and ace inhibitors or arb could be recommended either to reduce blood pressure or for other indications , indigo recommendations for these medications were not amenable to straightforward interpretation and were not analyzed in detail . secondary outcome measures included blood pressure and ldl cholesterol levels , bmi , and change in 5-year indigo - predicted risk of heart attack or stroke . secondary measures also included physician and patient perceptions of individualized guidelines ; data were collected during focus groups and a post - project email survey ( physicians ) and through a pre - post mail survey ( patients ) . we used difference - in - differences methods to evaluate changes in the secondary outcome measures between the intervention group and matched controls , assessing statistical significance with student 's t test . the kaiser permanente hawaii institutional review board reviewed the project and determined that it was quality improvement and not human subjects research . our quality improvement project used a matched control observational design in two clinics employing 10 primary care providers ( pcp ) in kaiser permanente 's hawaii region ( kphi ) . kphi serves 224 000 members with a hospital on oahu and 15 clinics across three islands . indigo individualized guidelines were implemented by automated daily extraction of relevant clinical information for all kphi adult members from kp health connect . after applying indigo calculations , risk and benefit scores were embedded in the panel support tool , a population management module of kp health connect , where pcp accessed them.2124 to encourage acceptance by practicing physicians and organizational leadership , algorithms were set such that all interventions that would be recommended by existing guidelines would also be recommended by indigo . the gui displayed each member 's current 5-year risk of heart attack or stroke and the comparable predicted risk after adopting and maintaining all identified interventions ( figure 1 ) . risk - reduction information was shown for each intervention ; selecting interventions as adopted dynamically changed the 5-year predicted risk display . gfr , glomerular filtration rate ; hba1c , hemoglobin a 1c ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein . patients were considered eligible for indigo use if they had a scheduled primary care visit and their indigo total benefit score was in the top third of the kphi population . pcp then selected individual patients for indigo use based on the patient 's chief complaint and availability of pcp time , and the extent to which pcp believed patients would be likely to accept treatment recommendations . physicians selected patients with primary care visits between november 2008 and april 2009 ; for the purposes of this project , patients were followed for 36 months . selected patients viewed a video explaining individualized guidelines , their benefits and limitations , and the gui . pcp and patients subsequently used indigo in 510-min shared decision - making sessions ; a printed summary displayed interventions chosen by the member and the predicted 5-year risk with no interventions , with selected interventions , and with all identified interventions . physicians made treatment recommendations based on existing guidelines , indigo benefit scores , and clinical judgment . for example , indigo modeling was based on specified medication doses , but physicians tailored doses to individual patient needs . the intervention described in this report thus includes three components : individualized guidelines , a gui to communicate risk - reduction options to patients and their physicians , and a brief face - to - face discussion of the risk - reduction options . we compared patients using indigo at the pilot clinics with those receiving usual care at other clinics . we identified the matched comparison cohort using nearest - neighbor propensity score matching with replacement . we estimated a logistic regression model with indigo use as the dependent variable and six matching variables : age ; gender ; pre - indigo history of cardiovascular disease , diabetes , or hypertension ; number of primary care visits during the 12 months before indigo use ; an indicator for the presence or absence of at least one health behavior benefit recommendation ; and an indicator for the presence or absence of at least one medication recommendation . the primary outcome measures were prescribing and dispensing of medications that have been shown to reduce the risk of heart attack or stroke : statins or other lipid - lowering medications , diuretics , calcium antagonists , -blockers , and ace inhibitors or angiotensin receptor blockers ( arb ) . because indigo often recommends multiple medications , we grouped them by likely indication to simplify the analysis and gauge any general effect on prescribing and dispensing rates . statins and other lipid - lowering medications were grouped , and we refer to them collectively as statins. medications and medication combinations intended primarily for hypertension control diuretics , calcium antagonists , and lisinopril / hydrochlorothiazide were collectively tracked as antihypertensives. we also included in this category recommendations for any of these medications in combination with a -blocker , ace inhibitor , or arb , which also have antihypertensive effect . we counted separately recommendations for -blockers alone and ace inhibitors or arb alone , because these may be indicated after a myocardial infarction and for chronic kidney disease , respectively . because -blockers and ace inhibitors or arb could be recommended either to reduce blood pressure or for other indications , indigo recommendations for these medications were not amenable to straightforward interpretation and were not analyzed in detail . secondary outcome measures included blood pressure and ldl cholesterol levels , bmi , and change in 5-year indigo - predicted risk of heart attack or stroke . secondary measures also included physician and patient perceptions of individualized guidelines ; data were collected during focus groups and a post - project email survey ( physicians ) and through a pre - post mail survey ( patients ) . we used difference - in - differences methods to evaluate changes in the secondary outcome measures between the intervention group and matched controls , assessing statistical significance with student 's t test . the kaiser permanente hawaii institutional review board reviewed the project and determined that it was quality improvement and not human subjects research . between november 2008 and april 2009 , 29 395 patients had a primary care visit and an indigo total benefit score that would make them eligible for indigo use ; 1915 ( 6.5% ) visited participating sites . of these , 489 ( 26% ) patients were selected for indigo use by their pcp . characteristics of patients using indigo and matched usual care comparison group indigo identified 1100 intervention recommendations , an average of 2.2 per patient . the average intervention benefit score among members with a non - zero benefit ranged from 3 to 39 . of indigo - identified interventions , 514 ( 47% ) matched guidelines already available as point - of - care decision supports . the proportion of unique to indigo identifications ranged from 0% for lifestyle interventions to 89% for adding a -blocker . frequency and type of recommendations for patients using indigo * indigo recommendations are based on adding simvastatin ( 40 mg ) to a patient 's treatment regimen . indigo recommendations are based on adding lisinopril ( 40 mg ) to a patient 's treatment regimen . recommendations that included an ace inhibitor or arb along with hydrochlorothiazide or amlodipine are counted under antiphypertensive agents. indigo recommendations are based on adding atenolol ( 50 mg ) to a patient 's treatment regimen . antiphypertensive agents. indigo recommendations are based on adding hydrochlorothiazide ( 25 mg ) , lisinopril / hydrochlorothiazide ( 20 mg/12.5 mg ) , or amlodipine ( 5 mg ) to a patient 's treatment regimen . patients who used indigo and had statins recommended received significantly more new statin prescriptions than did usual care patients ( 39% vs 8% , p<.01 ) . patients using indigo with new prescriptions for statins had a non - significant increase in the percentage that picked up at least one dispensing ( 72% vs 50% , p=0.10 ) . patients using indigo who had antihypertensive agents recommended did not receive more physician prescriptions ( 17% vs 15% , p>0.20 ) but were more likely to pick up at least one dispensing if prescribed ( 85% vs 67% , p=0.01 ) . no statistically significant differences existed in mean systolic blood pressure , ldl cholesterol , and bmi between baseline and follow - up for patients using indigo compared to matched usual care patients . the predicted 5-year risk of heart attack or stroke for patients using indigo decreased from 6.7% to 5.1% , a reduction of 1.6% , while predicted risk for usual care patients decreased by 1.0% ( table 3 ) . changes in medication prescribing , medication dispensing , physiological markers , and predicted 5-year risk of heart attack or stroke in 489 indigo - using patients versus 489 matched patients at usual care sites * antihypertensive agents include hydrochlorothiazide ( 25 mg ) , lisinopril / hydrochlorothiazide ( 20 mg/12.5 mg ) , or amlodipine ( 5 mg ) , as well as any of these in combination with atenolol or lisinopril . ldl and blood pressure values exclude 121 patients who were not reassessed between indigo use and the end of follow - up . the majority rated indigo as very useful for educating patients about health risks ( 80% ) , very helpful in involving patients in decision - making ( 60% ) , or in helping them make the best clinical decisions for patients ( 60% ) and usually or always helpful with treatment recommendations ( 60% ) . however , 90% of physicians reported they lacked time to use indigo with all patients who could benefit . pcp expressed belief in indigo 's long - term effectiveness at influencing patients towards needed behavior changes , and indicated that they found indigo most effective with patients who were non - adherent to medication recommendations , presented for care during a teachable moment , or rarely made in - person clinic visits . among 1005 members completing the pre - indigo survey , 817 ( 81% ) completed the post - survey ; 196 had used indigo and 621 had not . they did not differ significantly in demographics or self - reported health status . among indigo - using members , they were significantly more likely to report that their pcp or healthcare team had asked about their health goals , helped them with a specific plan for their care , or asked them to take a preventive medication , change their diet , exercise more , or stop smoking ( table 4 ) . no significant differences existed between groups in confidence to manage health conditions , belief in the importance of actively managing their healthcare , knowing how to prevent complications , knowing recommended lifestyle choices , having been asked to take insulin , perceived ability to follow recommended medication or lifestyle changes , assessment of how much their doctor helped them in the past 6 months , or overall rating of healthcare . statistically significant differences in patient experiences of care no significant differences were found between groups on nine other survey items , covering belief in the importance of actively managing their healthcare , knowing how to prevent complications , knowing recommended lifestyle choices , having been asked to take insulin , perceived ability to follow recommended medication or lifestyle changes , assessment of how much their doctor helped them in the past 6 months , or overall rating of healthcare . our project is , to the best of our knowledge , the first to document the implementation in primary care of individualized clinical guidelines derived from biomathematical simulation modeling . patients using indigo were 4.9 times more likely to receive a prescription for guideline - recommended statin medications , compared to a matched usual care comparison group , and those with new prescriptions were at least as likely to pick up their medications . patients using indigo were not significantly more likely to be prescribed an antihypertensive medication , but those with new prescriptions were 30% more likely to pick up at least one dispensing . no between - group differences were found in systolic blood pressure , ldl cholesterol , or bmi . the predicted 5-year risk of heart attack or stroke , if observed risk factor changes were maintained over that time , decreased significantly more among patients using indigo . the absolute reduction in predicted risk was modest , but meaningful in that it applies to leading causes of death . pcp reported benefits to indigo in terms of shared decision - making and patient education . compared to non - users , patients using indigo reported benefits in eight items related to goal setting , care planning , and medication and health behavior recommendations , but not in nine items assessing other aspects of their care experience . physicians selected patients for participation , which may have led to the inclusion of patients who were believed to be more amenable to new treatment recommendations or whose visits occurred during periods when physicians had more time for discussion . the matching resulted in significantly fewer obese patients and smokers in the usual care group , relative to indigo users . we can not distinguish between the effects of the individualized guidelines , the gui , and the 510-min discussions that ensued between physicians and patients . the measure of risk reduction was based on the same model as the individualized guideline recommendations , which could introduce confounding . sample size and project duration did not enable comparison of the stability of interventions over time or actual reductions in adverse events . our results shed some light on two hypothesized mechanisms by which individualized clinical guidelines may improve care , namely by enhancing shared decision - making and clinical decision support . patient participation in decision - making is pivotal to improved adherence , patient experience , and outcomes.2534 pcp and patients in our project both reported greater attention to shared decision - making when using indigo . greater engagement , such as the higher proportion of indigo - using patients who filled new prescriptions for antihypertensive medications , may have resulted from individualized information about the benefits of risk - reducing interventions . individualized guidelines constitute a further evolution in point - of - care clinical decision support , with patient - specific risk - reduction information based on all available information . clinical decision support improves guideline implementation by increasing user familiarity with guidelines and the underlying decision logic , overcoming inertia , and reducing guideline complexity.35 predicted , rather than speculative , risk - reduction estimates may have changed the nature of the pcp patient conversation . physicians estimation of patients cardiovascular risk , which has historically been primarily subjective , affects evidence - based pharmacotherapy recommendations.3638 well - documented improvements in prescribing rates have occurred for some preventive medications among patients with diabetes and hypertension.3944 the observed increase in statin prescribing rates in our project is consistent with these findings and suggests that the enhanced clinical decision support provided by indigo may be an effective mechanism for improving care . our limited - scale project demonstrates the feasibility of implementing individualized guidelines in clinical care and suggests benefits , but questions of both effectiveness and efficiency would need to be addressed and understood more fully before individualized guidelines are used on a widespread basis.7 a controlled trial of adequate size and duration would be required to address clinical issues , particularly whether indigo - predicted risk reductions translate into actual reductions in adverse events and whether any unanticipated side effects result . further assessment of individualized guidelines could also address practical issues we encountered , especially constraints on pcp time . alternative ways of using individualized guidelines might improve efficiency ; for example , nurse care managers instead of pcp could engage patients in using indigo . further efficiencies might result from mass customization : applying individualized guidelines to a large population , identifying patients with similar clinical profiles ( eg , those with borderline hypertension or hyperlipidemia accompanied by behavioral risk factors ) , scripting tailored patient and provider recommendations , and conducting systematic outreach for all patients who fit the profile . indigo individualized clinical guidelines were successfully implemented in primary care and were associated with increased prescribing and dispensing of some , but not all , cardioprotective medications . our assessment indicates that implementation of individualized guidelines in routine clinical care is feasible , and suggests that the intervention , including a gui and physician patient discussion , may result in care that reduces the risk of heart attack and stroke . an appropriate controlled trial would be needed to establish the clinical benefits of treatment guided by individualized guidelines , and alternative approaches could be developed to improve efficiency in their use .

objectiveto determine if indigo individualized clinical guidelines could be implemented in routine practice and assess their effects on care and care experience.methodsmatched comparison observational design . indigo individualized guidelines , based on a biomathematical simulation model , were used in shared decision - making . physicians and patients viewed risk estimates and tailored recommendations in a dynamic user interface and discussed them for 510 min . outcome measures were prescribing and dispensing of indigo - recommended medications , changes in physiological markers and predicted 5-year risk of heart attack and stroke , and physician and patient perceptions.results489 patients using indigo were 4.9 times more likely to receive a statin prescription than were matched usual care controls ( p=0.015 ) . no effect was observed on prescribing of antihypertensive medications , but indigo - using patients were more likely to pick up at least one dispensing ( p<0.05 ) . no significant changes were observed in blood pressure or serum lipid levels . predicted risk of heart attack or stroke decreased 1.6% among patients using indigo versus 1.0% among matched controls ( p<0.01 ) . physician and patient experiences were positive to neutral.limitationswe could not assess the separate effects of individualized guidelines , user interface , and physician patient discussions . patient selection could have influenced results . the measure of risk reduction was not independent of the individualized guidelines.conclusionsindigo individualized clinical guidelines were successfully implemented in primary care and were associated with increases in the use of cardioprotective medications and reduction in the predicted risk of adverse events , suggesting that a larger trial could be warranted .

Introduction Background and significance Materials and methods Design and setting Implementing IndiGO individualized guidelines Participants Propensity score matching Outcome measures and statistical analysis Results Discussion Conclusions

for example , the atp-3 dyslipidemia guideline uses comorbidities , risk factors , and coronary heart disease risk to sort individuals into risk categories.2 individualized clinical guidelines , taking into account individual risk factors and expected benefit from treatment , represent an even more precise application of evidence - based medicine.3 4 from the inception of modern evidence - based clinical guidelines , questions have consistently arisen about how best to disseminate and implement them.5 6 individualized guidelines heighten these concerns because of the requirement for detailed information about the person to whom they will be applied , for example , individual risk of morbidity or mortality and expected benefits of treatment . integration of guidelines in electronic health records ( ehr ) enables the ready availability of information necessary for personalized recommendations,7 but little is known about the use of individualized guidelines in routine clinical practice . demonstrating the potential benefits of individualized guidelines for a specific clinical problem is one challenge that must be met if they are to be used more broadly.7 a second challenge is clinician adoption . this report examines the feasibility of using individualized clinical guidelines in primary care and provides limited - scale observational data on medication use and cardiovascular risk . indigo individualized clinical guidelines are based on the archimedes model , a full - scale , rigorously validated biomathematical simulation model of human physiology , diseases , behavior , interventions , and healthcare systems.817 indigo uses regression equations to predict the likelihood of heart attack or stroke based on individual risk factors : cardiovascular disease history , comorbidities , medication history , biomarkers ( low - density lipoprotein ( ldl ) cholesterol , triglycerides , blood pressure , and body mass index ( bmi ) ) , and lifestyle behaviors . interventions modeled in indigo include smoking cessation , weight loss , and preventive medications : simvastatin , lisinopril , atenolol , hydrochlorthiazide , amlodipine , and lisinopril / hydrochlorthiazide . the score is a weighted combination of the decreased risk of adverse outcomes ; weights approximate the expected 5-year reduction in quality of life that would result from each outcome . finally , indigo predicts individualized 5-year risk of heart attack or stroke under three scenarios : ( 1 ) maintaining current interventions for 5 years ; ( 2 ) discontinuing all current interventions ; or ( 3 ) maintaining all identified interventions for 5 years . complete details of the predictive modeling have been published.18 automated data interchange between the ehr and the biomathematical model updates benefit scores daily , which are displayed in an interactive graphical user interface ( gui ) . indigo 's unique strength is identifying two types of individuals : those for whom traditional population - based guidelines indicate treatment but who would benefit too little to justify the side effects or risks , and those missed by population - based guidelines but who would derive considerable benefit . a simulation applying indigo to a secondary dataset found that individualized guidelines could lead to improved quality of care and lower costs for patients with hypertension , compared to traditional guidelines.3 we report here on the initial implementation of indigo individualized guidelines in primary care , in which we assessed the feasibility of use in routine clinical practice , integration into shared decision - making , patients and physicians experience with their use , and effects on medication use and cardiovascular risk in a small population over a short observation period . our quality improvement project used a matched control observational design in two clinics employing 10 primary care providers ( pcp ) in kaiser permanente 's hawaii region ( kphi ) . a comprehensive integrated ehr , kp health connect , contains all patient information.19 20 indigo individualized guidelines were implemented by automated daily extraction of relevant clinical information for all kphi adult members from kp health connect . after applying indigo calculations , risk and benefit scores were embedded in the panel support tool , a population management module of kp health connect , where pcp accessed them.2124 to encourage acceptance by practicing physicians and organizational leadership , algorithms were set such that all interventions that would be recommended by existing guidelines would also be recommended by indigo . the gui displayed each member 's current 5-year risk of heart attack or stroke and the comparable predicted risk after adopting and maintaining all identified interventions ( figure 1 ) . patients were considered eligible for indigo use if they had a scheduled primary care visit and their indigo total benefit score was in the top third of the kphi population . pcp then selected individual patients for indigo use based on the patient 's chief complaint and availability of pcp time , and the extent to which pcp believed patients would be likely to accept treatment recommendations . physicians selected patients with primary care visits between november 2008 and april 2009 ; for the purposes of this project , patients were followed for 36 months . selected patients viewed a video explaining individualized guidelines , their benefits and limitations , and the gui . pcp and patients subsequently used indigo in 510-min shared decision - making sessions ; a printed summary displayed interventions chosen by the member and the predicted 5-year risk with no interventions , with selected interventions , and with all identified interventions . physicians made treatment recommendations based on existing guidelines , indigo benefit scores , and clinical judgment . for example , indigo modeling was based on specified medication doses , but physicians tailored doses to individual patient needs . the intervention described in this report thus includes three components : individualized guidelines , a gui to communicate risk - reduction options to patients and their physicians , and a brief face - to - face discussion of the risk - reduction options . we compared patients using indigo at the pilot clinics with those receiving usual care at other clinics . we estimated a logistic regression model with indigo use as the dependent variable and six matching variables : age ; gender ; pre - indigo history of cardiovascular disease , diabetes , or hypertension ; number of primary care visits during the 12 months before indigo use ; an indicator for the presence or absence of at least one health behavior benefit recommendation ; and an indicator for the presence or absence of at least one medication recommendation . the primary outcome measures were prescribing and dispensing of medications that have been shown to reduce the risk of heart attack or stroke : statins or other lipid - lowering medications , diuretics , calcium antagonists , -blockers , and ace inhibitors or angiotensin receptor blockers ( arb ) . because indigo often recommends multiple medications , we grouped them by likely indication to simplify the analysis and gauge any general effect on prescribing and dispensing rates . medications and medication combinations intended primarily for hypertension control diuretics , calcium antagonists , and lisinopril / hydrochlorothiazide were collectively tracked as antihypertensives. because -blockers and ace inhibitors or arb could be recommended either to reduce blood pressure or for other indications , indigo recommendations for these medications were not amenable to straightforward interpretation and were not analyzed in detail . secondary outcome measures included blood pressure and ldl cholesterol levels , bmi , and change in 5-year indigo - predicted risk of heart attack or stroke . secondary measures also included physician and patient perceptions of individualized guidelines ; data were collected during focus groups and a post - project email survey ( physicians ) and through a pre - post mail survey ( patients ) . we used difference - in - differences methods to evaluate changes in the secondary outcome measures between the intervention group and matched controls , assessing statistical significance with student 's t test . our quality improvement project used a matched control observational design in two clinics employing 10 primary care providers ( pcp ) in kaiser permanente 's hawaii region ( kphi ) . indigo individualized guidelines were implemented by automated daily extraction of relevant clinical information for all kphi adult members from kp health connect . after applying indigo calculations , risk and benefit scores were embedded in the panel support tool , a population management module of kp health connect , where pcp accessed them.2124 to encourage acceptance by practicing physicians and organizational leadership , algorithms were set such that all interventions that would be recommended by existing guidelines would also be recommended by indigo . the gui displayed each member 's current 5-year risk of heart attack or stroke and the comparable predicted risk after adopting and maintaining all identified interventions ( figure 1 ) . patients were considered eligible for indigo use if they had a scheduled primary care visit and their indigo total benefit score was in the top third of the kphi population . pcp then selected individual patients for indigo use based on the patient 's chief complaint and availability of pcp time , and the extent to which pcp believed patients would be likely to accept treatment recommendations . physicians selected patients with primary care visits between november 2008 and april 2009 ; for the purposes of this project , patients were followed for 36 months . selected patients viewed a video explaining individualized guidelines , their benefits and limitations , and the gui . pcp and patients subsequently used indigo in 510-min shared decision - making sessions ; a printed summary displayed interventions chosen by the member and the predicted 5-year risk with no interventions , with selected interventions , and with all identified interventions . physicians made treatment recommendations based on existing guidelines , indigo benefit scores , and clinical judgment . for example , indigo modeling was based on specified medication doses , but physicians tailored doses to individual patient needs . the intervention described in this report thus includes three components : individualized guidelines , a gui to communicate risk - reduction options to patients and their physicians , and a brief face - to - face discussion of the risk - reduction options . we compared patients using indigo at the pilot clinics with those receiving usual care at other clinics . we estimated a logistic regression model with indigo use as the dependent variable and six matching variables : age ; gender ; pre - indigo history of cardiovascular disease , diabetes , or hypertension ; number of primary care visits during the 12 months before indigo use ; an indicator for the presence or absence of at least one health behavior benefit recommendation ; and an indicator for the presence or absence of at least one medication recommendation . the primary outcome measures were prescribing and dispensing of medications that have been shown to reduce the risk of heart attack or stroke : statins or other lipid - lowering medications , diuretics , calcium antagonists , -blockers , and ace inhibitors or angiotensin receptor blockers ( arb ) . because indigo often recommends multiple medications , we grouped them by likely indication to simplify the analysis and gauge any general effect on prescribing and dispensing rates . medications and medication combinations intended primarily for hypertension control diuretics , calcium antagonists , and lisinopril / hydrochlorothiazide were collectively tracked as antihypertensives. because -blockers and ace inhibitors or arb could be recommended either to reduce blood pressure or for other indications , indigo recommendations for these medications were not amenable to straightforward interpretation and were not analyzed in detail . secondary outcome measures included blood pressure and ldl cholesterol levels , bmi , and change in 5-year indigo - predicted risk of heart attack or stroke . secondary measures also included physician and patient perceptions of individualized guidelines ; data were collected during focus groups and a post - project email survey ( physicians ) and through a pre - post mail survey ( patients ) . we used difference - in - differences methods to evaluate changes in the secondary outcome measures between the intervention group and matched controls , assessing statistical significance with student 's t test . between november 2008 and april 2009 , 29 395 patients had a primary care visit and an indigo total benefit score that would make them eligible for indigo use ; 1915 ( 6.5% ) visited participating sites . characteristics of patients using indigo and matched usual care comparison group indigo identified 1100 intervention recommendations , an average of 2.2 per patient . of indigo - identified interventions , 514 ( 47% ) matched guidelines already available as point - of - care decision supports . frequency and type of recommendations for patients using indigo * indigo recommendations are based on adding simvastatin ( 40 mg ) to a patient 's treatment regimen . indigo recommendations are based on adding lisinopril ( 40 mg ) to a patient 's treatment regimen . patients who used indigo and had statins recommended received significantly more new statin prescriptions than did usual care patients ( 39% vs 8% , p<.01 ) . patients using indigo with new prescriptions for statins had a non - significant increase in the percentage that picked up at least one dispensing ( 72% vs 50% , p=0.10 ) . patients using indigo who had antihypertensive agents recommended did not receive more physician prescriptions ( 17% vs 15% , p>0.20 ) but were more likely to pick up at least one dispensing if prescribed ( 85% vs 67% , p=0.01 ) . no statistically significant differences existed in mean systolic blood pressure , ldl cholesterol , and bmi between baseline and follow - up for patients using indigo compared to matched usual care patients . the predicted 5-year risk of heart attack or stroke for patients using indigo decreased from 6.7% to 5.1% , a reduction of 1.6% , while predicted risk for usual care patients decreased by 1.0% ( table 3 ) . changes in medication prescribing , medication dispensing , physiological markers , and predicted 5-year risk of heart attack or stroke in 489 indigo - using patients versus 489 matched patients at usual care sites * antihypertensive agents include hydrochlorothiazide ( 25 mg ) , lisinopril / hydrochlorothiazide ( 20 mg/12.5 mg ) , or amlodipine ( 5 mg ) , as well as any of these in combination with atenolol or lisinopril . the majority rated indigo as very useful for educating patients about health risks ( 80% ) , very helpful in involving patients in decision - making ( 60% ) , or in helping them make the best clinical decisions for patients ( 60% ) and usually or always helpful with treatment recommendations ( 60% ) . pcp expressed belief in indigo 's long - term effectiveness at influencing patients towards needed behavior changes , and indicated that they found indigo most effective with patients who were non - adherent to medication recommendations , presented for care during a teachable moment , or rarely made in - person clinic visits . among indigo - using members , they were significantly more likely to report that their pcp or healthcare team had asked about their health goals , helped them with a specific plan for their care , or asked them to take a preventive medication , change their diet , exercise more , or stop smoking ( table 4 ) . no significant differences existed between groups in confidence to manage health conditions , belief in the importance of actively managing their healthcare , knowing how to prevent complications , knowing recommended lifestyle choices , having been asked to take insulin , perceived ability to follow recommended medication or lifestyle changes , assessment of how much their doctor helped them in the past 6 months , or overall rating of healthcare . statistically significant differences in patient experiences of care no significant differences were found between groups on nine other survey items , covering belief in the importance of actively managing their healthcare , knowing how to prevent complications , knowing recommended lifestyle choices , having been asked to take insulin , perceived ability to follow recommended medication or lifestyle changes , assessment of how much their doctor helped them in the past 6 months , or overall rating of healthcare . our project is , to the best of our knowledge , the first to document the implementation in primary care of individualized clinical guidelines derived from biomathematical simulation modeling . patients using indigo were 4.9 times more likely to receive a prescription for guideline - recommended statin medications , compared to a matched usual care comparison group , and those with new prescriptions were at least as likely to pick up their medications . patients using indigo were not significantly more likely to be prescribed an antihypertensive medication , but those with new prescriptions were 30% more likely to pick up at least one dispensing . no between - group differences were found in systolic blood pressure , ldl cholesterol , or bmi . the predicted 5-year risk of heart attack or stroke , if observed risk factor changes were maintained over that time , decreased significantly more among patients using indigo . the absolute reduction in predicted risk was modest , but meaningful in that it applies to leading causes of death . pcp reported benefits to indigo in terms of shared decision - making and patient education . compared to non - users , patients using indigo reported benefits in eight items related to goal setting , care planning , and medication and health behavior recommendations , but not in nine items assessing other aspects of their care experience . the matching resulted in significantly fewer obese patients and smokers in the usual care group , relative to indigo users . we can not distinguish between the effects of the individualized guidelines , the gui , and the 510-min discussions that ensued between physicians and patients . the measure of risk reduction was based on the same model as the individualized guideline recommendations , which could introduce confounding . sample size and project duration did not enable comparison of the stability of interventions over time or actual reductions in adverse events . our results shed some light on two hypothesized mechanisms by which individualized clinical guidelines may improve care , namely by enhancing shared decision - making and clinical decision support . patient participation in decision - making is pivotal to improved adherence , patient experience , and outcomes.2534 pcp and patients in our project both reported greater attention to shared decision - making when using indigo . greater engagement , such as the higher proportion of indigo - using patients who filled new prescriptions for antihypertensive medications , may have resulted from individualized information about the benefits of risk - reducing interventions . individualized guidelines constitute a further evolution in point - of - care clinical decision support , with patient - specific risk - reduction information based on all available information . clinical decision support improves guideline implementation by increasing user familiarity with guidelines and the underlying decision logic , overcoming inertia , and reducing guideline complexity.35 predicted , rather than speculative , risk - reduction estimates may have changed the nature of the pcp patient conversation . physicians estimation of patients cardiovascular risk , which has historically been primarily subjective , affects evidence - based pharmacotherapy recommendations.3638 well - documented improvements in prescribing rates have occurred for some preventive medications among patients with diabetes and hypertension.3944 the observed increase in statin prescribing rates in our project is consistent with these findings and suggests that the enhanced clinical decision support provided by indigo may be an effective mechanism for improving care . our limited - scale project demonstrates the feasibility of implementing individualized guidelines in clinical care and suggests benefits , but questions of both effectiveness and efficiency would need to be addressed and understood more fully before individualized guidelines are used on a widespread basis.7 a controlled trial of adequate size and duration would be required to address clinical issues , particularly whether indigo - predicted risk reductions translate into actual reductions in adverse events and whether any unanticipated side effects result . further assessment of individualized guidelines could also address practical issues we encountered , especially constraints on pcp time . alternative ways of using individualized guidelines might improve efficiency ; for example , nurse care managers instead of pcp could engage patients in using indigo . further efficiencies might result from mass customization : applying individualized guidelines to a large population , identifying patients with similar clinical profiles ( eg , those with borderline hypertension or hyperlipidemia accompanied by behavioral risk factors ) , scripting tailored patient and provider recommendations , and conducting systematic outreach for all patients who fit the profile . indigo individualized clinical guidelines were successfully implemented in primary care and were associated with increased prescribing and dispensing of some , but not all , cardioprotective medications . our assessment indicates that implementation of individualized guidelines in routine clinical care is feasible , and suggests that the intervention , including a gui and physician patient discussion , may result in care that reduces the risk of heart attack and stroke . an appropriate controlled trial would be needed to establish the clinical benefits of treatment guided by individualized guidelines , and alternative approaches could be developed to improve efficiency in their use .

the highest species diversity for most groups of organisms lies in the tropics . lichenized fungi do not appear to be an exception , as sipman & aptroot ( 2001 ) estimated that between one - third and one - half of the world s lichen diversity occurs there , and suggested that 50 % of the tropical lichen biota remained unknown . yet there have been few experimental studies on ascospore discharge , germination , development of mycelia , and physiology of the fungal partners ( mycobionts ) of tropical lichens compared with those on temperate species . this is a major gap in our understanding of even basic aspects of the biology of tropical lichens . the first cited studies on the isolation of lichen - forming fungi are generally those of tbler ( 1909 ) and thomas ( 1939 ) , although tbler was primarily interested in the re - synthesis of lichens from their individual symbionts ( turbin 1996 ) . however , werner ( 1927 ) , innovatively examined the effect of different media and additions on the growth of selected mycobionts from a range of lichens . subsequent workers have concentrated on the development of methods for lichen re - synthesis ( ahmadjian 1964 ) , and later ahmadjian et al . ( 1980 ) and ahmadjian & jacobs ( 1981 ) produced the two most successful protocols ( bubrick 1988 ) . ( 1995 ) were the first to attempt the isolation of a wide range of fungal partners of lichens , and also lichenicolous fungi , on a worldwide basis , although their material was predominantly from non - tropical regions . 2002 ) reviewed the protocols available for isolation and cultivation of fungal and algal partners of lichens , emphasising studies by japanese researchers , but again based largely on non - tropical material . a brief synopsis of methods used is provided by stocker - wrgtter & hager ( 2008 ) , with an emphasis on the production of extrolites ( lichen substances or secondary metabolites ) . the lack of basic information on the isolation and growth of the fungal partners of tropical lichens provided the rationale for the present study . we investigated ascospore discharge from a wide range of tropical lichens in order to make a preliminary assessment of the conditions under which discharge occurred , and whether there could be any seasonal correlations . observations on factors affecting germination and subsequent development on solid , or in liquid , growth media are also reported , since these are virtually undocumented for the fungal partners of tropical lichens . our studies were carried out to identify apparent trends and issues that merited in - depth investigations , as well as testing the efficacy of alternative culture methods . the collection of samples began in 1998 , and concentrated on khao yai national park ( ky ) , central thailand . the remainder were collected during field surveys to doi suthep ( 1849 n , 9953 e ) and chiang dao ( l940 n , 99 e ) in chiang mai province ( cm ) , mae fah luang arboretum ( 20 n , 99.5 e ) chiang rai province ( cr ) , sai yok district ( 14 n , 99 e ) kanjanaburi province ( kjb ) , khao sok national park ( 8 n , 99.5 e ) nakon srithammarat province ( ts and rpb respectively ) , phu kradueng national park , ( 16.8 n , 101.8 e ) loei province ( pkd ) , suan phueng district ( 13.5 n , 99 e ) ratchaburi province ( sp ) , and sakaeraj research station ( 14 n , 102 e ) nakhon ratchasima province ( skr ) . some collections from huai kha khaeng wildlife sanctuary kanjanaburi province , vietnam ( vn ) and cambodia ( cam ) were donated by colleagues and friends . khao yai national park was visited monthly during one year ( 19992000 ) for seasonal observations and experiments to explore the development of thalli , and also to ascertain if there were seasonal differences in ascospore discharge and spore viability samples were cut into pieces , wrapped in tissue paper , and placed in individual strong brown paper bags . these were then returned to a survey house workroom and cleaned of attached soil or other extraneous material . each sample was given a collection number , and information on the locality , substratum , and collection details were recorded . then . if the specimens could not be immediately transferred to the laboratory for pre - isolation treatment , they were either kept in a cool place , or ( where available ) a domestic refrigerator , until they could be transferred . in the laboratory , samples were air - dried at room temperature ( 30 c ) overnight , and then transferred to new paper envelopes with identification labels and stored in a domestic refrigerator at 4 c until the isolation protocol had been completed . specimens were identified as precisely as possible on the basis of their morphology , anatomy , and chemical constituents ( determined by standard thin - layer chromatographic methods ; orange et al . 2001 ) . in many cases it was not possible to fully determine the samples to species as identification remains a major problem in tropical lichenology . the basic monographic treatments required to provide a sound taxonomic basis for studies of lichen distribution , ecology , and physiology are still lacking for most lichen families and genera in the tropics . species that have not previously been described are also likely to be found ; homchantara & coppins ( 2002 ) described 26 species of thelotremataceae as new to science from thailand , and aptroot et al . ( 2007 ) added 300 tropical species to the national list , of which 12 were new to science . the number of collections made for each morphological type of lichen , together with their geographical locations , are summarized in table 1 , while full details of selected collections for which positive results were obtained are given in table 2 . a list of the material collected is included as supplementary information ( table s1 , online only ) and in sangvichien ( 2005 ) . voucher specimens are maintained in the lichen herbarium , ramkhamhaeng university , bangkok ( ramk ) . the specimens were removed from storage , and surface - cleaned with air from an aerosol camera blower to remove any remaining soil and debris . a sterile surgical blade ( gowlands no . 11 ) was used to dissect specimens to obtain small portions with ascomata , and the remainder of the samples were then returned to storage . the portions of lichen with ascomata , or occasionally only a single ascoma , were attached with a small quantity of petroleum jelly onto the inverted lid of a 9 cm diam petri dish ( sterilin ) . the spores were allowed to shoot upwards onto an overlying layer of tap water agar ( wa ; booth 1971 ) . the agar surface was examined daily with a stereozoom binocular microscope ( olympus sz11 ) , and once ascospores had been discharged , small agar blocks ( 35 mm ) with ascospores on the surface were excised and transferred to malt - yeast extract agar ( mya ; merck or oxoid ) . germination of ascospores was assessed under the stereozoom microscope ; observations were made daily for 7 d , and subsequently twice weekly . if no germ tubes had been observed after six weeks , then no - germination germinated ascospores were maintained at room temperature for further studies on growth and colony morphology , or were used as inoculum for liquid media . in order to investigate the seasonality and discharge of ascospores , thalli were collected each month from the same trees in khao yai national park over a one year period , and their discharge patterns and rates of germination were determined for each monthly sample following the protocol described above . the distance to which ascospores were discharged was studied in a representative sample of 15 species . clear plastic boxes 18 x 7.5 x 5 cm were used with a layer of tap water agar in the lid of the box . 0.3 mm diam , were attached to one vertical microscope slide ( 2 x 4 cm ) which was shallowly immersed in the agar layer . the box was then incubated on the laboratory bench at an ambient temperature of 2530 c , with approximately 12 h of daylight . the agar surface was examined under an olympus stereozoom microscope ( model sz 11 ) daily for 5 d. if no spores were discharged within 3 d , the procedure was repeated , and then , if after a second 3 d period no discharge was observed , the protocol was repeated for a third and final time . we also investigated the effects of relative humidity by incubating petri dishes in plastic moist chambers containing different saturated solutions to maintain the relative humidity at particular levels , following kaye & laby ( 1966 ) : potassium nitrate ( 92 % ) , ammonium sulphate ( 80 % ) , and sodium nitrate ( 65 % ) . we tried , but did not adopt , the surface sterilization protocol of crittenden et al . ( 1995 ) as we found it to be detrimental to ascospore discharge in the tropical lichens tested ; in consequence , untreated lichen samples were used throughout . mya ( see above ) was the medium of choice for all cultures of the fungal partners , but potato dextrose agar ( pda ) , corn meal agar ( cma ) , oatmeal agar ( oma ) , and czapek - dox agar ( cda ) , were also used to determine the optimum medium for growth . for recipes see booth ( 1971 ) . malt yeast extract broth ( myb ) was selected as the standard medium for studies of the isolated fungi in liquid culture , since good growth rates of several fungal partners had been observed on solid mya . myb has also been favoured by previous researchers ( e.g. hamada 1989 , honegger 1990 , yamamoto et al . static culture was most frequently used , and following initial trials with direct inoculation of pieces of the fungal cultures into the liquid medium , different types of physical support for the fungi on the surface of the liquid were tested . four types of material were evaluated : ( 1 ) stacked membrane filters ( pores 0.22 m diam ; polyvinylidene fluoride , pvdf ) were promising when tested first , but the slippery surface when floating on the liquid rendered them difficult to inoculate . ( 2 ) whatman no.1 filter papers were tested in order to overcome the problem of stacked layers . ( 3 ) kraft paper was tried as an alternative to whatman no.1 . and ( 4 ) , synthetic sponge ( polystyrene ) pieces 2.5 x 2.5 x 0.3 cm , together with pieces of fungal colonies cut from solid media of 0.4 x 0.4 cm , placed on the surface of these materials , and floated on the surface of 50 ml myb in 250 ml erlenmeyer flasks . observations were made twice daily and , at the same time , the flasks were gently swirled for 1015 s to circulate the medium . since poor aeration could be a factor limiting growth , the effect of increased aeration was tested in two ways . first , air was supplied by an aquarium air pump and passed through a sterile filter ( sartorious , sartofluor ; pores 0.2 m diam ) to prevent contamination . second , flasks were placed on an orbital shaker ( innova 4230 , new brunswick ) . shake cultures were prepared using inocula produced in the same way as for static cultures , and transferred to 250 ml erlenmeyer flasks containing 50 ml of myb . the orbital shaking incubator was set at a speed of 200 rpm , and at a temperature of 30 0.5 c . specimens for scanning electron microscopy ( sem ) , either intact ascomata or cultures of the isolated fungal partners , were fixed in 5 % glutaraldehyde and dehydrated in a graded ethyl alcohol series . the specimens were then attached to aluminium stubs using either dag metallic paint or adhesive carbon pads to prevent electron charging of the specimens . the samples were gold - coated using a sputter coating unit ( polaron ru - sc7620 ) and examined either with a jeol 840 sem , a jeol sem5410lv , or a leo 1455vp scanning electron microscope . digital images were produced using an image - capture system ( rentec ) or with accessories of leo 1455 vp . the collection of samples began in 1998 , and concentrated on khao yai national park ( ky ) , central thailand . the remainder were collected during field surveys to doi suthep ( 1849 n , 9953 e ) and chiang dao ( l940 n , 99 e ) in chiang mai province ( cm ) , mae fah luang arboretum ( 20 n , 99.5 e ) chiang rai province ( cr ) , sai yok district ( 14 n , 99 e ) kanjanaburi province ( kjb ) , khao sok national park ( 8 n , 99.5 e ) nakon srithammarat province ( ts and rpb respectively ) , phu kradueng national park , ( 16.8 n , 101.8 e ) loei province ( pkd ) , suan phueng district ( 13.5 n , 99 e ) ratchaburi province ( sp ) , and sakaeraj research station ( 14 n , 102 e ) nakhon ratchasima province ( skr ) . some collections from huai kha khaeng wildlife sanctuary kanjanaburi province , vietnam ( vn ) and cambodia ( cam ) were donated by colleagues and friends . khao yai national park was visited monthly during one year ( 19992000 ) for seasonal observations and experiments to explore the development of thalli , and also to ascertain if there were seasonal differences in ascospore discharge and spore viability samples were cut into pieces , wrapped in tissue paper , and placed in individual strong brown paper bags . these were then returned to a survey house workroom and cleaned of attached soil or other extraneous material . each sample was given a collection number , and information on the locality , substratum , and collection details were recorded . then . if the specimens could not be immediately transferred to the laboratory for pre - isolation treatment , they were either kept in a cool place , or ( where available ) a domestic refrigerator , until they could be transferred . in the laboratory , samples were air - dried at room temperature ( 30 c ) overnight , and then transferred to new paper envelopes with identification labels and stored in a domestic refrigerator at 4 c until the isolation protocol had been completed . specimens were identified as precisely as possible on the basis of their morphology , anatomy , and chemical constituents ( determined by standard thin - layer chromatographic methods ; orange et al . 2001 ) . in many cases it was not possible to fully determine the samples to species as identification remains a major problem in tropical lichenology . the basic monographic treatments required to provide a sound taxonomic basis for studies of lichen distribution , ecology , and physiology are still lacking for most lichen families and genera in the tropics . species that have not previously been described are also likely to be found ; homchantara & coppins ( 2002 ) described 26 species of thelotremataceae as new to science from thailand , and aptroot et al . ( 2007 ) added 300 tropical species to the national list , of which 12 were new to science . the number of collections made for each morphological type of lichen , together with their geographical locations , are summarized in table 1 , while full details of selected collections for which positive results were obtained are given in table 2 . a list of the material collected is included as supplementary information ( table s1 , online only ) and in sangvichien ( 2005 ) . voucher specimens are maintained in the lichen herbarium , ramkhamhaeng university , bangkok ( ramk ) . the specimens were removed from storage , and surface - cleaned with air from an aerosol camera blower to remove any remaining soil and debris . a sterile surgical blade ( gowlands no . 11 ) was used to dissect specimens to obtain small portions with ascomata , and the remainder of the samples were then returned to storage . the portions of lichen with ascomata , or occasionally only a single ascoma , were attached with a small quantity of petroleum jelly onto the inverted lid of a 9 cm diam petri dish ( sterilin ) . the spores were allowed to shoot upwards onto an overlying layer of tap water agar ( wa ; booth 1971 ) . the agar surface was examined daily with a stereozoom binocular microscope ( olympus sz11 ) , and once ascospores had been discharged , small agar blocks ( 35 mm ) with ascospores on the surface were excised and transferred to malt - yeast extract agar ( mya ; merck or oxoid ) . germination of ascospores was assessed under the stereozoom microscope ; observations were made daily for 7 d , and subsequently twice weekly . if no germ tubes had been observed after six weeks , then no - germination germinated ascospores were maintained at room temperature for further studies on growth and colony morphology , or were used as inoculum for liquid media . in order to investigate the seasonality and discharge of ascospores , thalli were collected each month from the same trees in khao yai national park over a one year period , and their discharge patterns and rates of germination were determined for each monthly sample following the protocol described above . the distance to which ascospores were discharged was studied in a representative sample of 15 species . clear plastic boxes 18 x 7.5 x 5 cm were used with a layer of tap water agar in the lid of the box . 0.3 mm diam , were attached to one vertical microscope slide ( 2 x 4 cm ) which was shallowly immersed in the agar layer . the box was then incubated on the laboratory bench at an ambient temperature of 2530 c , with approximately 12 h of daylight . the agar surface was examined under an olympus stereozoom microscope ( model sz 11 ) daily for 5 d. if no spores were discharged within 3 d , the procedure was repeated , and then , if after a second 3 d period no discharge was observed , the protocol was repeated for a third and final time . we also investigated the effects of relative humidity by incubating petri dishes in plastic moist chambers containing different saturated solutions to maintain the relative humidity at particular levels , following kaye & laby ( 1966 ) : potassium nitrate ( 92 % ) , ammonium sulphate ( 80 % ) , and sodium nitrate ( 65 % ) . we tried , but did not adopt , the surface sterilization protocol of crittenden et al . ( 1995 ) as we found it to be detrimental to ascospore discharge in the tropical lichens tested ; in consequence , untreated lichen samples were used throughout . mya ( see above ) was the medium of choice for all cultures of the fungal partners , but potato dextrose agar ( pda ) , corn meal agar ( cma ) , oatmeal agar ( oma ) , and czapek - dox agar ( cda ) , were also used to determine the optimum medium for growth . for recipes malt yeast extract broth ( myb ) was selected as the standard medium for studies of the isolated fungi in liquid culture , since good growth rates of several fungal partners had been observed on solid mya . myb has also been favoured by previous researchers ( e.g. hamada 1989 , honegger 1990 , yamamoto et al . static culture was most frequently used , and following initial trials with direct inoculation of pieces of the fungal cultures into the liquid medium , different types of physical support for the fungi on the surface of the liquid were tested . four types of material were evaluated : ( 1 ) stacked membrane filters ( pores 0.22 m diam ; polyvinylidene fluoride , pvdf ) were promising when tested first , but the slippery surface when floating on the liquid rendered them difficult to inoculate . ( 2 ) whatman no.1 filter papers were tested in order to overcome the problem of stacked layers . ( 3 ) kraft paper was tried as an alternative to whatman no.1 . and ( 4 ) , synthetic sponge ( polystyrene ) pieces 2.5 x 2.5 x 0.3 cm , together with pieces of fungal colonies cut from solid media of 0.4 x 0.4 cm , placed on the surface of these materials , and floated on the surface of 50 ml myb in 250 ml erlenmeyer flasks . observations were made twice daily and , at the same time , the flasks were gently swirled for 1015 s to circulate the medium . since poor aeration could be a factor limiting growth , the effect of increased aeration was tested in two ways . first , air was supplied by an aquarium air pump and passed through a sterile filter ( sartorious , sartofluor ; pores 0.2 m diam ) to prevent contamination . second , flasks were placed on an orbital shaker ( innova 4230 , new brunswick ) . shake cultures were prepared using inocula produced in the same way as for static cultures , and transferred to 250 ml erlenmeyer flasks containing 50 ml of myb . the orbital shaking incubator was set at a speed of 200 rpm , and at a temperature of 30 0.5 c . specimens for scanning electron microscopy ( sem ) , either intact ascomata or cultures of the isolated fungal partners , were fixed in 5 % glutaraldehyde and dehydrated in a graded ethyl alcohol series . the specimens were then attached to aluminium stubs using either dag metallic paint or adhesive carbon pads to prevent electron charging of the specimens . the samples were gold - coated using a sputter coating unit ( polaron ru - sc7620 ) and examined either with a jeol 840 sem , a jeol sem5410lv , or a leo 1455vp scanning electron microscope . digital images were produced using an image - capture system ( rentec ) or with accessories of leo 1455 vp . this study employed a large number of samples in order to gain an impression of possible general features of ascospore discharge and development in tropical lichens to provide a basis on which to determine directions future investigations might take . as replicates were not used for most of the species , the conclusions must be viewed as preliminary and treated with caution . nevertheless , some indications of trends emerged , although we recognize that further work may require their modification or refinement . this caveat must be borne in mind with respect to this discussion of our results . the number of crustose lichen collections made was much larger than that of foliose lichens ( table 1 ) . crustose lichens were preferentially selected for experimentation as preliminary studies suggested that their ascospores germinated more readily on artificial media . ascomata of erect shrubby ( fruticose ) and pendent lichens were much less common than crustose or foliose ones , especially at khao yai national park , and so could not be investigated further . of the 292 lichen samples collected ( table 1 ) , 170 samples liberated ascospores in the laboratory , and in several instances successive samples of the same lichen exhibited high percentage germination rates ( table 2 , fig.1 ) . in contrast , foliose lichen species ( e.g. heterodermia diademata ) exhibited a high discharge rate , but only a low percentage of ascospores germinated . eluteriae ( ky 66 ) , graphis elegans ( ky162),and g. rigidula ( ky165 ) , ascospores were discharged readily each month , and spores from each monthly sample also germinated . however , in contrast , cladonia submultiformis ( ky117 ) discharged ascospores only towards the end of the winter ( january - february ) , and in the summer ( april - june ) none were discharged ( fig . 2 ) . following germination , the ability of the isolated fungi to continue to develop and form colonies was investigated . in some common crustose lichen species , the fungal partners grew well and produced small colonies within a few months ( fig . species of trypethelium and laurera developed colonies readily , while in haematomma wattii and lecanora intumescens the spores germinated but growth was either very slow or soon ceased . in addition to growth on solid media , liquid culture under static growth conditions was tried , but generally growth was slow . further , when the fungi were inoculated onto the surface of membrane filters floating on the surface of myb , this was not satisfactory as the membranes often sank following inoculation , or tended to collapse after a period of growth . amongst the other physical carriers tested , were segments of unbleached kraft paper or whatman filter paper several layers thick . growth occurred on the surface of these , but it was only possible to assess this visually as it proved impossible to physically separate all the fungal material from their surfaces . in contrast , the sponge pieces tested as alternative carriers facilitated growth after incubation in the fungal partners tested ; in most cases , sponge proved to be superior to the other carriers tried ( fig . no contamination by spores from other fungi during discharge were encountered ; the distinctive ascospores from the lichens were deposited , either as small packets of spores or as single spores , and could easily be recognized for subculturing using a stereozoom microscope . our results suggest that high spore discharge rates are correlated with the freshness of the samples and season of collection , as well as the state of maturity of the ascomata . spore germination also appeared to be correlated with species distributions . widely distributed species , such as trypethelium eluteriae , laurea bengualensis , and most graphidaceae studied , exhibited relatively high rates of germination . there was , however , considerable variation in ascospore discharge between the species tested , and , also between different collections of the same species . in laurera bengualensis and l. madreporiformis , spores were readily discharged in all of the collections examined , but in l. meristospora , although discharge occurred , it was at a much lower rate . in trypethelium eluteriae , spore discharge occurred throughout the year , but in cladonia submultiformis , although ascospores were also readily discharged , only those from the end of the winter season ( february ) germinated . this suggests that in some lichen - fungi , seasonality is important even in the tropics . these observations of differences in spore discharge between species are in agreement with those of crittenden et al . the distance of discharge of ascospores is important for the dissemination of the species ( table 3 ) . of the 15 species tested , those of graphina sp . this figure compares with the maximum of 45 mm obtained for the temperate rhizocarpon umbilicatum ( bailey & garrett 1968 ) . however , in that study no information was given as to the distance attained by the majority of spores , which is perhaps the most pertinent parameter in relation to effective dispersal and establishment in nature . there was a wide variation even within the 75 % ranges of projection in many cases , and we speculate that this could be an adaptation to increase the probability of contact with a suitable new substrate . in nature , air turbulence currents and wind would also be expected to influence the final distance travelled . ( pkd3 ) and laurera subdiscreta ( skr1 ) , while at 45 c no discharge was observed in any species tested ( table 4 ) . as might have been expected for tropical species , most species discharged at 30 c and 35 c , but the mean optimum discharge temperature for all species was close to 25 c . relative humidity also appears to be important , with three of the four species investigated discharging at relative humidities from 65 % to 100 % ( fig . 4 ) . however , laurera subdiscreta ( skr1 ) , l. benguelensis ( ky 61 ) , pyrenula sp . ( ky95 ) , and graphis sp . 3 ( ky260 ) , discharged only at 100 % relative humidity . these results suggest that both temperature and relative humidity , which will vary with habitat and season , influence ascospore discharge in tropical lichens to different degrees , something that would be a major factor in their performance and occurrence in nature . widely distributed species such as trypetheium eluteriae and laurera bengualensis , together with most graphidaceae tested , exhibited relatively high rates of ascospore germination . ascospores of crustose lichens generally germinated readily , whilst those from shrubby and pendent species were much more difficult , or failed to germinate . ascospores of the different fungi exhibited several distinctive germination patterns ( fig.1a h ) : ( 1 ) multiple germination tubes developing from different regions of the spore ( e.g. pyrenula and arthopyrenia species , graphis cicatricosa , laurera benguelensis , graphina irabensis ; ( 2 ) bipolar germination ( e.g. trypethelium tropicum ) ; and ( 3 ) multiple germination tubes developing all over the spore from individual segments within them ( e.g. thelotremataceae , cyclographina platyleuca ky390/rpb3 ) . when germination was successful , fungal partners of most crustose species tested grew well on solid media , with small colonies developing within a few months . trypethelium and laurera species generally grew well , but haematomma wattii and lecanora intumescens developed very slowly and growth often ceased even though the ascospores germinated readily . growth in liquid culture was generally very slow , and static culture was found to be superior to shake culture for all species tested . however , growth on static liquid culture was much enhanced by the use of a physical carrier . while segments of kraft paper or whatman filter paper proved to be successful carriers , sponge pieces were superior in relation to visible enhanced growth . we consider that sponge pieces used as a carrier have a wide potential for studies on the physiology and development of lichen fungi as the colonies can be transferred without disruption to different liquid media . this means that , for example , the effect of different nutrients in the medium on the production of extrolites could be explored . culberson & armaleo ( 1992 ) , in their investigation of cladonia grayi , previously concluded that the production of compounds concentrated in the naturally occurring lichen was linked to the aerial growth habit . their conclusion was based on the finding that , following the transfer of lightly fragmented mycelia from liquid to solid media , there was a subsequent proliferation of aerial hyphae and extrolite production . although only a limited investigation of the chemical products of the isolated fungal partners of the tropical lichens was undertaken in our study , comparison of extrolites from the whole lichen thallus with those produced by the fungal partner alone indicated that in some cases more compounds were produced by the whole thallus than in the isolated fungal cultures . there were also few differences between the compounds produced by the fungus cultured under static conditions compared to those grown in shake culture . in a few cases , however , some additional compounds were detected in the shake culture extracts . ( 1995 ) in particular , demonstrate that , contrary to a general belief of recalcitrance to grow on artificial media , it is possible to obtain many lichen - forming fungi in isolated culture provided that recently collected material is used . further , our results on ascospore discharge show that the seasonal behaviour and discharge distances of the ascospores of tropical lichens recalls that of those in temperate regions . we also suspect that the short distances over which ascospores are discharged , especially where these are multicelled and large , contributes to the inability of many to spread into secondary environments from old - growth native forests and so facilitates their utility as bioindicators of ecological stability ( wolesley et al . 1994 ) . we hope that this preliminary study will encourage more experimental work on the factors affecting the reproductive biology of tropical lichens , which are crucial to an understanding of their ecology and distribution especially at local scales .

a total of 292 lichen samples , representing over 200 species and at least 65 genera and 26 families , were collected , mainly in thailand ; 170 of the specimens discharged ascospores in the laboratory . generally , crustose lichens exhibited the highest discharge rates and percentage germination . in contrast , foliose lichen samples , although having a high discharge rate , had a lower percentage germination than crustose species tested . a correlation with season was indicated for a number of species . continued development of germinated ascospores into recognizable colonies in pure culture was followed for a selection of species . the most successful medium tried was 2 % malt - yeast extract agar ( mya ) , and under static conditions using a liquid culture medium , a sponge proved to be the best of several physical carriers tested ; this novel method has considerable potential for experimental work with lichen mycobionts .

INTRODUCTION MATERIALS AND METHODS Taxon sampling Spore discharge and germination Fungal culture on solid media Fungal culture in liquid media Scanning electron microscopy RESULTS AND DISCUSSION

the highest species diversity for most groups of organisms lies in the tropics . lichenized fungi do not appear to be an exception , as sipman & aptroot ( 2001 ) estimated that between one - third and one - half of the world s lichen diversity occurs there , and suggested that 50 % of the tropical lichen biota remained unknown . yet there have been few experimental studies on ascospore discharge , germination , development of mycelia , and physiology of the fungal partners ( mycobionts ) of tropical lichens compared with those on temperate species . the first cited studies on the isolation of lichen - forming fungi are generally those of tbler ( 1909 ) and thomas ( 1939 ) , although tbler was primarily interested in the re - synthesis of lichens from their individual symbionts ( turbin 1996 ) . however , werner ( 1927 ) , innovatively examined the effect of different media and additions on the growth of selected mycobionts from a range of lichens . subsequent workers have concentrated on the development of methods for lichen re - synthesis ( ahmadjian 1964 ) , and later ahmadjian et al . ( 1995 ) were the first to attempt the isolation of a wide range of fungal partners of lichens , and also lichenicolous fungi , on a worldwide basis , although their material was predominantly from non - tropical regions . we investigated ascospore discharge from a wide range of tropical lichens in order to make a preliminary assessment of the conditions under which discharge occurred , and whether there could be any seasonal correlations . the collection of samples began in 1998 , and concentrated on khao yai national park ( ky ) , central thailand . the remainder were collected during field surveys to doi suthep ( 1849 n , 9953 e ) and chiang dao ( l940 n , 99 e ) in chiang mai province ( cm ) , mae fah luang arboretum ( 20 n , 99.5 e ) chiang rai province ( cr ) , sai yok district ( 14 n , 99 e ) kanjanaburi province ( kjb ) , khao sok national park ( 8 n , 99.5 e ) nakon srithammarat province ( ts and rpb respectively ) , phu kradueng national park , ( 16.8 n , 101.8 e ) loei province ( pkd ) , suan phueng district ( 13.5 n , 99 e ) ratchaburi province ( sp ) , and sakaeraj research station ( 14 n , 102 e ) nakhon ratchasima province ( skr ) . khao yai national park was visited monthly during one year ( 19992000 ) for seasonal observations and experiments to explore the development of thalli , and also to ascertain if there were seasonal differences in ascospore discharge and spore viability samples were cut into pieces , wrapped in tissue paper , and placed in individual strong brown paper bags . if the specimens could not be immediately transferred to the laboratory for pre - isolation treatment , they were either kept in a cool place , or ( where available ) a domestic refrigerator , until they could be transferred . in the laboratory , samples were air - dried at room temperature ( 30 c ) overnight , and then transferred to new paper envelopes with identification labels and stored in a domestic refrigerator at 4 c until the isolation protocol had been completed . specimens were identified as precisely as possible on the basis of their morphology , anatomy , and chemical constituents ( determined by standard thin - layer chromatographic methods ; orange et al . the basic monographic treatments required to provide a sound taxonomic basis for studies of lichen distribution , ecology , and physiology are still lacking for most lichen families and genera in the tropics . species that have not previously been described are also likely to be found ; homchantara & coppins ( 2002 ) described 26 species of thelotremataceae as new to science from thailand , and aptroot et al . the specimens were removed from storage , and surface - cleaned with air from an aerosol camera blower to remove any remaining soil and debris . 11 ) was used to dissect specimens to obtain small portions with ascomata , and the remainder of the samples were then returned to storage . the spores were allowed to shoot upwards onto an overlying layer of tap water agar ( wa ; booth 1971 ) . the agar surface was examined daily with a stereozoom binocular microscope ( olympus sz11 ) , and once ascospores had been discharged , small agar blocks ( 35 mm ) with ascospores on the surface were excised and transferred to malt - yeast extract agar ( mya ; merck or oxoid ) . germination of ascospores was assessed under the stereozoom microscope ; observations were made daily for 7 d , and subsequently twice weekly . in order to investigate the seasonality and discharge of ascospores , thalli were collected each month from the same trees in khao yai national park over a one year period , and their discharge patterns and rates of germination were determined for each monthly sample following the protocol described above . clear plastic boxes 18 x 7.5 x 5 cm were used with a layer of tap water agar in the lid of the box . 0.3 mm diam , were attached to one vertical microscope slide ( 2 x 4 cm ) which was shallowly immersed in the agar layer . the agar surface was examined under an olympus stereozoom microscope ( model sz 11 ) daily for 5 d. if no spores were discharged within 3 d , the procedure was repeated , and then , if after a second 3 d period no discharge was observed , the protocol was repeated for a third and final time . we also investigated the effects of relative humidity by incubating petri dishes in plastic moist chambers containing different saturated solutions to maintain the relative humidity at particular levels , following kaye & laby ( 1966 ) : potassium nitrate ( 92 % ) , ammonium sulphate ( 80 % ) , and sodium nitrate ( 65 % ) . ( 1995 ) as we found it to be detrimental to ascospore discharge in the tropical lichens tested ; in consequence , untreated lichen samples were used throughout . mya ( see above ) was the medium of choice for all cultures of the fungal partners , but potato dextrose agar ( pda ) , corn meal agar ( cma ) , oatmeal agar ( oma ) , and czapek - dox agar ( cda ) , were also used to determine the optimum medium for growth . malt yeast extract broth ( myb ) was selected as the standard medium for studies of the isolated fungi in liquid culture , since good growth rates of several fungal partners had been observed on solid mya . static culture was most frequently used , and following initial trials with direct inoculation of pieces of the fungal cultures into the liquid medium , different types of physical support for the fungi on the surface of the liquid were tested . and ( 4 ) , synthetic sponge ( polystyrene ) pieces 2.5 x 2.5 x 0.3 cm , together with pieces of fungal colonies cut from solid media of 0.4 x 0.4 cm , placed on the surface of these materials , and floated on the surface of 50 ml myb in 250 ml erlenmeyer flasks . shake cultures were prepared using inocula produced in the same way as for static cultures , and transferred to 250 ml erlenmeyer flasks containing 50 ml of myb . the orbital shaking incubator was set at a speed of 200 rpm , and at a temperature of 30 0.5 c . specimens for scanning electron microscopy ( sem ) , either intact ascomata or cultures of the isolated fungal partners , were fixed in 5 % glutaraldehyde and dehydrated in a graded ethyl alcohol series . the specimens were then attached to aluminium stubs using either dag metallic paint or adhesive carbon pads to prevent electron charging of the specimens . the samples were gold - coated using a sputter coating unit ( polaron ru - sc7620 ) and examined either with a jeol 840 sem , a jeol sem5410lv , or a leo 1455vp scanning electron microscope . the collection of samples began in 1998 , and concentrated on khao yai national park ( ky ) , central thailand . the remainder were collected during field surveys to doi suthep ( 1849 n , 9953 e ) and chiang dao ( l940 n , 99 e ) in chiang mai province ( cm ) , mae fah luang arboretum ( 20 n , 99.5 e ) chiang rai province ( cr ) , sai yok district ( 14 n , 99 e ) kanjanaburi province ( kjb ) , khao sok national park ( 8 n , 99.5 e ) nakon srithammarat province ( ts and rpb respectively ) , phu kradueng national park , ( 16.8 n , 101.8 e ) loei province ( pkd ) , suan phueng district ( 13.5 n , 99 e ) ratchaburi province ( sp ) , and sakaeraj research station ( 14 n , 102 e ) nakhon ratchasima province ( skr ) . khao yai national park was visited monthly during one year ( 19992000 ) for seasonal observations and experiments to explore the development of thalli , and also to ascertain if there were seasonal differences in ascospore discharge and spore viability samples were cut into pieces , wrapped in tissue paper , and placed in individual strong brown paper bags . if the specimens could not be immediately transferred to the laboratory for pre - isolation treatment , they were either kept in a cool place , or ( where available ) a domestic refrigerator , until they could be transferred . in the laboratory , samples were air - dried at room temperature ( 30 c ) overnight , and then transferred to new paper envelopes with identification labels and stored in a domestic refrigerator at 4 c until the isolation protocol had been completed . the basic monographic treatments required to provide a sound taxonomic basis for studies of lichen distribution , ecology , and physiology are still lacking for most lichen families and genera in the tropics . species that have not previously been described are also likely to be found ; homchantara & coppins ( 2002 ) described 26 species of thelotremataceae as new to science from thailand , and aptroot et al . the specimens were removed from storage , and surface - cleaned with air from an aerosol camera blower to remove any remaining soil and debris . 11 ) was used to dissect specimens to obtain small portions with ascomata , and the remainder of the samples were then returned to storage . the portions of lichen with ascomata , or occasionally only a single ascoma , were attached with a small quantity of petroleum jelly onto the inverted lid of a 9 cm diam petri dish ( sterilin ) . the agar surface was examined daily with a stereozoom binocular microscope ( olympus sz11 ) , and once ascospores had been discharged , small agar blocks ( 35 mm ) with ascospores on the surface were excised and transferred to malt - yeast extract agar ( mya ; merck or oxoid ) . in order to investigate the seasonality and discharge of ascospores , thalli were collected each month from the same trees in khao yai national park over a one year period , and their discharge patterns and rates of germination were determined for each monthly sample following the protocol described above . clear plastic boxes 18 x 7.5 x 5 cm were used with a layer of tap water agar in the lid of the box . 0.3 mm diam , were attached to one vertical microscope slide ( 2 x 4 cm ) which was shallowly immersed in the agar layer . the agar surface was examined under an olympus stereozoom microscope ( model sz 11 ) daily for 5 d. if no spores were discharged within 3 d , the procedure was repeated , and then , if after a second 3 d period no discharge was observed , the protocol was repeated for a third and final time . we also investigated the effects of relative humidity by incubating petri dishes in plastic moist chambers containing different saturated solutions to maintain the relative humidity at particular levels , following kaye & laby ( 1966 ) : potassium nitrate ( 92 % ) , ammonium sulphate ( 80 % ) , and sodium nitrate ( 65 % ) . ( 1995 ) as we found it to be detrimental to ascospore discharge in the tropical lichens tested ; in consequence , untreated lichen samples were used throughout . mya ( see above ) was the medium of choice for all cultures of the fungal partners , but potato dextrose agar ( pda ) , corn meal agar ( cma ) , oatmeal agar ( oma ) , and czapek - dox agar ( cda ) , were also used to determine the optimum medium for growth . for recipes malt yeast extract broth ( myb ) was selected as the standard medium for studies of the isolated fungi in liquid culture , since good growth rates of several fungal partners had been observed on solid mya . static culture was most frequently used , and following initial trials with direct inoculation of pieces of the fungal cultures into the liquid medium , different types of physical support for the fungi on the surface of the liquid were tested . and ( 4 ) , synthetic sponge ( polystyrene ) pieces 2.5 x 2.5 x 0.3 cm , together with pieces of fungal colonies cut from solid media of 0.4 x 0.4 cm , placed on the surface of these materials , and floated on the surface of 50 ml myb in 250 ml erlenmeyer flasks . shake cultures were prepared using inocula produced in the same way as for static cultures , and transferred to 250 ml erlenmeyer flasks containing 50 ml of myb . the orbital shaking incubator was set at a speed of 200 rpm , and at a temperature of 30 0.5 c . specimens for scanning electron microscopy ( sem ) , either intact ascomata or cultures of the isolated fungal partners , were fixed in 5 % glutaraldehyde and dehydrated in a graded ethyl alcohol series . the specimens were then attached to aluminium stubs using either dag metallic paint or adhesive carbon pads to prevent electron charging of the specimens . the samples were gold - coated using a sputter coating unit ( polaron ru - sc7620 ) and examined either with a jeol 840 sem , a jeol sem5410lv , or a leo 1455vp scanning electron microscope . crustose lichens were preferentially selected for experimentation as preliminary studies suggested that their ascospores germinated more readily on artificial media . ascomata of erect shrubby ( fruticose ) and pendent lichens were much less common than crustose or foliose ones , especially at khao yai national park , and so could not be investigated further . of the 292 lichen samples collected ( table 1 ) , 170 samples liberated ascospores in the laboratory , and in several instances successive samples of the same lichen exhibited high percentage germination rates ( table 2 , fig.1 ) . in contrast , foliose lichen species ( e.g. heterodermia diademata ) exhibited a high discharge rate , but only a low percentage of ascospores germinated . eluteriae ( ky 66 ) , graphis elegans ( ky162),and g. rigidula ( ky165 ) , ascospores were discharged readily each month , and spores from each monthly sample also germinated . however , in contrast , cladonia submultiformis ( ky117 ) discharged ascospores only towards the end of the winter ( january - february ) , and in the summer ( april - june ) none were discharged ( fig . following germination , the ability of the isolated fungi to continue to develop and form colonies was investigated . in addition to growth on solid media , liquid culture under static growth conditions was tried , but generally growth was slow . amongst the other physical carriers tested , were segments of unbleached kraft paper or whatman filter paper several layers thick . in contrast , the sponge pieces tested as alternative carriers facilitated growth after incubation in the fungal partners tested ; in most cases , sponge proved to be superior to the other carriers tried ( fig . no contamination by spores from other fungi during discharge were encountered ; the distinctive ascospores from the lichens were deposited , either as small packets of spores or as single spores , and could easily be recognized for subculturing using a stereozoom microscope . our results suggest that high spore discharge rates are correlated with the freshness of the samples and season of collection , as well as the state of maturity of the ascomata . there was , however , considerable variation in ascospore discharge between the species tested , and , also between different collections of the same species . in laurera bengualensis and l. madreporiformis , spores were readily discharged in all of the collections examined , but in l. meristospora , although discharge occurred , it was at a much lower rate . in trypethelium eluteriae , spore discharge occurred throughout the year , but in cladonia submultiformis , although ascospores were also readily discharged , only those from the end of the winter season ( february ) germinated . of the 15 species tested , those of graphina sp . however , in that study no information was given as to the distance attained by the majority of spores , which is perhaps the most pertinent parameter in relation to effective dispersal and establishment in nature . ( pkd3 ) and laurera subdiscreta ( skr1 ) , while at 45 c no discharge was observed in any species tested ( table 4 ) . relative humidity also appears to be important , with three of the four species investigated discharging at relative humidities from 65 % to 100 % ( fig . ( ky95 ) , and graphis sp . when germination was successful , fungal partners of most crustose species tested grew well on solid media , with small colonies developing within a few months . growth in liquid culture was generally very slow , and static culture was found to be superior to shake culture for all species tested . however , growth on static liquid culture was much enhanced by the use of a physical carrier . while segments of kraft paper or whatman filter paper proved to be successful carriers , sponge pieces were superior in relation to visible enhanced growth . we consider that sponge pieces used as a carrier have a wide potential for studies on the physiology and development of lichen fungi as the colonies can be transferred without disruption to different liquid media . culberson & armaleo ( 1992 ) , in their investigation of cladonia grayi , previously concluded that the production of compounds concentrated in the naturally occurring lichen was linked to the aerial growth habit . although only a limited investigation of the chemical products of the isolated fungal partners of the tropical lichens was undertaken in our study , comparison of extrolites from the whole lichen thallus with those produced by the fungal partner alone indicated that in some cases more compounds were produced by the whole thallus than in the isolated fungal cultures . there were also few differences between the compounds produced by the fungus cultured under static conditions compared to those grown in shake culture . further , our results on ascospore discharge show that the seasonal behaviour and discharge distances of the ascospores of tropical lichens recalls that of those in temperate regions .

in addition to being the first cause of mortality for persons under the age of 45 , injury represents the disease with the highest financial burden . furthermore , the demographic shift along with the incessant development of new and costly technologies have led to an explosion in the costs of trauma care.[13 ] in the light of evidence suggesting that acute clinical trauma care is suboptimal and that patient outcomes vary across trauma centers , health care authorities worldwide are expressing the urgent need for information about health care performance . performance in health care is widely measured using donabedian 's structure - process - outcome model . structure refers to the physical environment of the health care facility including human resources , process refers to clinical interventions in individual patients , and outcome refers to the status of the patient at the end of an episode of care . according to this model , improvements in structure influence process and ultimately performance evaluation in trauma began in 1976 , when the american college of surgeons committee on trauma ( acs - cot ) introduced a series of 12 audit filters , updated to 22 in 1993 . however , these audit filters are based uniquely on clinical processes and patient outcome . in organised trauma systems , structure is traditionally evaluated in a qualitative fashion with on - site accreditation visits by a committee of external experts who verify adherence to a series of criteria recommended by acs - cot . no quantitative indicator of structural performance has been proposed for trauma center evaluation despite the widespread use of accreditation procedures . the objective of this study was to develop and validate a structural performance indicator that can be ascertained from accreditation visit data and used to drive performance improvement efforts . the study was based on the inclusive trauma system of the province of quebec , canada . the quebec trauma system was instated in 1993 and involves regionalized care from urban level i trauma centers through to rural community hospitals . at the end of the study period , the system included 6 level i ( including 2 pediatric ) , 4 level ii , 21 level iii , and 28 level iv centers . standardized prehospital protocols ensure that major trauma cases are taken to these hospitals and standing agreements regulate interhospital transfers within the system . data were extracted from accreditation reports completed during on - site visits performed at each trauma center between 1994 and 2005 . the quebec accreditation process was performed by an independent trauma medical counsel comprising 30 medical experts from provincial trauma centers . the medical counsel evaluated each center using a checklist of items based on acs - cot criteria . after each site visit , the accreditation committee made a final recommendation to either : 1 ) maintain the accreditation level without modification , ( 2 ) maintain current level pending specific modifications without an on - site control visit , 3 ) maintain current level pending on modifications with an on - site control visit , or 4 ) revoke the designation status . two waves of accreditation visits were performed by the trauma medical counsel after the initial designation process in 1993 the first between 1994 and 1999 and the second between 1998 and 2005 . analyses were based on the second wave of visits because they were conducted after the system run - in period and they coincide with the collection of trauma registry data . to describe the patient population of the quebec trauma system , data were extracted from the quebec trauma registry ( 1999 - 2006 ) . this registry is mandatory for all 59 provincially designated trauma centers and includes all deaths following injury , intensive care unit admissions , hospital stays 2 days , and interhospital transfers . we first developed a standardized evaluation grid to extract qualitative information from the accreditation reports ( appendix ) . this grid included 73 items grouped under three themes commitment , trauma program , and procedural protocols that were based on the acs - cot checklist . a preliminary series of 10 randomly selected reports were transposed independently onto the grid by five experts from the trauma medical counsel to standardize the transposition process . after this run - in period , a single evaluator transposed all reports onto the evaluation grid . a series of 12 randomly selected reports were duplicated and inserted under a fictitious hospital among the reports to verify intraobserver reliability . to generate a quantitative score from the evaluation grid , each item was scored either on a four - point likert scale ( 0-very negative to 4-very positive ) or on a binary scale ( 0-absent/4-present ) . weights were then assigned to each item of the grid by group of 10 experts from the trauma medical counsel according to perceived importance . the structural performance score was calculated for each trauma center as the weighted sum of the 73 items , standardized to range between 0 and 100 ( see appendix ) . a higher score thus represents better structural performance ; a score of 0 would be given to a trauma center with all items absent or very negative whereas a score of 100 would be given to a trauma center with all items present or very positive . trauma center accreditation grid : items and their individual weights ( sum = 100 points ) since the accreditation reports were written in short form , elements of the evaluation grid were not all thoroughly documented in each report . in addition , some items were not scored because they were not applicable to a particular level of trauma center ( see appendix ) . sensitivity analyses to verify the robustness of results to this treatment of missing data are presented in the results section . structural performance scores are presented using modified rank plots whereby trauma centers are ordered by designation level and volume . because data are based on only one accreditation visit , confidence intervals ( ci ) we , therefore , identified outliers by plotting 2 standard deviation around the global mean . the structural performance indicator was evaluated in terms of intra - observer reliability , discrimination between trauma centers , construct validity , and forecasting according to recommendations for evaluating composite indicators proposed by the agency for healthcare research and quality . intraobserver reliability was evaluated by calculating quadratic weighted kappa statistics on each item for a sample of 12 randomly selected reports that were coded twice by the same evaluator . discrimination was defined as the ability of the indicator to differentiate performance between trauma centers and was evaluated with the coefficient of variation , calculated as the ratio of the standard deviation of the structural performance scores to their mean multiplied by 100 . the ci of the coefficient of variation was generated using boostrap resampling ( n = 500 ) . coefficients of variation are expressed as a percentage where higher values indicate higher between - center variation . a coefficient of variation with a lower 95% ci above 10% was considered to reflect a distribution with high variation , synonym of good between - center discrimination . construct validity was defined as the degree of association between the structural performance indicator and other measures of quality and was assessed by evaluating the correlation of the structure scores with the following : i ) designation levels , ii ) trauma patient volume , iii ) accreditation recommendations . forecasting was defined as the ability of the structural performance indicator to predict structural performance over time . for this analysis we calculated structural performance scores using accreditation reports completed between 1994 and 1999 during the run - in period of the quebec trauma system ( data not used in any other analyses ) . we then evaluated the correlation between structural performance scores generated from the two waves of accreditation visits ( 1994 - 1999 and 1998 - 2005 ) . since the first wave of visits was only available for 57/59 trauma centers , the correlation coefficient was based on n = 57 . sensitivity analyses were performed to assess the robustness of the structural performance scores to the treatment of missing data items and the weighting scheme . for missing data items , we evaluated the correlation in trauma center structural performance scores when missing data values were coded 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) instead of 2 ( neutral ) . for the weighting scheme , we evaluated the correlation in trauma center ranks when equal weights were used over consensus - based weights . the study was based on the inclusive trauma system of the province of quebec , canada . the quebec trauma system was instated in 1993 and involves regionalized care from urban level i trauma centers through to rural community hospitals . at the end of the study period , the system included 6 level i ( including 2 pediatric ) , 4 level ii , 21 level iii , and 28 level iv centers . standardized prehospital protocols ensure that major trauma cases are taken to these hospitals and standing agreements regulate interhospital transfers within the system . data were extracted from accreditation reports completed during on - site visits performed at each trauma center between 1994 and 2005 . the quebec accreditation process was performed by an independent trauma medical counsel comprising 30 medical experts from provincial trauma centers . the medical counsel evaluated each center using a checklist of items based on acs - cot criteria . after each site visit , the accreditation committee made a final recommendation to either : 1 ) maintain the accreditation level without modification , ( 2 ) maintain current level pending specific modifications without an on - site control visit , 3 ) maintain current level pending on modifications with an on - site control visit , or 4 ) revoke the designation status . two waves of accreditation visits were performed by the trauma medical counsel after the initial designation process in 1993 the first between 1994 and 1999 and the second between 1998 and 2005 . analyses were based on the second wave of visits because they were conducted after the system run - in period and they coincide with the collection of trauma registry data . to describe the patient population of the quebec trauma system , data were extracted from the quebec trauma registry ( 1999 - 2006 ) . this registry is mandatory for all 59 provincially designated trauma centers and includes all deaths following injury , intensive care unit admissions , hospital stays 2 days , and interhospital transfers . data were extracted from accreditation reports completed during on - site visits performed at each trauma center between 1994 and 2005 . the quebec accreditation process was performed by an independent trauma medical counsel comprising 30 medical experts from provincial trauma centers . the medical counsel evaluated each center using a checklist of items based on acs - cot criteria . after each site visit , the accreditation committee made a final recommendation to either : 1 ) maintain the accreditation level without modification , ( 2 ) maintain current level pending specific modifications without an on - site control visit , 3 ) maintain current level pending on modifications with an on - site control visit , or 4 ) revoke the designation status . two waves of accreditation visits were performed by the trauma medical counsel after the initial designation process in 1993 the first between 1994 and 1999 and the second between 1998 and 2005 . analyses were based on the second wave of visits because they were conducted after the system run - in period and they coincide with the collection of trauma registry data . to describe the patient population of the quebec trauma system , data were extracted from the quebec trauma registry ( 1999 - 2006 ) . this registry is mandatory for all 59 provincially designated trauma centers and includes all deaths following injury , intensive care unit admissions , hospital stays 2 days , and interhospital transfers . we first developed a standardized evaluation grid to extract qualitative information from the accreditation reports ( appendix ) . this grid included 73 items grouped under three themes commitment , trauma program , and procedural protocols that were based on the acs - cot checklist . a preliminary series of 10 randomly selected reports were transposed independently onto the grid by five experts from the trauma medical counsel to standardize the transposition process . after this run - in period , a single evaluator transposed all reports onto the evaluation grid . a series of 12 randomly selected reports were duplicated and inserted under a fictitious hospital among the reports to verify intraobserver reliability . to generate a quantitative score from the evaluation grid , each item was scored either on a four - point likert scale ( 0-very negative to 4-very positive ) or on a binary scale ( 0-absent/4-present ) . weights were then assigned to each item of the grid by group of 10 experts from the trauma medical counsel according to perceived importance . the structural performance score was calculated for each trauma center as the weighted sum of the 73 items , standardized to range between 0 and 100 ( see appendix ) . a higher score thus represents better structural performance ; a score of 0 would be given to a trauma center with all items absent or very negative whereas a score of 100 would be given to a trauma center with all items present or very positive . trauma center accreditation grid : items and their individual weights ( sum = 100 points ) since the accreditation reports were written in short form , elements of the evaluation grid were not all thoroughly documented in each report . in addition , some items were not scored because they were not applicable to a particular level of trauma center ( see appendix ) . sensitivity analyses to verify the robustness of results to this treatment of missing data are presented in the results section . structural performance scores are presented using modified rank plots whereby trauma centers are ordered by designation level and volume . because data are based on only one accreditation visit , confidence intervals ( ci ) we , therefore , identified outliers by plotting 2 standard deviation around the global mean . the structural performance indicator was evaluated in terms of intra - observer reliability , discrimination between trauma centers , construct validity , and forecasting according to recommendations for evaluating composite indicators proposed by the agency for healthcare research and quality . intraobserver reliability was evaluated by calculating quadratic weighted kappa statistics on each item for a sample of 12 randomly selected reports that were coded twice by the same evaluator . discrimination was defined as the ability of the indicator to differentiate performance between trauma centers and was evaluated with the coefficient of variation , calculated as the ratio of the standard deviation of the structural performance scores to their mean multiplied by 100 . the ci of the coefficient of variation was generated using boostrap resampling ( n = 500 ) . coefficients of variation are expressed as a percentage where higher values indicate higher between - center variation . a coefficient of variation with a lower 95% ci above 10% was considered to reflect a distribution with high variation , synonym of good between - center discrimination . construct validity was defined as the degree of association between the structural performance indicator and other measures of quality and was assessed by evaluating the correlation of the structure scores with the following : i ) designation levels , ii ) trauma patient volume , iii ) accreditation recommendations . forecasting was defined as the ability of the structural performance indicator to predict structural performance over time . for this analysis we calculated structural performance scores using accreditation reports completed between 1994 and 1999 during the run - in period of the quebec trauma system ( data not used in any other analyses ) . we then evaluated the correlation between structural performance scores generated from the two waves of accreditation visits ( 1994 - 1999 and 1998 - 2005 ) . since the first wave of visits was only available for 57/59 trauma centers , the correlation coefficient was based on n = 57 . sensitivity analyses were performed to assess the robustness of the structural performance scores to the treatment of missing data items and the weighting scheme . for missing data items , we evaluated the correlation in trauma center structural performance scores when missing data values were coded 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) instead of 2 ( neutral ) . for the weighting scheme , we evaluated the correlation in trauma center ranks when equal weights were used over consensus - based weights . level i trauma centers admitted on average 900 patients per year that met trauma registry criteria whereas mean volume was below 40 for rural level iv centers [ table 1 ] . level i / ii centers admitted more seriously injured patients who more frequently presented with a head injury than lower level centers . characteristics of trauma centers in the quebec trauma system ( 1999 - 2006 ) the mean structural performance score among the 59 trauma centers was 47.4/100 ( 95% ci : 43.6 - 51.1 ) . most points were lost on trauma program ( mean of 12.8/33.4 ) , followed by procedural protocols ( 20.3/44.4 ) , and lastly commitment ( 15.6/22.2 ) . there were two institutional outliers - one level iii center was a high performer whereas one level iv center was a low performer [ figure 1 ] . structural performance scores for the 59 trauma centers in the quebec trauma system ( 1998 - 2005 ) intraobserver agreement was almost perfect ( kappa > 0.8 ) for 66 of the 73 items ( 90% ) in the evaluation grid . between - center discrimination of structural performance was good . the coefficient of variation of the structural performance scores was 31.2% with a bootstrap 95% ci of 25.5 - 37.6% , indicating that the distribution of structural performance scores had high variation . of the 59 trauma centers in the system , 20 ( 33.9% ) maintained their designation status as is , 6 ( 10.2% ) were asked to make specific changes with no on - site control visit , 15 ( 25.4% ) were asked to make changes with a control visit and 18 ( 30.5% ) had their designation level revoked . in terms of construct validity , accreditation decisions had a strong correlation with structural performance scores [ table 2 ] ; median scores were 52/100 for those that conserved their status as is , 47/100 for centers with changes to make but no control visit , 43/100 for centers with changes and a control visit , and 35/100 for centers with revoked status ( p = 0.0008 ) . the indicator of structural performance also correlated with trauma center designation level [ table 2 ] . median structural performance scores for level i through iv were 63/100 , 52/100 , 54/100 , and 40/100 , respectively , showing decreasing scores with decreasing level of accreditation ( p = 0.002 ) . we observed a weaker but statistically significant correlation with patient volume whereby higher volumes were associated with higher structural performance scores ( p = 0.05 ) . no correlation was observed between structural performance scores in the first wave of accreditation visits , performed just after implementation of the trauma system in 1993 , and those in the second wave performed between 1998 and 2005 [ figure 2 ] . association of structural performance scores with the accreditation decision , designation level , and trauma center volume ( 1998 - 2005 ) correlation between structural performance scores derived from accreditation visits performed in the first wave ( 1994 - 1999 ) and the second wave ( 1998 - 2005 ) structural performance scores calculated by attributing a value of 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) to all missing data items had a high correlation with scores calculated by attributing a value of 2 [ table 3 ] . between - center variation was unchanged , as expected . correlations with accreditation decision , designation level and volume were similar as was the correlation between the two waves of visits . the correlation between structural performance scores based on consensus weights and equal weights for each item of the grid was almost perfect : r = 0.99 ( 95% ci 0.98 - 0.99 ) . sensitivity analyses : comparison of study results when missing data values were coded 2 ( neutral ) with study results when missing data values were coded 0 ( very negative / absent ) or 4 ( very positive / present ) structural performance scores calculated by attributing a value of 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) to all missing data items had a high correlation with scores calculated by attributing a value of 2 [ table 3 ] correlations with accreditation decision , designation level and volume were similar as was the correlation between the two waves of visits . the correlation between structural performance scores based on consensus weights and equal weights for each item of the grid was almost perfect : r = 0.99 ( 95% ci 0.98 - 0.99 ) . sensitivity analyses : comparison of study results when missing data values were coded 2 ( neutral ) with study results when missing data values were coded 0 ( very negative / absent ) or 4 ( very positive / present ) in this study , we have demonstrated the feasibility of deriving a quantitative composite indicator of structural performance using trauma system accreditation reports based on asc - cot criteria . the proposed indicator shows good reliability , discriminates between centers , and correlates well with designation level , volume , and accreditation status . lack of correlation over time may suggest that accreditation reports need to be filled out in a more standardized manner but may also be an indication that structural performance fluctuates over time . the proposed indicator can be used to describe structural performance and identify trauma center outliers to drive quality improvement efforts . indicators of structural performance are an essential part of trauma center performance evaluation . in addition , they are easier to assess than process or outcome indicators as they are collected at the hospital rather than the patient level and they may also be more easily actionable . data used to calculate the proposed indicator are already routinely collected in most trauma systems on periodic accreditation visits . furthermore , many items could be collected at a distance rather than at on - site visits . the observed association of the proposed structural performance indicator with designation level and volume is an indication of construct validity and suggests that the indicator is likely to have good predictive criterion validity as both designation level and volume have been shown to have a negative correlation with risk - adjusted rates of mortality , severe disability at discharge , and hospital length of stay . in addition , the near perfect correlation between structural performance scores based on consensus and equal weights suggests that the latter could be used with no impact on results . this would simplify calculation of structural performance scores as while weights allow administrators to account for the perceived importance of structural aspects , they require periodic updating according to changes in best practise guidelines and may not be exportable across trauma systems . the observed inability of the score to forecast performance at a later time could be due to the lack of standardization of the accreditation process between the first visit during the run - in period and the second visit , performed when the system had had a chance to mature . this and the presence of missing data suggest that the accreditation process would benefit from better standardization . indeed , in our system , the upcoming round of visits will be based on mandatory electronic completion of all items in the evaluation grid . however , the observed lack of temporal correlation may also be explained by important changes in structural performance over time . of interest , among the themes that are part of the structural score , we observed better forecasting for procedural protocols ( r = 0.19 ; 95% ci -0.08;0.43 ) than for the trauma program ( r = 0.009 ; 95% ci -0.25;0.27 ) or commitment ( r = -0.05 ; 95% ci -0.30;0.22 ) . however , a recent scoping review and systematic review on trauma quality indicators did not identify any studies that implemented or validated a composite indicator of structural performance for trauma care . in other health care sectors , few studies have looked at quantifying hospital structural performance . daley et al . used on - site visits to measure structural performance for veteran 's affairs medical centers and developed a quantitative score using consensus - based ratings . they demonstrated a relationship between this indicator of structural performance and risk - adjusted mortality / morbidity rates . the index of hospital quality includes a composite indicator of structure for 12 medical specialities derived by a one - factor solution from principal components analysis that is used by the us news and world report ( national opinion research center ) to generate us hospital rankings . although trauma care is not evaluated per se in us hospital rankings , the structural indicator includes an indicator of the presence and level of trauma care . this study was based on accreditation data from a mature integrated trauma system that follows acs - cot designation criteria . in addition , we were able to evaluate intra - rater reliability and partly address the internal validity of the score . however , limitations include the lack of standardized data collection , problems related to composite scores , and generalizability beyond the quebec trauma system . the accreditation visit reports used in the present study lacked standardization because they were not performed in the context of quantitative performance evaluation . we addressed this problem by retrospectively extracting qualitative information from reports and transposing it onto a standardized evaluation grid . however , missing data items were frequent , which may have influenced the final structural performance scores . a value of 2 ( neutral ) was attributed to missing data items of the evaluation grid . this would have led to an underestimation of between - center variation and is likely to have led to an underestimation of associations of structural performance scores with designation level , volume , and accreditation status . however , the results of sensitivity analyses suggest that the strategy used to address missing data items was adequate . to address this problem , future accreditation visits will be based on mandatory prospective electronic completion of all items in the evaluation grid . the proposed indicator of structural performance is a composite measure and while it is a useful indicator of global performance , it is associated with the limitations inherent to composite scores . it may mask item - specific differences across centers and may make action difficult as it is hard to identify the specific causes of high / low performance . for this reason , composite scores should always be accompanied by item - specific data . there are several different methods for deriving composite scores and there is no clear consensus on which method is optimal . we used a simple weighted average ( consensus or equal - weights ) over more complex methods such as principal components analysis or latent variable models partly because these methods would have required at least 10 observations ( trauma centers ) per grid item ( i.e. , 10 84 trauma centers ) to generate accurate results . finally , the evaluation grid used in our trauma system is based on acs - cot criteria but evaluation items are likely to vary across systems . therefore , while this study provides evidence of the feasibility of generating a quantitative structural performance indicator from report data , methods will have to be adapted to different reporting formats . in addition , the validity and reliability of the proposed composite indicator of trauma center structural performance will need to be assessed in other trauma systems . this study was based on accreditation data from a mature integrated trauma system that follows acs - cot designation criteria . in addition , we were able to evaluate intra - rater reliability and partly address the internal validity of the score . however , limitations include the lack of standardized data collection , problems related to composite scores , and generalizability beyond the quebec trauma system . the accreditation visit reports used in the present study lacked standardization because they were not performed in the context of quantitative performance evaluation . we addressed this problem by retrospectively extracting qualitative information from reports and transposing it onto a standardized evaluation grid . however , missing data items were frequent , which may have influenced the final structural performance scores . a value of 2 ( neutral ) was attributed to missing data items of the evaluation grid . this would have led to an underestimation of between - center variation and is likely to have led to an underestimation of associations of structural performance scores with designation level , volume , and accreditation status . however , the results of sensitivity analyses suggest that the strategy used to address missing data items was adequate . to address this problem , future accreditation visits will be based on mandatory prospective electronic completion of all items in the evaluation grid . the proposed indicator of structural performance is a composite measure and while it is a useful indicator of global performance , it is associated with the limitations inherent to composite scores . it may mask item - specific differences across centers and may make action difficult as it is hard to identify the specific causes of high / low performance . for this reason , composite scores should always be accompanied by item - specific data . there are several different methods for deriving composite scores and there is no clear consensus on which method is optimal . we used a simple weighted average ( consensus or equal - weights ) over more complex methods such as principal components analysis or latent variable models partly because these methods would have required at least 10 observations ( trauma centers ) per grid item ( i.e. , 10 84 trauma centers ) to generate accurate results . finally , the evaluation grid used in our trauma system is based on acs - cot criteria but evaluation items are likely to vary across systems . therefore , while this study provides evidence of the feasibility of generating a quantitative structural performance indicator from report data , methods will have to be adapted to different reporting formats . in addition , the validity and reliability of the proposed composite indicator of trauma center structural performance will need to be assessed in other trauma systems . in summary , we have proposed a structural performance indicator that can be derived from trauma center accreditation visit reports and we have provided evidence of its reliability and internal validity . the indicator can be used to describe performance , identify institutional outliers , and inform accreditation decisions with the goal of driving performance improvement efforts . further research needs to evaluate the influence of structural performance measured by the proposed indicator on clinical process performance and ultimately , on patient outcome . the observed variability in structural performance across centers and the change in structural scores over time underline the importance of evaluating structural performance in trauma systems at regular intervals to drive quality improvement efforts .

background : indicators of structure , process , and outcome are required to evaluate the performance of trauma centers to improve the quality and efficiency of care . while periodic external accreditation visits are part of most trauma systems , a quantitative indicator of structural performance has yet to be proposed . the objective of this study was to develop and validate a trauma center structural performance indicator using accreditation report data.materials and methods : analyses were based on accreditation reports completed during on - site visits in the quebec trauma system ( 1994 - 2005 ) . qualitative report data was retrospectively transposed onto an evaluation grid and the weighted average of grid items was used to quantify performance . the indicator of structural performance was evaluated in terms of test - retest reliability ( kappa statistic ) , discrimination between centers ( coefficient of variation ) , content validity ( correlation with accreditation decision , designation level , and patient volume ) and forecasting ( correlation between visits performed in 1994 - 1999 and 1998 - 2005).results : kappa statistics were > 0.8 for 66 of the 73 ( 90% ) grid items . mean structural performance score over 59 trauma centers was 47.4 ( 95% ci : 43.6 - 51.1 ) . two centers were flagged as outliers and the coefficient of variation was 31.2% ( 95% ci : 25.5% to 37.6% ) , showing good discrimination . correlation coefficients of associations with accreditation decision , designation level , and volume were all statistically significant ( r = 0.61 , -0.40 , and 0.24 , respectively ) . no correlation was observed over time ( r = 0.03).conclusion : this study demonstrates the feasibility of quantifying trauma center structural performance using accreditation reports . the proposed performance indicator shows good test - retest reliability , between - center discrimination , and construct validity . the observed variability in structural performance across centers and over - time underlines the importance of evaluating structural performance in trauma systems at regular intervals to drive quality improvement efforts .

INTRODUCTION MATERIALS AND METHODS Study population Data Accreditation data Patient-level data Development of the structural performance indicator Validation of the structural performance indicator Sensitivity analyses RESULTS Sensitivity analyses DISCUSSION Strengths and limitations CONCLUSION

structure refers to the physical environment of the health care facility including human resources , process refers to clinical interventions in individual patients , and outcome refers to the status of the patient at the end of an episode of care . in organised trauma systems , structure is traditionally evaluated in a qualitative fashion with on - site accreditation visits by a committee of external experts who verify adherence to a series of criteria recommended by acs - cot . no quantitative indicator of structural performance has been proposed for trauma center evaluation despite the widespread use of accreditation procedures . the objective of this study was to develop and validate a structural performance indicator that can be ascertained from accreditation visit data and used to drive performance improvement efforts . the study was based on the inclusive trauma system of the province of quebec , canada . the quebec trauma system was instated in 1993 and involves regionalized care from urban level i trauma centers through to rural community hospitals . at the end of the study period , the system included 6 level i ( including 2 pediatric ) , 4 level ii , 21 level iii , and 28 level iv centers . data were extracted from accreditation reports completed during on - site visits performed at each trauma center between 1994 and 2005 . after each site visit , the accreditation committee made a final recommendation to either : 1 ) maintain the accreditation level without modification , ( 2 ) maintain current level pending specific modifications without an on - site control visit , 3 ) maintain current level pending on modifications with an on - site control visit , or 4 ) revoke the designation status . two waves of accreditation visits were performed by the trauma medical counsel after the initial designation process in 1993 the first between 1994 and 1999 and the second between 1998 and 2005 . analyses were based on the second wave of visits because they were conducted after the system run - in period and they coincide with the collection of trauma registry data . to describe the patient population of the quebec trauma system , data were extracted from the quebec trauma registry ( 1999 - 2006 ) . this registry is mandatory for all 59 provincially designated trauma centers and includes all deaths following injury , intensive care unit admissions , hospital stays 2 days , and interhospital transfers . this grid included 73 items grouped under three themes commitment , trauma program , and procedural protocols that were based on the acs - cot checklist . the structural performance score was calculated for each trauma center as the weighted sum of the 73 items , standardized to range between 0 and 100 ( see appendix ) . a higher score thus represents better structural performance ; a score of 0 would be given to a trauma center with all items absent or very negative whereas a score of 100 would be given to a trauma center with all items present or very positive . trauma center accreditation grid : items and their individual weights ( sum = 100 points ) since the accreditation reports were written in short form , elements of the evaluation grid were not all thoroughly documented in each report . structural performance scores are presented using modified rank plots whereby trauma centers are ordered by designation level and volume . the structural performance indicator was evaluated in terms of intra - observer reliability , discrimination between trauma centers , construct validity , and forecasting according to recommendations for evaluating composite indicators proposed by the agency for healthcare research and quality . discrimination was defined as the ability of the indicator to differentiate performance between trauma centers and was evaluated with the coefficient of variation , calculated as the ratio of the standard deviation of the structural performance scores to their mean multiplied by 100 . the ci of the coefficient of variation was generated using boostrap resampling ( n = 500 ) . coefficients of variation are expressed as a percentage where higher values indicate higher between - center variation . a coefficient of variation with a lower 95% ci above 10% was considered to reflect a distribution with high variation , synonym of good between - center discrimination . construct validity was defined as the degree of association between the structural performance indicator and other measures of quality and was assessed by evaluating the correlation of the structure scores with the following : i ) designation levels , ii ) trauma patient volume , iii ) accreditation recommendations . forecasting was defined as the ability of the structural performance indicator to predict structural performance over time . for this analysis we calculated structural performance scores using accreditation reports completed between 1994 and 1999 during the run - in period of the quebec trauma system ( data not used in any other analyses ) . we then evaluated the correlation between structural performance scores generated from the two waves of accreditation visits ( 1994 - 1999 and 1998 - 2005 ) . sensitivity analyses were performed to assess the robustness of the structural performance scores to the treatment of missing data items and the weighting scheme . for missing data items , we evaluated the correlation in trauma center structural performance scores when missing data values were coded 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) instead of 2 ( neutral ) . the study was based on the inclusive trauma system of the province of quebec , canada . the quebec trauma system was instated in 1993 and involves regionalized care from urban level i trauma centers through to rural community hospitals . at the end of the study period , the system included 6 level i ( including 2 pediatric ) , 4 level ii , 21 level iii , and 28 level iv centers . data were extracted from accreditation reports completed during on - site visits performed at each trauma center between 1994 and 2005 . after each site visit , the accreditation committee made a final recommendation to either : 1 ) maintain the accreditation level without modification , ( 2 ) maintain current level pending specific modifications without an on - site control visit , 3 ) maintain current level pending on modifications with an on - site control visit , or 4 ) revoke the designation status . two waves of accreditation visits were performed by the trauma medical counsel after the initial designation process in 1993 the first between 1994 and 1999 and the second between 1998 and 2005 . analyses were based on the second wave of visits because they were conducted after the system run - in period and they coincide with the collection of trauma registry data . to describe the patient population of the quebec trauma system , data were extracted from the quebec trauma registry ( 1999 - 2006 ) . this registry is mandatory for all 59 provincially designated trauma centers and includes all deaths following injury , intensive care unit admissions , hospital stays 2 days , and interhospital transfers . data were extracted from accreditation reports completed during on - site visits performed at each trauma center between 1994 and 2005 . after each site visit , the accreditation committee made a final recommendation to either : 1 ) maintain the accreditation level without modification , ( 2 ) maintain current level pending specific modifications without an on - site control visit , 3 ) maintain current level pending on modifications with an on - site control visit , or 4 ) revoke the designation status . two waves of accreditation visits were performed by the trauma medical counsel after the initial designation process in 1993 the first between 1994 and 1999 and the second between 1998 and 2005 . analyses were based on the second wave of visits because they were conducted after the system run - in period and they coincide with the collection of trauma registry data . to describe the patient population of the quebec trauma system , data were extracted from the quebec trauma registry ( 1999 - 2006 ) . this registry is mandatory for all 59 provincially designated trauma centers and includes all deaths following injury , intensive care unit admissions , hospital stays 2 days , and interhospital transfers . this grid included 73 items grouped under three themes commitment , trauma program , and procedural protocols that were based on the acs - cot checklist . the structural performance score was calculated for each trauma center as the weighted sum of the 73 items , standardized to range between 0 and 100 ( see appendix ) . a higher score thus represents better structural performance ; a score of 0 would be given to a trauma center with all items absent or very negative whereas a score of 100 would be given to a trauma center with all items present or very positive . trauma center accreditation grid : items and their individual weights ( sum = 100 points ) since the accreditation reports were written in short form , elements of the evaluation grid were not all thoroughly documented in each report . structural performance scores are presented using modified rank plots whereby trauma centers are ordered by designation level and volume . the structural performance indicator was evaluated in terms of intra - observer reliability , discrimination between trauma centers , construct validity , and forecasting according to recommendations for evaluating composite indicators proposed by the agency for healthcare research and quality . discrimination was defined as the ability of the indicator to differentiate performance between trauma centers and was evaluated with the coefficient of variation , calculated as the ratio of the standard deviation of the structural performance scores to their mean multiplied by 100 . the ci of the coefficient of variation was generated using boostrap resampling ( n = 500 ) . coefficients of variation are expressed as a percentage where higher values indicate higher between - center variation . a coefficient of variation with a lower 95% ci above 10% was considered to reflect a distribution with high variation , synonym of good between - center discrimination . construct validity was defined as the degree of association between the structural performance indicator and other measures of quality and was assessed by evaluating the correlation of the structure scores with the following : i ) designation levels , ii ) trauma patient volume , iii ) accreditation recommendations . forecasting was defined as the ability of the structural performance indicator to predict structural performance over time . for this analysis we calculated structural performance scores using accreditation reports completed between 1994 and 1999 during the run - in period of the quebec trauma system ( data not used in any other analyses ) . we then evaluated the correlation between structural performance scores generated from the two waves of accreditation visits ( 1994 - 1999 and 1998 - 2005 ) . sensitivity analyses were performed to assess the robustness of the structural performance scores to the treatment of missing data items and the weighting scheme . for missing data items , we evaluated the correlation in trauma center structural performance scores when missing data values were coded 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) instead of 2 ( neutral ) . characteristics of trauma centers in the quebec trauma system ( 1999 - 2006 ) the mean structural performance score among the 59 trauma centers was 47.4/100 ( 95% ci : 43.6 - 51.1 ) . structural performance scores for the 59 trauma centers in the quebec trauma system ( 1998 - 2005 ) intraobserver agreement was almost perfect ( kappa > 0.8 ) for 66 of the 73 items ( 90% ) in the evaluation grid . between - center discrimination of structural performance was good . the coefficient of variation of the structural performance scores was 31.2% with a bootstrap 95% ci of 25.5 - 37.6% , indicating that the distribution of structural performance scores had high variation . of the 59 trauma centers in the system , 20 ( 33.9% ) maintained their designation status as is , 6 ( 10.2% ) were asked to make specific changes with no on - site control visit , 15 ( 25.4% ) were asked to make changes with a control visit and 18 ( 30.5% ) had their designation level revoked . in terms of construct validity , accreditation decisions had a strong correlation with structural performance scores [ table 2 ] ; median scores were 52/100 for those that conserved their status as is , 47/100 for centers with changes to make but no control visit , 43/100 for centers with changes and a control visit , and 35/100 for centers with revoked status ( p = 0.0008 ) . the indicator of structural performance also correlated with trauma center designation level [ table 2 ] . median structural performance scores for level i through iv were 63/100 , 52/100 , 54/100 , and 40/100 , respectively , showing decreasing scores with decreasing level of accreditation ( p = 0.002 ) . we observed a weaker but statistically significant correlation with patient volume whereby higher volumes were associated with higher structural performance scores ( p = 0.05 ) . no correlation was observed between structural performance scores in the first wave of accreditation visits , performed just after implementation of the trauma system in 1993 , and those in the second wave performed between 1998 and 2005 [ figure 2 ] . association of structural performance scores with the accreditation decision , designation level , and trauma center volume ( 1998 - 2005 ) correlation between structural performance scores derived from accreditation visits performed in the first wave ( 1994 - 1999 ) and the second wave ( 1998 - 2005 ) structural performance scores calculated by attributing a value of 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) to all missing data items had a high correlation with scores calculated by attributing a value of 2 [ table 3 ] . between - center variation was unchanged , as expected . correlations with accreditation decision , designation level and volume were similar as was the correlation between the two waves of visits . the correlation between structural performance scores based on consensus weights and equal weights for each item of the grid was almost perfect : r = 0.99 ( 95% ci 0.98 - 0.99 ) . sensitivity analyses : comparison of study results when missing data values were coded 2 ( neutral ) with study results when missing data values were coded 0 ( very negative / absent ) or 4 ( very positive / present ) structural performance scores calculated by attributing a value of 0 ( very negative / absent : worst case scenario ) or 4 ( very positive / present : best case scenario ) to all missing data items had a high correlation with scores calculated by attributing a value of 2 [ table 3 ] correlations with accreditation decision , designation level and volume were similar as was the correlation between the two waves of visits . the correlation between structural performance scores based on consensus weights and equal weights for each item of the grid was almost perfect : r = 0.99 ( 95% ci 0.98 - 0.99 ) . sensitivity analyses : comparison of study results when missing data values were coded 2 ( neutral ) with study results when missing data values were coded 0 ( very negative / absent ) or 4 ( very positive / present ) in this study , we have demonstrated the feasibility of deriving a quantitative composite indicator of structural performance using trauma system accreditation reports based on asc - cot criteria . the proposed indicator shows good reliability , discriminates between centers , and correlates well with designation level , volume , and accreditation status . lack of correlation over time may suggest that accreditation reports need to be filled out in a more standardized manner but may also be an indication that structural performance fluctuates over time . the proposed indicator can be used to describe structural performance and identify trauma center outliers to drive quality improvement efforts . indicators of structural performance are an essential part of trauma center performance evaluation . data used to calculate the proposed indicator are already routinely collected in most trauma systems on periodic accreditation visits . the observed association of the proposed structural performance indicator with designation level and volume is an indication of construct validity and suggests that the indicator is likely to have good predictive criterion validity as both designation level and volume have been shown to have a negative correlation with risk - adjusted rates of mortality , severe disability at discharge , and hospital length of stay . in addition , the near perfect correlation between structural performance scores based on consensus and equal weights suggests that the latter could be used with no impact on results . this would simplify calculation of structural performance scores as while weights allow administrators to account for the perceived importance of structural aspects , they require periodic updating according to changes in best practise guidelines and may not be exportable across trauma systems . the observed inability of the score to forecast performance at a later time could be due to the lack of standardization of the accreditation process between the first visit during the run - in period and the second visit , performed when the system had had a chance to mature . indeed , in our system , the upcoming round of visits will be based on mandatory electronic completion of all items in the evaluation grid . however , the observed lack of temporal correlation may also be explained by important changes in structural performance over time . of interest , among the themes that are part of the structural score , we observed better forecasting for procedural protocols ( r = 0.19 ; 95% ci -0.08;0.43 ) than for the trauma program ( r = 0.009 ; 95% ci -0.25;0.27 ) or commitment ( r = -0.05 ; 95% ci -0.30;0.22 ) . however , a recent scoping review and systematic review on trauma quality indicators did not identify any studies that implemented or validated a composite indicator of structural performance for trauma care . used on - site visits to measure structural performance for veteran 's affairs medical centers and developed a quantitative score using consensus - based ratings . they demonstrated a relationship between this indicator of structural performance and risk - adjusted mortality / morbidity rates . although trauma care is not evaluated per se in us hospital rankings , the structural indicator includes an indicator of the presence and level of trauma care . this study was based on accreditation data from a mature integrated trauma system that follows acs - cot designation criteria . however , limitations include the lack of standardized data collection , problems related to composite scores , and generalizability beyond the quebec trauma system . this would have led to an underestimation of between - center variation and is likely to have led to an underestimation of associations of structural performance scores with designation level , volume , and accreditation status . to address this problem , future accreditation visits will be based on mandatory prospective electronic completion of all items in the evaluation grid . the proposed indicator of structural performance is a composite measure and while it is a useful indicator of global performance , it is associated with the limitations inherent to composite scores . finally , the evaluation grid used in our trauma system is based on acs - cot criteria but evaluation items are likely to vary across systems . therefore , while this study provides evidence of the feasibility of generating a quantitative structural performance indicator from report data , methods will have to be adapted to different reporting formats . in addition , the validity and reliability of the proposed composite indicator of trauma center structural performance will need to be assessed in other trauma systems . this study was based on accreditation data from a mature integrated trauma system that follows acs - cot designation criteria . however , limitations include the lack of standardized data collection , problems related to composite scores , and generalizability beyond the quebec trauma system . this would have led to an underestimation of between - center variation and is likely to have led to an underestimation of associations of structural performance scores with designation level , volume , and accreditation status . to address this problem , future accreditation visits will be based on mandatory prospective electronic completion of all items in the evaluation grid . the proposed indicator of structural performance is a composite measure and while it is a useful indicator of global performance , it is associated with the limitations inherent to composite scores . finally , the evaluation grid used in our trauma system is based on acs - cot criteria but evaluation items are likely to vary across systems . therefore , while this study provides evidence of the feasibility of generating a quantitative structural performance indicator from report data , methods will have to be adapted to different reporting formats . in addition , the validity and reliability of the proposed composite indicator of trauma center structural performance will need to be assessed in other trauma systems . in summary , we have proposed a structural performance indicator that can be derived from trauma center accreditation visit reports and we have provided evidence of its reliability and internal validity . the indicator can be used to describe performance , identify institutional outliers , and inform accreditation decisions with the goal of driving performance improvement efforts . further research needs to evaluate the influence of structural performance measured by the proposed indicator on clinical process performance and ultimately , on patient outcome . the observed variability in structural performance across centers and the change in structural scores over time underline the importance of evaluating structural performance in trauma systems at regular intervals to drive quality improvement efforts .

les enqutes portant sur les complications associes aux interventions anesthsiques sont une mthode de contrle de la qualit qui identifie les domaines o les soins cliniques et la scurit des patients peuvent tre amliors . la plupart des recherches se sont jusquici bases sur les registres des plaintes rgles et les comptes rendus anonymes , ce qui entrane certaines limites spcifiques . par consquent , afin dvaluer le systme de dclaration des incidents dun hpital , notre tude a t conue de faon dcrire les incidents critiques que les anesthsiologistes ont rapport de faon volontaire et non anonyme via un systme de gestion de linformation en anesthsie . il sagit dune tude de cohorte observationnelle historique portant sur des patients ( gs de plus de 18 ans ) subissant des interventions anesthsiques dans un hpital central de soins tertiaires . une liste de complications comprenant 20 lments , telle que mise au point par la socit nerlandaise des anesthsiologistes , a t complte de faon prospective lors de chaque intervention . tous les incidents critiques enregistrs dans le systme de gestion de linformation en anesthsie ont ensuite t reclasss en 92 incidents critiques diffrents dune manire reproductible . au total , 110 310 interventions ont t ralises chez 65 985 patients , et aprs avoir exclus 158 comptes rendus qui ne dcrivaient pas dincident critique , il restait 3904 incidents critiques dans le cadre de 3807 ( 3,5 % ) interventions anesthsiques . les difficults techniques lies lanesthsie rgionale ( n = 445 ; 40 par 10 000 anesthsies ; intervalle de confiance [ ic ] 95 % , 36 44 ) , lhypotension ( n = 432 ; 39 par 10 000 anesthsies ; ic 95 % , 35 43 ) , et les intubations difficiles non anticipes ( n = 216 ; 20 par 10 000 anesthsies ; ic 95 % , 18 23 ) constituaient les incidents critiques les plus frquemment documents . la mesure prcise et la surveillance des incidents critiques sont essentielles la scurit des patients . malgr le risque de sous - documentation et de mauvaise classification probable des systmes de dclaration manuels , nos rsultats donnent une vue densemble complte concernant la survenue dincidents critiques lis lanesthsie et rapports de faon volontaire . cette vue densemble peut guider la mise au point dun nouveau systme de dclaration des incidents et de stratgies de prvention afin de rduire la survenue future dincidents critiques . the institutional review board ( irb ) of the university medical center utrecht reviewed the study protocol and found that it was not subject to the dutch medical research in human subjects act . therefore , the irb waived the need for informed consent ( 11 - 271/c ; july 5 , 2011 ) . this observational study describes prospectively reported critical incidents and complications relating to anesthesia in patients 18 years and older undergoing any type of anesthetic procedure in a tertiary referral university hospital ( university medical center utrecht , the netherlands ) from january 1 , 2005 to may 18 , 2011 . anesthesiologists and anesthesia registrars voluntarily reported complications and critical incidents on a non - anonymous basis via the 20-item complication list of the netherlands society of anesthesiologists . the reporting system was implemented in september 2004 ; therefore , we chose to evaluate critical incidents reported as of january 1 , 2005 to allow an optimization period of three months . we defined a critical incident as an event that could have led ( if not discovered or corrected in time ) or did lead to an undesirable outcome , i.e. , ranging from increased length of hospital stay to death or permanent disability . we included all anesthesia - related critical incidents that occurred at a time when the patient was under the care of an anesthesiologist and were described in clear detail by a person who either observed or was involved in the critical incident . we included critical incidents that not only seemed preventable ( i.e. , inadequate preoperative screening ) or involved human error ( i.e. , medication error)21 but also were non - preventable ( i.e. , unexpected difficult intubation).3,15,22 critical incidents were reported by anesthesiologists and anesthesia registrars ( reporters ) in the aims on a voluntary and non - anonymous basis . during every anesthetic procedure , a menu item in the aims termed complication is presented by pressing the standard event key start skin closure , at which time , a reporter can complete a standardized computerized audit form . if a critical incident is reported , a drop down menu displays the 20-item complication list ( with miscellaneous as an additional option ) developed by the netherlands society of anesthesiologists ( table 1 ) . thereafter , the incident s grade of severity can be reported and , if deemed necessary , free text can be added . if the complication report is not entered into the database by the end of the day , the anesthesiologist involved receives a reminder e - mail . upon completion , the critical incident report is stored in a database within the aims along with the patient characteristics . the registry also includes a means to assign a pop - up warning for subsequent anesthetic procedures ( i.e. , difficult intubation).table 1classification of critical incidents according to netherlands society of anesthesiologists complication no . incidents ( % of total number of incidents , n = 4,062 ) aspiration54 ( 1.3%)laryngospasm374 ( 9.2%)hypoxemia226 ( 5.6%)hypoventilation195 ( 4.8%)hypertension76 ( 1.9%)hypotension700 ( 17.2%)myocardial ischemia / infarction114 ( 2.8%)cardiac arrhythmia302 ( 7.4%)acute cardiac decompensation26 ( 0.6%)dental lesion36 ( 0.9%)nerve / skin / cornea lesion141 ( 3.5%)lesion through needle puncture127 ( 3.1%)hypothermia79 ( 1.9%)conversion of regional anesthesia / inadequate block416 ( 10.2%)urinary retention19 ( 0.5%)inadequate postoperative analgesia12 ( 0.3%)postoperative agitation18 ( 1.7%)awareness51 ( 1.3%)allergic reaction111 ( 2.7%)transfusion / mediation error106 ( 2.6%)miscellaneous879 ( 21.6% ) classification of critical incidents according to netherlands society of anesthesiologists the currently used 20-item complication list of the netherlands society of anesthesiologists facilitates a generalized classification of critical incidents . after reviewing the critical incident reports , we concluded that we could not base firm conclusions on the classification system as it was too generalized ; therefore , we reclassified all critical incidents . based on the initial classification and comments added by the reporter , we reclassified the critical incidents in keeping with a classification system of the german society of anaesthesiology and intensive care13 which is a more detailed classification system on which to base our conclusions . if no comment was available or the comment was unclear , we consulted the aims to investigate the critical incident in detail . when information was inconsistent , consensus was reached by discussion with two researchers ( j.d.g . and b.v.z . ) . if more than one category was possible for one critical incident , the most appropriate or most severe category was chosen . if different critical incidents occurred during one anesthetic procedure , these were categorized as separate critical incidents . all reports involving death as grade of severity were discussed with all observers ( k.m . chicago , il , usa ) , except for the calculation of the 95% confidence interval ( ci ) according to wilson s formula ( epitools : http://epitools.ausvet.com.au ) . where appropriate , a chi square test or an independent samples student s t test was carried out to display differences between groups . the institutional review board ( irb ) of the university medical center utrecht reviewed the study protocol and found that it was not subject to the dutch medical research in human subjects act . therefore , the irb waived the need for informed consent ( 11 - 271/c ; july 5 , 2011 ) . this observational study describes prospectively reported critical incidents and complications relating to anesthesia in patients 18 years and older undergoing any type of anesthetic procedure in a tertiary referral university hospital ( university medical center utrecht , the netherlands ) from january 1 , 2005 to may 18 , 2011 . anesthesiologists and anesthesia registrars voluntarily reported complications and critical incidents on a non - anonymous basis via the 20-item complication list of the netherlands society of anesthesiologists . the reporting system was implemented in september 2004 ; therefore , we chose to evaluate critical incidents reported as of january 1 , 2005 to allow an optimization period of three months . we defined a critical incident as an event that could have led ( if not discovered or corrected in time ) or did lead to an undesirable outcome , i.e. , ranging from increased length of hospital stay to death or permanent disability . we included all anesthesia - related critical incidents that occurred at a time when the patient was under the care of an anesthesiologist and were described in clear detail by a person who either observed or was involved in the critical incident . we included critical incidents that not only seemed preventable ( i.e. , inadequate preoperative screening ) or involved human error ( i.e. , medication error)21 but also were non - preventable ( i.e. , unexpected difficult intubation).3,15,22 critical incidents were reported by anesthesiologists and anesthesia registrars ( reporters ) in the aims on a voluntary and non - anonymous basis . during every anesthetic procedure , a menu item in the aims termed complication is presented by pressing the standard event key start skin closure , at which time , a reporter can complete a standardized computerized audit form . if a critical incident is reported , a drop down menu displays the 20-item complication list ( with miscellaneous as an additional option ) developed by the netherlands society of anesthesiologists ( table 1 ) . thereafter , the incident s grade of severity can be reported and , if deemed necessary , free text can be added . if the complication report is not entered into the database by the end of the day , the anesthesiologist involved receives a reminder e - mail . upon completion , the critical incident report is stored in a database within the aims along with the patient characteristics . the registry also includes a means to assign a pop - up warning for subsequent anesthetic procedures ( i.e. , difficult intubation).table 1classification of critical incidents according to netherlands society of anesthesiologists complication no . incidents ( % of total number of incidents , n = 4,062 ) aspiration54 ( 1.3%)laryngospasm374 ( 9.2%)hypoxemia226 ( 5.6%)hypoventilation195 ( 4.8%)hypertension76 ( 1.9%)hypotension700 ( 17.2%)myocardial ischemia / infarction114 ( 2.8%)cardiac arrhythmia302 ( 7.4%)acute cardiac decompensation26 ( 0.6%)dental lesion36 ( 0.9%)nerve / skin / cornea lesion141 ( 3.5%)lesion through needle puncture127 ( 3.1%)hypothermia79 ( 1.9%)conversion of regional anesthesia / inadequate block416 ( 10.2%)urinary retention19 ( 0.5%)inadequate postoperative analgesia12 ( 0.3%)postoperative agitation18 ( 1.7%)awareness51 ( 1.3%)allergic reaction111 ( 2.7%)transfusion / mediation error106 ( 2.6%)miscellaneous879 ( 21.6% ) classification of critical incidents according to netherlands society of anesthesiologists the currently used 20-item complication list of the netherlands society of anesthesiologists facilitates a generalized classification of critical incidents . after reviewing the critical incident reports , we concluded that we could not base firm conclusions on the classification system as it was too generalized ; therefore , we reclassified all critical incidents . based on the initial classification and comments added by the reporter , we reclassified the critical incidents in keeping with a classification system of the german society of anaesthesiology and intensive care13 which is a more detailed classification system on which to base our conclusions . if no comment was available or the comment was unclear , we consulted the aims to investigate the critical incident in detail . when information was inconsistent , consensus was reached by discussion with two researchers ( j.d.g . and b.v.z . ) . if more than one category was possible for one critical incident , the most appropriate or most severe category was chosen . if different critical incidents occurred during one anesthetic procedure , these were categorized as separate critical incidents . all reports involving death as grade of severity were discussed with all observers ( k.m . , j.d.g . , and b.v.z . ) . all statistical analyses were performed using spss 17.0 for windows ( spss inc . , chicago , il , usa ) , except for the calculation of the 95% confidence interval ( ci ) according to wilson s formula ( epitools : http://epitools.ausvet.com.au ) . where appropriate , a chi square test or an independent samples student s t test was carried out to display differences between groups . the complication status of 104,133 ( 94.4% ) of 110,310 anesthetic procedures was known ( 95% ci , 94.2 to 94.5 ) ( figure ) . in total , 4,062 events were reported in the aims , and 158 ( 3.9% ) reports were classified as not being a critical incident ( 95% ci , 3.3 to 4.5 ) because they consisted of surgical complications and warnings for a subsequent anesthetic procedure . the remaining 3,904 critical incidents were found in 3,807 of the 110,310 anesthetic procedures ( 354 per 10,000 anesthetics ; 95% ci , 343 to 365 ) . the 3,904 critical incidents consisted of one single critical incident in 3,715 ( 97.6% ) anesthetic procedures , two critical incidents in 87 ( 2.3% ) anesthetic procedures , and three critical incidents in five ( 0.1% ) anesthetic procedures . table 2 shows demographic data of the study population ; no clinically significant differences were found . the largest critical incident categories were cardiovascular incidents , with 1,164 incidents ( 106 per 10,000 anesthetics ) , respiratory problems with 851 incidents ( 77 per 10,000 anesthetics ) , and lesions with 820 incidents ( 74 per 10,000 anesthetics ) ( table 3 ) . the cardiovascular critical incidents consisted mainly of hypotension ; the respiratory problems critical incidents consisted mainly of difficulties to ventilate ( with or without hypoxemia ) , difficulties to intubate , bronchospasm , and laryngospasm ; and the lesion critical incidents consisted mainly of technical difficulties with regional anesthesia ( table 4 ) . the largest groups of reported critical incidents were technical difficulties with regional anesthesia ( 40 per 10,000 anesthetics ) and hypotension ( 39 per 10,000 anesthetics ) ( table 5).figureflow diagram for reclassification of critical incidentstable 2characteristics of study populationtotal number of anestheticsanesthetics with critical incidentscritical incidents per 10,000 anesthetics ( 95% ci ) p value110,3103,807 mean age 52 ( 95% ci , 33 to 69)55 ( 95% ci , 38 to 72)<0.001 sex 0.001 male53,741 ( 48.7%)1,903 ( 50.0% ) female56,569 ( 51.3%)1,904 ( 50.0% ) asa classification < 0.001 i22,148687310 ( 288 to 334 ) ii31,9481,387434 ( 412 to 457 ) iii7,274434597 ( 545 to 654 ) iv25318711 ( 454 to 1,096 ) v100 ( 0 to 7,935 ) not specified48,6861,281263 ( 249 to 278 ) urgent surgery < 0.001 elective89,9233,138349 ( 337 to 361 ) emergency21,210669315 ( 292 to 339)asa = american society of anesthesiologists ; ci = confidence intervaltable 3incidence of critical incidents in the different categories with reported consequence consequence of critical incident critical incident categoryno consequencetemporary consequence without interventionrecovery after intervention(probable ) permanent damagedeathdeath from another causenone specifiedtotal ( per 10,000 anesthetics ; 99% ci)largest individual contribution of an anesthesiologist ( % ) respiratory43828115420111851 ( 77 ; 71 to 84)49 ( 5.8)cardiovascular39737640414002701,164 ( 106 ; 98 to 114)99 ( 8.5)laboratory results620000311 ( 1 ; 0 to 2)4 ( 36.4)central nervous system4541000026112 ( 10 ; 8 to 13)11 ( 9.8)equipment / organization141803102102329 ( 30 ; 26 to 34)30 ( 9.1)detriment / injury44322111700138820 ( 74 ; 68 to 81)36 ( 4.3)medication16795210058323 ( 29 ; 25 to 34)18 ( 5.6)miscellaneous113135111043294 ( 27 ; 23 to 31)44 ( 15.0)total ( per 10,000 anesthetics ; 99% ci)1,750 ( 159 ; 149 to 169)1,231 ( 112 ; 104 to 120)72 ( 7 ; 5 to 9)55 ( 5 ; 4 to 7)43 ( 4 ; 3 to 6)2 ( 0.2 ; 0 to 0.9)751 ( 68 ; 62 to 75)3,904 ( 354 ; 340 to 369)288 ( 7.4)largest individual contribution of an anesthesiologist ( % ) 88 ( 5.0)143 ( 11.6)8 ( 11.3)7 ( 13.7)4 ( 9.3)1 ( 50.0)111 ( 14.7)288 ( 7.4)-the results are limited to those critical incidents for which no greater than 15.0% of the reported incidents were attributed to one anesthesiologistci = confidence intervaltable 4incidence of the various critical incidents after reclassification critical incident no consequencetemporary consequence without interventionrecovery after intervention(probable ) permanent damagedeathdeath from another causenone specifiedtotal ( per 10,000 anesthetics ) respiratory 438 281 15 4 2 0 111 851 ( 77 ) disconnection70000007 ( 1 ) kinking of tube32000016 ( 1 ) accidental extubation1850000225 ( 2 ) unexpected difficult intubation13338100044216 ( 20 ) impossible intubation1374010328 ( 3 ) failed intubation1261000221 ( 2 ) mainstem intubation23000016 ( 1 ) re - intubation262000313 ( 1 ) laryngospasm49431000699 ( 9 ) bronchospasm314100001082 ( 7 ) aspiration7160110833 ( 3 ) hypoventilation / hypoxemia5865110012137 ( 12 ) difficult ventilation * 27241100760 ( 5 ) pulmonary edema01100013 ( 0 ) vomiting with laryngeal mask airway * 560000112 ( 1 ) failure of laryngeal mask airway * 56110000673 ( 7 ) other respiratory disturbances1683100432 ( 3 ) cardiovascular 397 376 40 41 40 0 270 1,164 ( 106 ) hypotension1681219300131432 ( 39 ) hypertension181221001548 ( 4 ) arrhythmia5251203035143 ( 13 ) tachycardia36000009 ( 1 ) bradycardia25110000137 ( 3 ) hypovolemia2468171217025163 ( 15 ) heart failure2132440227 ( 2 ) pulmonary embolism00011002 ( 0.2 ) circulatory arrest40362125014109 ( 10 ) myocardial infarction1635501333 ( 3 ) temporary st - segmental changes * 274131002597 ( 9 ) hemodynamic instability due to sepsis * 330240517 ( 2 ) vagal response to needle puncture * 3380000445 ( 4 ) other cardiovascular disturbances00001001 ( 0.1 ) laboratory results 6 2 0 0 0 0 3 11 ( 0.3 ) anemia10000001 ( 0.1 ) disturbances of electrolytes30000025 ( 0.5 ) disturbances of serum glucose12000003 ( 0.3 ) other disturbances in laboratory results10000012 ( 0.2 ) central nervous system 45 41 0 0 0 0 26 112 ( 10 ) central anticholinergic syndrome01000001 ( 0.1 ) cerebral ischemia10000012 ( 0.2 ) seizure680000519 ( 2 ) awareness * 22000037 ( 1 ) postoperative agitation * 990000321 ( 2 ) reaction of patient during anesthesia without awareness * 242000001357 ( 5 ) transurethral resection ( tur ) syndrome * 21000014 ( 0.4 ) other neurological disturbances10000001 ( 0.1 ) equipment / organization 141 80 3 1 0 2 102 329 ( 30 ) anesthetic machine1860000226 ( 2 ) ecg - monitor10000001 ( 0.1 ) monitor of blood pressure420101917 ( 2 ) external pacemaker10000001 ( 0.1 ) pulse oximeter00000011 ( 0.1 ) intubation set21000014 ( 0.4 ) drug application23000027 ( 1 ) insufficient documentation * 14400001634 ( 3 ) inadequate preoperative screening * 15500001535 ( 3 ) inadequate preoperative preparation * 252200001966 ( 6 ) organizational problem * 3830300129101 ( 9 ) failure of electronic anesthesia information management system * 820000111 ( 1 ) electricity failure * 32000016 ( 1 ) other kind of equipment1030000619 ( 2 ) detriment / injury 443 221 11 7 0 0 138 820 ( 74 ) technical difficulties with regional anesthesia29573200075445 ( 40 ) total spinal * 31100000243 ( 4 ) spinal tap * 19240000447 ( 4 ) failed or repeated puncture ( blood vessels)1570000830 ( 3 ) accidental puncture of artery * 24132000746 ( 4 ) teeth475600527 ( 2 ) vessels43000029 ( 1 ) muscles / soft tissue330000410 ( 1 ) skin / lip17250000547(4 ) airway23000016 ( 1 ) eyes03010026 ( 1 ) epistaxis15150000333 ( 3 ) pneumothorax / hemothorax05100017 ( 1 ) nerves080000412 ( 1 ) failed urinary catheter * 6110000825 ( 2 ) accidental removal of intravenous catheter * 780000419 ( 2 ) other detriment / injury12100037 ( 1 ) medication 167 95 2 1 0 0 58 323 ( 29 ) inappropriate drug * 1840000527 ( 2 ) overdosage * 32150000451 ( 5 ) side effect * 450000615 ( 1 ) wrong drug * 2160000431 ( 3 ) intravenous injection given subcutaneously * 343711001487 ( 8) inadequate administration of medication * 301210001861 ( 6 ) residual muscle paralysis after extubation * 19110000333 ( 3 ) accidental intravenous administration of local anesthetic * 41000005 ( 0.5 ) other * 540000413 ( 1 ) miscellaneous 113 135 1 1 1 0 43 294 ( 27 ) nausea / vomiting1160000825 ( 2 ) anaphylactic shock2210010327 ( 2 ) allergic reaction * 453810001195 ( 9 ) shivering10000001 ( 0.1 ) hypothermia28470000479 ( 7 ) transfusion reaction14000027 ( 1 ) oliguria / acute renal failure10000012 ( 0.2 ) urinary retention * 460000616 ( 1 ) insufficient postoperative pain management * 25000029 ( 1 ) positioning * 1140000015 ( 1 ) failed gavage * 11000002 ( 0.2 ) other630100616 ( 1 ) total 1,750 1,231 72 55 43 2 751 3,904 ( 354 ) ecg = electrocardiogram*categories added to the critical incident list of the german society of anaesthesiology and intensive caretable 5top ten most frequently reported critical incidentscritical incidentcritical incident categoryno of critical incidents ( per 10,000 anesthetics ; 99% ci ) n = 3,904technical difficulties with regional anesthesiadetriment / injury445 ( 40 ; 36 to 6)hypotensioncardiovascular432 ( 39 ; 35 to 44)unexpected difficult intubationrespiratory216 ( 20 ; 16 to 23)hypovolemiacardiovascular163 ( 15 ; 12 to 18)arrhythmiacardiovascular143 ( 13 ; 11 to 16)hypoventilation / hypoxemiarespiratory137 ( 12 ; 10 to 16)circulatory arrestcardiovascular109 ( 10 ; 8 to 13)organizational problemequipment / organization101 ( 9 ; 7 to 12)laryngospasmrespiratory99 ( 9 ; 7 to 12)allergic reactionmiscellaneous95 ( 9 ; 7 to 11)ci = confidence interval flow diagram for reclassification of critical incidents characteristics of study population asa = american society of anesthesiologists ; ci = confidence interval incidence of critical incidents in the different categories with reported consequence the results are limited to those critical incidents for which no greater than 15.0% of the reported incidents were attributed to one anesthesiologist ci = confidence interval incidence of the various critical incidents after reclassification ecg = electrocardiogram * categories added to the critical incident list of the german society of anaesthesiology and intensive care top ten most frequently reported critical incidents ci = confidence interval critical incidents with ( probable ) permanent damage consisted primarily of respiratory and cardiovascular critical incidents . forty - three ( 1.1% of all critical incidents ) critical incidents led to the death of a patient receiving an anesthetic procedure ; forty of those critical incidents comprised a cardiovascular incident ranging from arrhythmia to myocardial infarction ( table 6).table 6critical incidents with ( probable ) permanent damage or death as consequencecritical incidentpermanent damage ( per 10,000 anesthetics)death ( per 10,000 anesthetics ) respiratory 4 ( 0.4)2 ( 0.2 ) impossible intubation01 ( 0.1 ) aspiration1 ( 0.1)1 ( 0.1 ) hypoventilation / hypoxemia1 ( 0.1)0 difficult ventilation1 ( 0.1)0 other respiratory disturbances1 ( 0.1)0 cardiovascular 41 ( 3.7)40 ( 3.6 ) hypotension3 ( 0.3)0 hypertension1 ( 0.1)0 arrhythmia03 ( 0.3 ) hypovolemia12 ( 1.1)17 ( 1.5 ) heart failure4 ( 0.4)4 ( 0.4 ) pulmonary embolism1 ( 0.1)1 ( 0.1 ) circulatory arrest12 ( 1.1)5 ( 0.5 ) myocardial infarction5 ( 0.5)5 ( 0.5 ) temporary st - segmental changes1 ( 0.1)0 hemodynamic instability due to sepsis2 ( 0.2)4 ( 0.4 ) other cardiovascular disturbances01 ( 0.1 ) equipment / organization 1 ( 0.1)0 monitor of blood pressure1 ( 0.1)0 detriment / injury 7 ( 0.6)0 teeth6 ( 0.5)0 eyes1 ( 0.1)0 medication 1 ( 0.1)0 intravenous injection given subcutaneously1 ( 0.1)0 miscellaneous 1 ( 0.1)1 ( 0.1 ) anaphylactic shock01 ( 0.1 ) other1 ( 0.1)0 total ( per 10,000 anesthetics ; 99% ci ) 55 ( 5 ; 4 to 7)43 ( 4 ; 3 to 6)ci = confidence interval critical incidents with ( probable ) permanent damage or death as consequence ci = confidence interval the voluntary and non - anonymous critical incident registration system in this study proved to be very effective ( response rate 94.4% ) . this high response was achieved by way of a reminder in the aims for reporting during skin closure and an e - mail reminder after completion of the anesthetic procedure . furthermore , the non - anonymous registration allowed feedback through a twice weekly complication meeting in which action regarding a critical incident was discussed and initiated , thereby encouraging clinicians to report critical incidents . in 3.5% ( 354 per 10,000 anesthetics ; 95% ci , 343 to 365 ) of anesthetic procedures a critical incident was reported , which is similar to the incidence reported in children using the same methodology.23 the present voluntary and non - anonymous reporting system is unique and has its advantages and disadvantages . voluntarily reported critical incidents may suffer from underreporting.3,24 previous studies have shown a low level of compliance when voluntary reporting was compared with automatically detected critical incidents,25 - 27 and in a different study , an incidence of 28% was reached when researchers completed a retrospective evaluation of all anesthetic procedures.13 nevertheless , the response rate in the present system was very high ( 94% ) , and the advantage of the present system is the fact that anesthesiologists reported only those critical incidents considered to be clinically relevant . the non - anonymous system of reporting may also cause underreporting because a reporter might refrain from reporting due to fear of consequences.6,10,24 nevertheless , a strong advantage of non - anonymous reporting is the ability to discuss the critical incident with detailed information from the involved anesthesiologist , which can lead to a teaching moment.3 the reporting system used in this study was based on the 20-item complication list of the netherlands society of anesthesiologists . this 20-item complication list was not sufficient for detailed analysis and required extension as 21.6% of reported events could not be classified within the original list and were reported as miscellaneous ( tables 1 and 4 ) . nevertheless , the limited number of items in the classification system of the netherlands society of anesthesiologists and the large amount of critical incidents in the miscellaneous category might have induced underreporting of the items not in the original list . for example , some might judge certain events as a critical incident , while others might judge the same event as not being a critical incident , and vice versa.24,28 it could be argued that not every critical incident that we present is truly a critical incident , e.g. , technical difficulties with regional anesthesia are an inevitable occurrence when performing regional anesthesia . furthermore , for the present study , all critical incidents were reclassified retrospectively to allow detailed analyses , and lack of information may have caused misclassification . cardiovascular incidents ( 106 per 10,000 anesthetics ) , in particular hypotension ( 40 per 10,000 anesthetics ) , comprised the majority of critical incidents ( tables 3 and 5 ) . previous studies showed the same level of cardiovascular incidents,13,29 whereas some studies indicated that difficulty with airway management11,14,16,29,30 or wrong drug / wrong drug - dose / wrong drug - labelling14 was the critical incident that occurred most frequently . this variance in number and type of critical incident might be due to the diversity of methods in the reporting systems and differences in definitions . for example , closed claims studies report death ( 26% ) , nerve injuries ( 22% ) , and permanent brain damage ( 9% ) as the most common complications.11 the present study identified the most frequently reported and most severe anesthetic critical incidents in our hospital on which to base future improvements for patient safety . the technical difficulties with regional anesthesia ( table 5 ) are being addressed in part by implementation of ultrasound guidance,31 but we propose a thorough investigation to determine which regional technique results in the most technical difficulties . furthermore , the administration of the wrong drug ( table 4 ) is being tackled by strictly double - checking medication before administration.32 in conclusion , the present study shows that the present reporting system in aims along with e - mail feedback leads to a very high response rate in reporting critical incidents . even so , the complication lists of the netherlands society of anesthesiologists proved to be too limited , and therefore , the present list of complications can be used as an alternative .

purposeinvestigation of adverse events associated with anesthetic procedures is a method of quality control that identifies topics to improve clinical care and patient safety . most research to date has been based on closed claim registries and anonymous reports which have specific limitations . therefore , to evaluate a hospital s reporting system , the present study was designed to describe critical incidents that anesthesiologists voluntarily and non - anonymously reported through an anesthesia information management system.methodsthis is a historical observational cohort study on patients ( age > 18 yr ) undergoing anesthetic procedures in a tertiary referral hospital . a 20-item list of complications , as developed by the netherlands society of anesthesiologists , was prospectively completed for each procedure . all critical incidents registered in the anesthesia information management system were then reclassified into 95 different critical incidents in a reproducible way.resultsthere were 110,310 procedures performed in 65,985 patients , and after excluding 158 reports that did not depict a critical incident , 3,904 critical incidents in 3,807 ( 3.5% ) anesthetic procedures remained . technical difficulties with regional anesthesia ( n = 445 ; 40 per 10,000 anesthetics ; 95% confidence interval [ ci ] , 36 to 44 ) , hypotension ( n = 432 ; 39 per 10,000 anesthetics ; 95% ci , 35 to 43 ) , and unexpected difficult intubation ( n = 216 ; 20 per 10,000 anesthetics ; 95% ci , 18 to 23 ) were the most frequently documented critical incidents.conclusionaccurate measurement and monitoring of critical incidents is crucial for patient safety . despite the risk of underreporting and probable misclassification of manual reporting systems , our results give a comprehensive overview on the occurrence of voluntarily reported anesthesia - related critical incidents . this overview can direct development of a new reporting system and preventive strategies to decrease the future occurrence of critical incidents .

Objectif Mthode Rsultats Conclusion Methods Study design Definitions Data acquisition Statistical analysis Results Discussion Funding Conflicts of interest

les difficults techniques lies lanesthsie rgionale ( n = 445 ; 40 par 10 000 anesthsies ; intervalle de confiance [ ic ] 95 % , 36 44 ) , lhypotension ( n = 432 ; 39 par 10 000 anesthsies ; ic 95 % , 35 43 ) , et les intubations difficiles non anticipes ( n = 216 ; 20 par 10 000 anesthsies ; ic 95 % , 18 23 ) constituaient les incidents critiques les plus frquemment documents . incidents ( % of total number of incidents , n = 4,062 ) aspiration54 ( 1.3%)laryngospasm374 ( 9.2%)hypoxemia226 ( 5.6%)hypoventilation195 ( 4.8%)hypertension76 ( 1.9%)hypotension700 ( 17.2%)myocardial ischemia / infarction114 ( 2.8%)cardiac arrhythmia302 ( 7.4%)acute cardiac decompensation26 ( 0.6%)dental lesion36 ( 0.9%)nerve / skin / cornea lesion141 ( 3.5%)lesion through needle puncture127 ( 3.1%)hypothermia79 ( 1.9%)conversion of regional anesthesia / inadequate block416 ( 10.2%)urinary retention19 ( 0.5%)inadequate postoperative analgesia12 ( 0.3%)postoperative agitation18 ( 1.7%)awareness51 ( 1.3%)allergic reaction111 ( 2.7%)transfusion / mediation error106 ( 2.6%)miscellaneous879 ( 21.6% ) classification of critical incidents according to netherlands society of anesthesiologists the currently used 20-item complication list of the netherlands society of anesthesiologists facilitates a generalized classification of critical incidents . the remaining 3,904 critical incidents were found in 3,807 of the 110,310 anesthetic procedures ( 354 per 10,000 anesthetics ; 95% ci , 343 to 365 ) . the largest groups of reported critical incidents were technical difficulties with regional anesthesia ( 40 per 10,000 anesthetics ) and hypotension ( 39 per 10,000 anesthetics ) ( table 5).figureflow diagram for reclassification of critical incidentstable 2characteristics of study populationtotal number of anestheticsanesthetics with critical incidentscritical incidents per 10,000 anesthetics ( 95% ci ) p value110,3103,807 mean age 52 ( 95% ci , 33 to 69)55 ( 95% ci , 38 to 72)<0.001 sex 0.001 male53,741 ( 48.7%)1,903 ( 50.0% ) female56,569 ( 51.3%)1,904 ( 50.0% ) asa classification < 0.001 i22,148687310 ( 288 to 334 ) ii31,9481,387434 ( 412 to 457 ) iii7,274434597 ( 545 to 654 ) iv25318711 ( 454 to 1,096 ) v100 ( 0 to 7,935 ) not specified48,6861,281263 ( 249 to 278 ) urgent surgery < 0.001 elective89,9233,138349 ( 337 to 361 ) emergency21,210669315 ( 292 to 339)asa = american society of anesthesiologists ; ci = confidence intervaltable 3incidence of critical incidents in the different categories with reported consequence consequence of critical incident critical incident categoryno consequencetemporary consequence without interventionrecovery after intervention(probable ) permanent damagedeathdeath from another causenone specifiedtotal ( per 10,000 anesthetics ; 99% ci)largest individual contribution of an anesthesiologist ( % ) respiratory43828115420111851 ( 77 ; 71 to 84)49 ( 5.8)cardiovascular39737640414002701,164 ( 106 ; 98 to 114)99 ( 8.5)laboratory results620000311 ( 1 ; 0 to 2)4 ( 36.4)central nervous system4541000026112 ( 10 ; 8 to 13)11 ( 9.8)equipment / organization141803102102329 ( 30 ; 26 to 34)30 ( 9.1)detriment / injury44322111700138820 ( 74 ; 68 to 81)36 ( 4.3)medication16795210058323 ( 29 ; 25 to 34)18 ( 5.6)miscellaneous113135111043294 ( 27 ; 23 to 31)44 ( 15.0)total ( per 10,000 anesthetics ; 99% ci)1,750 ( 159 ; 149 to 169)1,231 ( 112 ; 104 to 120)72 ( 7 ; 5 to 9)55 ( 5 ; 4 to 7)43 ( 4 ; 3 to 6)2 ( 0.2 ; 0 to 0.9)751 ( 68 ; 62 to 75)3,904 ( 354 ; 340 to 369)288 ( 7.4)largest individual contribution of an anesthesiologist ( % ) 88 ( 5.0)143 ( 11.6)8 ( 11.3)7 ( 13.7)4 ( 9.3)1 ( 50.0)111 ( 14.7)288 ( 7.4)-the results are limited to those critical incidents for which no greater than 15.0% of the reported incidents were attributed to one anesthesiologistci = confidence intervaltable 4incidence of the various critical incidents after reclassification critical incident no consequencetemporary consequence without interventionrecovery after intervention(probable ) permanent damagedeathdeath from another causenone specifiedtotal ( per 10,000 anesthetics ) respiratory 438 281 15 4 2 0 111 851 ( 77 ) disconnection70000007 ( 1 ) kinking of tube32000016 ( 1 ) accidental extubation1850000225 ( 2 ) unexpected difficult intubation13338100044216 ( 20 ) impossible intubation1374010328 ( 3 ) failed intubation1261000221 ( 2 ) mainstem intubation23000016 ( 1 ) re - intubation262000313 ( 1 ) laryngospasm49431000699 ( 9 ) bronchospasm314100001082 ( 7 ) aspiration7160110833 ( 3 ) hypoventilation / hypoxemia5865110012137 ( 12 ) difficult ventilation * 27241100760 ( 5 ) pulmonary edema01100013 ( 0 ) vomiting with laryngeal mask airway * 560000112 ( 1 ) failure of laryngeal mask airway * 56110000673 ( 7 ) other respiratory disturbances1683100432 ( 3 ) cardiovascular 397 376 40 41 40 0 270 1,164 ( 106 ) hypotension1681219300131432 ( 39 ) hypertension181221001548 ( 4 ) arrhythmia5251203035143 ( 13 ) tachycardia36000009 ( 1 ) bradycardia25110000137 ( 3 ) hypovolemia2468171217025163 ( 15 ) heart failure2132440227 ( 2 ) pulmonary embolism00011002 ( 0.2 ) circulatory arrest40362125014109 ( 10 ) myocardial infarction1635501333 ( 3 ) temporary st - segmental changes * 274131002597 ( 9 ) hemodynamic instability due to sepsis * 330240517 ( 2 ) vagal response to needle puncture * 3380000445 ( 4 ) other cardiovascular disturbances00001001 ( 0.1 ) laboratory results 6 2 0 0 0 0 3 11 ( 0.3 ) anemia10000001 ( 0.1 ) disturbances of electrolytes30000025 ( 0.5 ) disturbances of serum glucose12000003 ( 0.3 ) other disturbances in laboratory results10000012 ( 0.2 ) central nervous system 45 41 0 0 0 0 26 112 ( 10 ) central anticholinergic syndrome01000001 ( 0.1 ) cerebral ischemia10000012 ( 0.2 ) seizure680000519 ( 2 ) awareness * 22000037 ( 1 ) postoperative agitation * 990000321 ( 2 ) reaction of patient during anesthesia without awareness * 242000001357 ( 5 ) transurethral resection ( tur ) syndrome * 21000014 ( 0.4 ) other neurological disturbances10000001 ( 0.1 ) equipment / organization 141 80 3 1 0 2 102 329 ( 30 ) anesthetic machine1860000226 ( 2 ) ecg - monitor10000001 ( 0.1 ) monitor of blood pressure420101917 ( 2 ) external pacemaker10000001 ( 0.1 ) pulse oximeter00000011 ( 0.1 ) intubation set21000014 ( 0.4 ) drug application23000027 ( 1 ) insufficient documentation * 14400001634 ( 3 ) inadequate preoperative screening * 15500001535 ( 3 ) inadequate preoperative preparation * 252200001966 ( 6 ) organizational problem * 3830300129101 ( 9 ) failure of electronic anesthesia information management system * 820000111 ( 1 ) electricity failure * 32000016 ( 1 ) other kind of equipment1030000619 ( 2 ) detriment / injury 443 221 11 7 0 0 138 820 ( 74 ) technical difficulties with regional anesthesia29573200075445 ( 40 ) total spinal * 31100000243 ( 4 ) spinal tap * 19240000447 ( 4 ) failed or repeated puncture ( blood vessels)1570000830 ( 3 ) accidental puncture of artery * 24132000746 ( 4 ) teeth475600527 ( 2 ) vessels43000029 ( 1 ) muscles / soft tissue330000410 ( 1 ) skin / lip17250000547(4 ) airway23000016 ( 1 ) eyes03010026 ( 1 ) epistaxis15150000333 ( 3 ) pneumothorax / hemothorax05100017 ( 1 ) nerves080000412 ( 1 ) failed urinary catheter * 6110000825 ( 2 ) accidental removal of intravenous catheter * 780000419 ( 2 ) other detriment / injury12100037 ( 1 ) medication 167 95 2 1 0 0 58 323 ( 29 ) inappropriate drug * 1840000527 ( 2 ) overdosage * 32150000451 ( 5 ) side effect * 450000615 ( 1 ) wrong drug * 2160000431 ( 3 ) intravenous injection given subcutaneously * 343711001487 ( 8) inadequate administration of medication * 301210001861 ( 6 ) residual muscle paralysis after extubation * 19110000333 ( 3 ) accidental intravenous administration of local anesthetic * 41000005 ( 0.5 ) other * 540000413 ( 1 ) miscellaneous 113 135 1 1 1 0 43 294 ( 27 ) nausea / vomiting1160000825 ( 2 ) anaphylactic shock2210010327 ( 2 ) allergic reaction * 453810001195 ( 9 ) shivering10000001 ( 0.1 ) hypothermia28470000479 ( 7 ) transfusion reaction14000027 ( 1 ) oliguria / acute renal failure10000012 ( 0.2 ) urinary retention * 460000616 ( 1 ) insufficient postoperative pain management * 25000029 ( 1 ) positioning * 1140000015 ( 1 ) failed gavage * 11000002 ( 0.2 ) other630100616 ( 1 ) total 1,750 1,231 72 55 43 2 751 3,904 ( 354 ) ecg = electrocardiogram*categories added to the critical incident list of the german society of anaesthesiology and intensive caretable 5top ten most frequently reported critical incidentscritical incidentcritical incident categoryno of critical incidents ( per 10,000 anesthetics ; 99% ci ) n = 3,904technical difficulties with regional anesthesiadetriment / injury445 ( 40 ; 36 to 6)hypotensioncardiovascular432 ( 39 ; 35 to 44)unexpected difficult intubationrespiratory216 ( 20 ; 16 to 23)hypovolemiacardiovascular163 ( 15 ; 12 to 18)arrhythmiacardiovascular143 ( 13 ; 11 to 16)hypoventilation / hypoxemiarespiratory137 ( 12 ; 10 to 16)circulatory arrestcardiovascular109 ( 10 ; 8 to 13)organizational problemequipment / organization101 ( 9 ; 7 to 12)laryngospasmrespiratory99 ( 9 ; 7 to 12)allergic reactionmiscellaneous95 ( 9 ; 7 to 11)ci = confidence interval flow diagram for reclassification of critical incidents characteristics of study population asa = american society of anesthesiologists ; ci = confidence interval incidence of critical incidents in the different categories with reported consequence the results are limited to those critical incidents for which no greater than 15.0% of the reported incidents were attributed to one anesthesiologist ci = confidence interval incidence of the various critical incidents after reclassification ecg = electrocardiogram * categories added to the critical incident list of the german society of anaesthesiology and intensive care top ten most frequently reported critical incidents ci = confidence interval critical incidents with ( probable ) permanent damage consisted primarily of respiratory and cardiovascular critical incidents . forty - three ( 1.1% of all critical incidents ) critical incidents led to the death of a patient receiving an anesthetic procedure ; forty of those critical incidents comprised a cardiovascular incident ranging from arrhythmia to myocardial infarction ( table 6).table 6critical incidents with ( probable ) permanent damage or death as consequencecritical incidentpermanent damage ( per 10,000 anesthetics)death ( per 10,000 anesthetics ) respiratory 4 ( 0.4)2 ( 0.2 ) impossible intubation01 ( 0.1 ) aspiration1 ( 0.1)1 ( 0.1 ) hypoventilation / hypoxemia1 ( 0.1)0 difficult ventilation1 ( 0.1)0 other respiratory disturbances1 ( 0.1)0 cardiovascular 41 ( 3.7)40 ( 3.6 ) hypotension3 ( 0.3)0 hypertension1 ( 0.1)0 arrhythmia03 ( 0.3 ) hypovolemia12 ( 1.1)17 ( 1.5 ) heart failure4 ( 0.4)4 ( 0.4 ) pulmonary embolism1 ( 0.1)1 ( 0.1 ) circulatory arrest12 ( 1.1)5 ( 0.5 ) myocardial infarction5 ( 0.5)5 ( 0.5 ) temporary st - segmental changes1 ( 0.1)0 hemodynamic instability due to sepsis2 ( 0.2)4 ( 0.4 ) other cardiovascular disturbances01 ( 0.1 ) equipment / organization 1 ( 0.1)0 monitor of blood pressure1 ( 0.1)0 detriment / injury 7 ( 0.6)0 teeth6 ( 0.5)0 eyes1 ( 0.1)0 medication 1 ( 0.1)0 intravenous injection given subcutaneously1 ( 0.1)0 miscellaneous 1 ( 0.1)1 ( 0.1 ) anaphylactic shock01 ( 0.1 ) other1 ( 0.1)0 total ( per 10,000 anesthetics ; 99% ci ) 55 ( 5 ; 4 to 7)43 ( 4 ; 3 to 6)ci = confidence interval critical incidents with ( probable ) permanent damage or death as consequence ci = confidence interval the voluntary and non - anonymous critical incident registration system in this study proved to be very effective ( response rate 94.4% ) . in 3.5% ( 354 per 10,000 anesthetics ; 95% ci , 343 to 365 ) of anesthetic procedures a critical incident was reported , which is similar to the incidence reported in children using the same methodology.23 the present voluntary and non - anonymous reporting system is unique and has its advantages and disadvantages . voluntarily reported critical incidents may suffer from underreporting.3,24 previous studies have shown a low level of compliance when voluntary reporting was compared with automatically detected critical incidents,25 - 27 and in a different study , an incidence of 28% was reached when researchers completed a retrospective evaluation of all anesthetic procedures.13 nevertheless , the response rate in the present system was very high ( 94% ) , and the advantage of the present system is the fact that anesthesiologists reported only those critical incidents considered to be clinically relevant . the non - anonymous system of reporting may also cause underreporting because a reporter might refrain from reporting due to fear of consequences.6,10,24 nevertheless , a strong advantage of non - anonymous reporting is the ability to discuss the critical incident with detailed information from the involved anesthesiologist , which can lead to a teaching moment.3 the reporting system used in this study was based on the 20-item complication list of the netherlands society of anesthesiologists . for example , closed claims studies report death ( 26% ) , nerve injuries ( 22% ) , and permanent brain damage ( 9% ) as the most common complications.11 the present study identified the most frequently reported and most severe anesthetic critical incidents in our hospital on which to base future improvements for patient safety . even so , the complication lists of the netherlands society of anesthesiologists proved to be too limited , and therefore , the present list of complications can be used as an alternative .

in recent years , biology has been undergoing profound changes that are happening so quickly that biologists have not had the time to integrate these conceptual changes into intellectual and experimental frameworks ( dinger et al . , along with this increasingly rapid accumulation of data from research , biological education is not keeping pace with either conceptual or factual learning . educational methodologies such as conceptual learning , hands - on learning , problem - based learning , and student - based learning have been recommended but are not universally implemented ( klymkowsky , 2005 ) . one common theme throughout the literature is the need for increased instruction in quantitative biology ( brent 2004 ; gross , 2004 ; hoy , 2004 ) . the bio2010 report ( national research council , 2003 ) emphasizes a more quantitative approach to teaching the biological sciences preferably at the introductory level . additional calls for change are the suggested competencies for premed students and physicians ( see the association of american medical colleges [ aamc ] and the howard hughes medical institute [ hhmi ] report ) , to be reflected in proposed changes to the medical college admission test . since the advent of project 2061 ( american association for the advancement of science , 1993 ) , curriculum reform , student achievement , and the assessment of and improving student outcomes in the sciences has been a focus of k12 education and the accrediting commissions . if we want students to be successful in science , mathematics , and technology in higher education , we must shift from a professor - centered lecture paradigm to a student - centered learning paradigm ( harkness , unpublished , paper at 101st american political science association annual conference , washington , dc ) . science , technology , engineering , and mathematics talent expansion program ( nsf - step ) grant , were the pilot group for the initial implementation of the three - semester integrated curriculum . the first semester ( symbiosis i : biology and statistics ) also has been taught for the state of tennessee governor 's school : scientific models and data analysis summer program since 2008 . designing of the curriculum material and the pre- and posttest concept questions was a concerted effort of faculty from the departments of biological sciences , mathematics and statistics , and curriculum and instruction . the results of these assessments demonstrated that the symbiosis material could be used in introducing college and precollege students to an integrated approach to quantitative biology . a large - scale assessment protocol has been developed to establish a baseline of the effect of instruction on biology students ' understanding of integrated biology and mathematical concepts using the traditional biology curriculum as a comparison to the symbiosis curriculum . although a review of funded grants shows most programs develop integrated courses at the junior , senior , and graduate levels , we developed an integrated curriculum for the introductory level . during this initial development ( 20052006 ) , the departments received an nsf step grant allowing us to recruit freshmen as a logical pilot group to take the integrative courses . each symbiosis module includes the biology concept(s ) and the math skills needed to analyze data sets that examined the concepts . a 2-wk laboratory , one for collecting data and the other for analyzing data , were developed for each module . the availability of data sets to support specific biology concepts became our overriding criteria . to match the current biology and math curriculum ( www.etsu.edu/reg/catalog/undergraduate.aspx ) , the courses were designed to contain 5 h of lecture and 2 h of laboratory each week . many graduate and professional schools are not currently set up to recognize integrated courses as meeting the admission requirements . it was therefore critical that the names of these courses reflect the content on the students ' transcripts . the courses were named as follows : integrated biology and statistics , integrated biology and calculus , and integrated biology and discrete math , and a separate rubric ( ibms ) for the courses was obtained . the curriculum committee also worked with the east tennessee state university ( etsu ) quillen college of medicine admission committee and the office of medical professions advisement at etsu to ensure that the application process through the american medical college application service recognizes the course content in the integrated curriculum . once the initial logistics were complete , faculty from math and biology submitted an hhmi educational grant with the vision to create a three - semester introductory curriculum of integrated biology / math where the student would receive credit for three semesters of introductory biology for majors , a semester of statistics , and a semester of calculus . the 4-yr grant was funded in fall 2006 , and the course was first taught in fall 2007 . as in many institutions , our standard biology for majors curriculum involves a large lecture format ( sections of 300 + students ) and smaller lab sections of 25 students . the material has largely been based on a typical textbook in terms of content but seems to promote passive learning with superficial retention of the material and little integration of conceptual learning ( armbruster et al . , 2009 ) . symbiosis is a unique approach where each semester is made up of six 2-wk modules defining both the biology and mathematics or statistical material . each module consists of 10 h of classroom and two 2-h labs consisting of an experimental and analytical lab . a team consisting of at least two faculty members , a biologist and a mathematician or statistician , developed and team - taught each module . over time , the faculty has become more comfortable with each other 's field . statistics and biology make a relatively easy pairing , both conceptually , as well as operationally ( joplin et al . , 2010 ) . modern biology pedagogy is based , to a large extent , on a pseudological framework of going from small - to - big and is rooted in what biology has done rather than why or how it is done ( moore et al . , 2010 ) . an examination of modern biology textbooks supports this contention because they are not quantitative and are primarily encyclopedic in content with very little integration of material between chapters . noticeably missing are equations , data sets , and graphs , which demonstrate the richness of variation that is a major component of biology . in traditional pedagogy , this lack of quantitative analysis leads the student to be bombarded with an unending series of facts that have no logical connection to the whole . could this old approach be one reason why professors have observed that students retain little introductory material in upper - level courses ( bransford et al . a literature search reveals very little development of an integrated math biology curriculum at the introductory level . the criteria that have guided the development of the statistical component of the first semester of symbiosis are as follows : an early introduction of inference to be able to answer research questions from the beginning , examples of the sequence rationale - algorithm - computer program , use of a problem - oriented approach presenting statistical methods when they are needed , emphasis on the study of variability , emphasis on a multivariate view whenever possible , and inclusion of topics at the elementary level that can serve as a preparation to later understand the language and methods of biostatistics / bioinformatics , including exposure to statistical software . active learning and critical thinking are promoted through class discussion and activities , homework , and assignments . the statistical analysis of real biological data drives the students into discovering facts instead of listening to facts passively . a complete description of the material in the curriculum is beyond the space of the article . version can be found at our website at www.etsu.edu/cas/symbiosis/default.aspx . a short description of model i in symbiosis i is presented with the remainder modules just outlined . in symbiosis i , students start with the scientific method ( module i ) and statistical hypotheses testing during the first week of the course , even though the classical topics of sampling distributions have yet not been covered . this was just the first example of how we have adjusted course material by using randomization methods ( permutations test ) to test hypotheses about the means of two populations . to test hypotheses about a population proportion , basics of probability and the binomial distribution are introduced and the exact test appears as a simple application of the binomial distribution . the scientific method is taught using case studies such as von helmont 's tree growing experiment , stanley pruissner 's development of the prion theory , the methodology of identification of mosquito vectors of arboborne diseases , introduction of human immunodeficiency virus ( hiv ) as the etiology of aids , before asking the question as a class project of whether mosquitoes could transmit hiv . the answer to this question is the use of statistics to look at the bel glade study in florida by using epidemiology ( centers for disease control and prevention , 1986 ) . the module ends with a description of the five basic concepts of biology : evolution ; membrane - bound cellular organization ; energy metabolism ; response to the environment ; and growth , reproduction , and development . each of these concepts is explicitly covered in each of the modules that maintain a connection between the topics of the course . module ii covers the cell by using data sets of abnormal and normal cells ; red blood cell counts ; and the dimensions of cells to introduce descriptive statistics , including correlation and statistical graphs . the study of relationships between variables is extended by the study of functions by using scaling to study allometry and isometry ( module iii ) . genetics and probability ( module iv ) are also a natural fit because copying alleles from parents to offspring involves randomness and provides a perfect motivation for the study of probability , along with conditional probability , independence , and the corresponding test of independence . dna genetics ( module v ) and introductory bioinformatics ( module vi ) provides the opportunity to apply probability and testing hypotheses to molecular biology , such as calculating the probability of sequences in dna data sets . classic topics of statistical inference ( e.g. , confidence interval estimation , test of hypotheses for proportions using large samples , t tests ) from introductory statistics also are included . these methods are used to compare mitochondrial sequences of insect species with the drosophila mitochondria as a reference . the students discuss the differences between species at the dna level . in the following semester , symbiosis ii the biological topics were rearranged from our normal progression to use rate of change for the calculus . the models are as follows : populations , ecology , behavioral ecology , chronobiology , structured populations , energy , and enzymes . one of the goals of the symbiosis project is to cover a first - semester calculus course by the end of symbiosis ii . thus , the calculus in symbiosis ii is actually a continuation of math concepts begun in symbiosis i. a student who successfully completes symbiosis i and ii will have covered all the topics that constitute a rigorous , sciences majors ' statistics and calculus course . the coverage of calculus has been extended across two semesters , allowing students to acclimate to the calculus at a more deliberate pace than they would in other calculus courses . in addition , the overwhelming majority of the calculus that is covered is introduced and developed in contexts that are important to the study and practice of biology . the result is a symbiotic relationship between calculus and biology biology benefits from the rich variety of analytical models that calculus permits , whereas calculus benefits from the rich variety of settings and motivations that biology provides . moreover , weaker students benefit from having an entire year to study material that is typically covered in a one - semester course . the mathematics component is assessed by the students taking the math department 's gateway exam that demonstrates competency in calculus concepts to receive credit for the calculus course . all of the symbiosis students who have taken this exam have passed the gateway exam . in symbiosis iii , the emphasis of the quantitative component includes calculus ; matrices ; graph theory ; and advanced statistical topics such as nonlinear estimation , multivariate methods , and an introduction to bioinformatics . transitioning from class sizes of < 20 in the pilot study to multiple sections of 250300 for biology and 5075 for math and statistics means adopting a different pedagogical approach . the recognized challenges include the large block of time required , the faculty load / credit assignment issues , and the differences in instructional necessities . the first two semesters of biology will be taught using the symbiosis integrated material with specifically designed mathematics and statistics courses as corequisites . thus , symbiosis i modules will be taught as biology i ( ibms 1110 ) and statistics ( ibms 1530 ) in the first semester . symbiosis ii modules will be taught as biology ii ( ibms 1120 ) and calculus ( ibms 1910 ) in the second semester . symbiosis iii ( ibms 1130 ) modules will maintain the integrated approach during the third semester . the mathematics component in ibms 1130 is an extension of differential and integral calculus , introduction to linear algebra , and matrices and analysis of variance ( anova ) . this approach maintains our commitment to an integrated presentation of biology , statistics , and mathematics . to maintain this integration , regularly scheduled meetings of the entire instructional team will be held to ensure all faculty , instructors , and lab instructors are staying on track with the course and lab materials . recently , hhmi aamc published a set of learning competencies for premedical students ( 2009 ) . careful consideration of the objectives of the symbiosis modules enabled us to map the modules to these competencies ( table 1 ) . matching the symbiosis modules to the list of competencies ( e ) for premedical undergraduate students with the hhmi the first set of competencies ( e1 ) concerns demonstration of quantitative skills to data analysis . a major component of our curriculum involves students examining data sets by using statistical skills . students participate in data analysis in both the classroom and laboratory . in one example , of the cell module , students are introduced to data related to the cell area of erythrocytes in birds ( www.genomesize.com/cellsize ) . students use these data to learn approaches to data visualization as well as supporting concepts related to cell volume , cell size , and species variation . pedagogical approaches such as these are used throughout the symbiosis curriculum to assist students in developing higher - level thinking skills ( bloom , 1956 ; metz , 2008 ) . these problem - solving opportunities support several learning objectives outlined under competency e1 ( pages 22 and 23 ) in the hhmi this is significant because a large proportion of etsu students who enroll in the freshman biology program are preprofessional , many destined for a career in medicine . these competencies and objectives will serve as a guide and framework for us using a backward design and teaching for understanding ( wiggins and mctighe , 2005 ) approach in the ongoing development of this curriculum . in the evolution of the symbiosis curriculum , we are committed to having students learn how biologists do science by focusing on having students develop critical - thinking skills ( context ) and in becoming intentional learners as they collect and analyze data and model biological systems ( process ) . this takes a nonlinear approach to biological and math material away from concentrating solely on the content material . in fall 2007 , a group of students took the interdisciplinary biology and mathematics & statistics course , symbiosis i consisting of modules 16 , with wet and dry labs to reinforce concepts as well as to develop and apply analytical skills . as an institutional review board ( irb)-approved study , students were tested on biology concepts and mathematics / statistics skills before and after each module . the pretest questions , with identical wording and format , were imbedded in each module exam . overall , their scores increased from 30.9% for the pretests to 71.33% for the posttests . the gain ( g ) for statistics concept questions was 0.56 , and the g for the biology items was 0.61 ( table 2 ) . assessment of symbiosis i a similar curriculum was taught for the east tennessee state university governor 's school : scientific models and data analysis during summer 2009 . their scores increased from a mean of 34% for the pretests to a mean of 65% for the posttests for the subset of concept questions . in spring 2008 , a comprehensive concept pretest to cover modules 712 in the interdisciplinary course symbiosis ii was developed . these items were identified as either predominantly math , predominantly biology , or as truly integrated conceptually . the final for this course included 33 of the items from the pretest and were used in the analysis . the scores for the biology concept questions increased from 27.3 to 43.6% , the scores for the predominantly math items increased from 26.3 to 65.7% , and the scores for the integrated items increased tremendously from 28.6 to 83.1% for a normalized g of 0.76 . combined scores went from 27.7% for the pretest to 66.2% for the posttest ( table 3 ) . similar results were obtained for cohort 2 in spring 2009 as shown in table 3 . assessment of symbiosis ii the first cohort continued their interdisciplinary biology program with the six modules of symbiosis iii in fall 2008 ; it consisted of lecture units with wet and dry labs to reinforce concepts ( table 4 ) . assessment of symbiosis iii the test items along with the curriculum were developed by the teaching faculty . to substantiate these gains as being significant , we have developed an assessment tool that will allow us to compare symbiosis students with students completing the current , more traditional curriculum . in a project of this scope , assessment is a major consideration to decide if the results of the symbiosis project are an improvement to students ' conceptual learning over the standard curriculum . the general purposes of assessment vary from attempting to measure how well students are learning content material to assessing teaching pedagogy to improve teaching effectiveness . posttest assessment has been widely used across many disciplines to measure basic course knowledge of core concepts and course objectives . although it is evident that the pretest posttest assessments can demonstrate evidence of course content learning and assess instructional effectiveness , it also measures entering student preparedness for course material . furthermore , the instrument can be used to assess knowledge retention from sequential course prerequisites ( s.j . our multifaceted plan has the unique opportunity to collect a large data set from the standard lecture - based introductory biology courses over a 2-yr period . it is important to note that perhaps the best way to address the shortcomings of a pre-,mid- , and posttest study is to use a control group ( colosi and dunifon , 2006 ) . several cohorts of the etsu biology ( ibms 1110 , 1120 , 1130 ) students , beginning in 2009 , have been used as a comparison group . these courses will be compared with the symbiosis ( ibms 1110 , 1130 , 1120 ) courses beginning in the 2011 school year . although this aspect was not envisioned in our initial objectives , it will be a landmark assessment study in biology curriculum development considering the inclusion of the large comparison group . the purpose of this protocol is to investigate the effect of instruction on biology students ' understanding of integrated biology and mathematical concepts in both the traditional biology curriculum and the implemented symbiosis curriculum . to avoid the danger of assessment pushing teaching and learning in undesirable directions that are counterproductive ( britton and schneider , 2007 ) , the instructor of record does not and will not have access to the assessment items . this program is an etsu irb approved protocol with no known associated risks to the participants , and all members of the interdisciplinary assessment team are collaborative institutional training initiative trained . the assessment test consists of 60 multiple - choice questions , with 20 questions applicable to each of the three semesters . multiple - choice questions are advantageous in a large - scale study of this nature for efficiency reasons , especially because we are doing an item analysis of each question . in our desire to create a test that measures higher - level objectives , we followed the following steps : use of questions with a variety of answer types , e.g. , moving away from single answer to multiple response to combined response ; and use of questions that included functional distractors to make them more complex than routine questions . a pool of several hundred questions written by the interdisciplinary team was reviewed to choose the top 100 most appropriate questions that ran the scope of the curricular material . from this pool of 100 questions , 20 questions relating to each of the three semesters was chosen to produce the 60-item assessment test . these items are unique to the curriculum and also there is no standardized pool of biology concept questions in the research literature , especially at the quantitative level for introductory biology . to find the reliability of this 60-item multiple - choice test ( 55 questions have five alternative answers ; four questions have four alternative answers ; and one question has two alternative answers ) , we determined the probability of scoring 70% or higher by guessing alone . data collection , rausch analyses , computation of scale scores , and statistical tests ( t tests , anovas , analyses of covariance , and normalized gain ) will be used to determine students ' changes in content knowledge . the inherent difficulty with raw scores is that not all of the items are equally difficult . a student who correctly answers the five hardest items on a test probably knows more than the student who answered the five easiest items correctly . to address this issue , several scaling methods can be used , the most well known being the rausch model . the rausch item analysis will be used to allow the calculation of a scale score for a more meaningful and rigorous comparison of student abilities as measured with this particular test . other parameters include pre- and postcourse surveys such as the sure iii , sure follow up survey , cure projects of the grinnell model ( www.grinnell.edu/academic/psychology/faculty/dl/sure&cure ) , in - class clicker technology , and self - assessment journals . in higher education , there are no empirical data showing student gains from integrated mathematics and science curriculum projects . achieving successful curricular change was driven by our willingness to embrace the vision of bio2010 . during the development of the courses , we came to the conclusion that the approach of teaching biology with math has greatly added to the conceptual richness of biology instruction while giving the math a contextual basis for instruction . our ability to distinguish and overcome obstacles and our willingness to be open - minded , along with cooperation , communication , and collaboration , were essential to the success of the program . the new curriculum is a shift from the secure and familiar traditional educational methodologies to a more student - centered approach . the complete six - phase , multiyear project covers a lot of material and will be one of the most complete , controlled comparisons of teaching methodology that has been reported . the assessment protocol will provide the teaching faculty in biology and mathematics with valuable information on current curriculum and teaching practices compared with the efficacy of the symbiosis curriculum , document student achievement of learning outcomes , and identify effective and ineffective practices . as such , the departments will be able to direct the necessary resources and interventions that will ensure the success of the implementation of the symbiosis curriculum .

it takes a lot of courage to release the familiar and seemingly secure , to embrace the new . but there is no real security in what is no longer meaningful . there is more security in the adventurous and exciting , for in movement there is life , and in change there is power.alan cohen ( used by permission . all rights reserved . for more information on alan cohen 's books and programs , see ( www.alancohen.com.)with the support of the east tennessee state university ( etsu ) administration and a grant from howard hughes medical institute , the departments of biological sciences , mathematics and statistics , and curriculum and instruction have developed a biology math integrated curriculum . an interdisciplinary faculty team , charged with teaching the 18 curriculum modules , designed this three - semester curriculum , known as symbiosis . this curriculum was piloted to two student cohorts during the developmental stage . the positive feedback and assessment results of this project have given us the foundation to implement the symbiosis curriculum as a replacement for the standard biology majors curriculum at the introductory level . this article addresses the history and development of the curriculum , previous assessment results and current assessment protocol , and the future of etsu 's approach to implementing the symbiosis curriculum .

INTRODUCTION DEVELOPMENT OF SYMBIOSIS CURRICULUM SYMBIOSIS CURRICULUM CURRICULUM IMPLEMENTATION OF COUPLED COURSES PREVIOUS ASSESSMENT RESULTS CURRENT ASSESSMENT PROTOCOL CONCLUSIONS

in recent years , biology has been undergoing profound changes that are happening so quickly that biologists have not had the time to integrate these conceptual changes into intellectual and experimental frameworks ( dinger et al . , along with this increasingly rapid accumulation of data from research , biological education is not keeping pace with either conceptual or factual learning . educational methodologies such as conceptual learning , hands - on learning , problem - based learning , and student - based learning have been recommended but are not universally implemented ( klymkowsky , 2005 ) . one common theme throughout the literature is the need for increased instruction in quantitative biology ( brent 2004 ; gross , 2004 ; hoy , 2004 ) . the bio2010 report ( national research council , 2003 ) emphasizes a more quantitative approach to teaching the biological sciences preferably at the introductory level . additional calls for change are the suggested competencies for premed students and physicians ( see the association of american medical colleges [ aamc ] and the howard hughes medical institute [ hhmi ] report ) , to be reflected in proposed changes to the medical college admission test . since the advent of project 2061 ( american association for the advancement of science , 1993 ) , curriculum reform , student achievement , and the assessment of and improving student outcomes in the sciences has been a focus of k12 education and the accrediting commissions . if we want students to be successful in science , mathematics , and technology in higher education , we must shift from a professor - centered lecture paradigm to a student - centered learning paradigm ( harkness , unpublished , paper at 101st american political science association annual conference , washington , dc ) . science , technology , engineering , and mathematics talent expansion program ( nsf - step ) grant , were the pilot group for the initial implementation of the three - semester integrated curriculum . the first semester ( symbiosis i : biology and statistics ) also has been taught for the state of tennessee governor 's school : scientific models and data analysis summer program since 2008 . designing of the curriculum material and the pre- and posttest concept questions was a concerted effort of faculty from the departments of biological sciences , mathematics and statistics , and curriculum and instruction . the results of these assessments demonstrated that the symbiosis material could be used in introducing college and precollege students to an integrated approach to quantitative biology . a large - scale assessment protocol has been developed to establish a baseline of the effect of instruction on biology students ' understanding of integrated biology and mathematical concepts using the traditional biology curriculum as a comparison to the symbiosis curriculum . although a review of funded grants shows most programs develop integrated courses at the junior , senior , and graduate levels , we developed an integrated curriculum for the introductory level . during this initial development ( 20052006 ) , the departments received an nsf step grant allowing us to recruit freshmen as a logical pilot group to take the integrative courses . each symbiosis module includes the biology concept(s ) and the math skills needed to analyze data sets that examined the concepts . a 2-wk laboratory , one for collecting data and the other for analyzing data , were developed for each module . to match the current biology and math curriculum ( www.etsu.edu/reg/catalog/undergraduate.aspx ) , the courses were designed to contain 5 h of lecture and 2 h of laboratory each week . the courses were named as follows : integrated biology and statistics , integrated biology and calculus , and integrated biology and discrete math , and a separate rubric ( ibms ) for the courses was obtained . the curriculum committee also worked with the east tennessee state university ( etsu ) quillen college of medicine admission committee and the office of medical professions advisement at etsu to ensure that the application process through the american medical college application service recognizes the course content in the integrated curriculum . once the initial logistics were complete , faculty from math and biology submitted an hhmi educational grant with the vision to create a three - semester introductory curriculum of integrated biology / math where the student would receive credit for three semesters of introductory biology for majors , a semester of statistics , and a semester of calculus . the 4-yr grant was funded in fall 2006 , and the course was first taught in fall 2007 . as in many institutions , our standard biology for majors curriculum involves a large lecture format ( sections of 300 + students ) and smaller lab sections of 25 students . the material has largely been based on a typical textbook in terms of content but seems to promote passive learning with superficial retention of the material and little integration of conceptual learning ( armbruster et al . a team consisting of at least two faculty members , a biologist and a mathematician or statistician , developed and team - taught each module . over time , the faculty has become more comfortable with each other 's field . modern biology pedagogy is based , to a large extent , on a pseudological framework of going from small - to - big and is rooted in what biology has done rather than why or how it is done ( moore et al . an examination of modern biology textbooks supports this contention because they are not quantitative and are primarily encyclopedic in content with very little integration of material between chapters . noticeably missing are equations , data sets , and graphs , which demonstrate the richness of variation that is a major component of biology . in traditional pedagogy , this lack of quantitative analysis leads the student to be bombarded with an unending series of facts that have no logical connection to the whole . could this old approach be one reason why professors have observed that students retain little introductory material in upper - level courses ( bransford et al . a literature search reveals very little development of an integrated math biology curriculum at the introductory level . the criteria that have guided the development of the statistical component of the first semester of symbiosis are as follows : an early introduction of inference to be able to answer research questions from the beginning , examples of the sequence rationale - algorithm - computer program , use of a problem - oriented approach presenting statistical methods when they are needed , emphasis on the study of variability , emphasis on a multivariate view whenever possible , and inclusion of topics at the elementary level that can serve as a preparation to later understand the language and methods of biostatistics / bioinformatics , including exposure to statistical software . active learning and critical thinking are promoted through class discussion and activities , homework , and assignments . a complete description of the material in the curriculum is beyond the space of the article . a short description of model i in symbiosis i is presented with the remainder modules just outlined . in symbiosis i , students start with the scientific method ( module i ) and statistical hypotheses testing during the first week of the course , even though the classical topics of sampling distributions have yet not been covered . this was just the first example of how we have adjusted course material by using randomization methods ( permutations test ) to test hypotheses about the means of two populations . to test hypotheses about a population proportion , basics of probability and the binomial distribution are introduced and the exact test appears as a simple application of the binomial distribution . the scientific method is taught using case studies such as von helmont 's tree growing experiment , stanley pruissner 's development of the prion theory , the methodology of identification of mosquito vectors of arboborne diseases , introduction of human immunodeficiency virus ( hiv ) as the etiology of aids , before asking the question as a class project of whether mosquitoes could transmit hiv . the answer to this question is the use of statistics to look at the bel glade study in florida by using epidemiology ( centers for disease control and prevention , 1986 ) . the module ends with a description of the five basic concepts of biology : evolution ; membrane - bound cellular organization ; energy metabolism ; response to the environment ; and growth , reproduction , and development . each of these concepts is explicitly covered in each of the modules that maintain a connection between the topics of the course . module ii covers the cell by using data sets of abnormal and normal cells ; red blood cell counts ; and the dimensions of cells to introduce descriptive statistics , including correlation and statistical graphs . the study of relationships between variables is extended by the study of functions by using scaling to study allometry and isometry ( module iii ) . genetics and probability ( module iv ) are also a natural fit because copying alleles from parents to offspring involves randomness and provides a perfect motivation for the study of probability , along with conditional probability , independence , and the corresponding test of independence . classic topics of statistical inference ( e.g. these methods are used to compare mitochondrial sequences of insect species with the drosophila mitochondria as a reference . the students discuss the differences between species at the dna level . in the following semester , symbiosis ii the biological topics were rearranged from our normal progression to use rate of change for the calculus . the models are as follows : populations , ecology , behavioral ecology , chronobiology , structured populations , energy , and enzymes . one of the goals of the symbiosis project is to cover a first - semester calculus course by the end of symbiosis ii . thus , the calculus in symbiosis ii is actually a continuation of math concepts begun in symbiosis i. a student who successfully completes symbiosis i and ii will have covered all the topics that constitute a rigorous , sciences majors ' statistics and calculus course . in addition , the overwhelming majority of the calculus that is covered is introduced and developed in contexts that are important to the study and practice of biology . moreover , weaker students benefit from having an entire year to study material that is typically covered in a one - semester course . the mathematics component is assessed by the students taking the math department 's gateway exam that demonstrates competency in calculus concepts to receive credit for the calculus course . all of the symbiosis students who have taken this exam have passed the gateway exam . in symbiosis iii , the emphasis of the quantitative component includes calculus ; matrices ; graph theory ; and advanced statistical topics such as nonlinear estimation , multivariate methods , and an introduction to bioinformatics . transitioning from class sizes of < 20 in the pilot study to multiple sections of 250300 for biology and 5075 for math and statistics means adopting a different pedagogical approach . the recognized challenges include the large block of time required , the faculty load / credit assignment issues , and the differences in instructional necessities . the first two semesters of biology will be taught using the symbiosis integrated material with specifically designed mathematics and statistics courses as corequisites . thus , symbiosis i modules will be taught as biology i ( ibms 1110 ) and statistics ( ibms 1530 ) in the first semester . symbiosis ii modules will be taught as biology ii ( ibms 1120 ) and calculus ( ibms 1910 ) in the second semester . symbiosis iii ( ibms 1130 ) modules will maintain the integrated approach during the third semester . the mathematics component in ibms 1130 is an extension of differential and integral calculus , introduction to linear algebra , and matrices and analysis of variance ( anova ) . this approach maintains our commitment to an integrated presentation of biology , statistics , and mathematics . to maintain this integration , regularly scheduled meetings of the entire instructional team will be held to ensure all faculty , instructors , and lab instructors are staying on track with the course and lab materials . careful consideration of the objectives of the symbiosis modules enabled us to map the modules to these competencies ( table 1 ) . matching the symbiosis modules to the list of competencies ( e ) for premedical undergraduate students with the hhmi the first set of competencies ( e1 ) concerns demonstration of quantitative skills to data analysis . students participate in data analysis in both the classroom and laboratory . in one example , of the cell module , students are introduced to data related to the cell area of erythrocytes in birds ( www.genomesize.com/cellsize ) . students use these data to learn approaches to data visualization as well as supporting concepts related to cell volume , cell size , and species variation . pedagogical approaches such as these are used throughout the symbiosis curriculum to assist students in developing higher - level thinking skills ( bloom , 1956 ; metz , 2008 ) . these problem - solving opportunities support several learning objectives outlined under competency e1 ( pages 22 and 23 ) in the hhmi this is significant because a large proportion of etsu students who enroll in the freshman biology program are preprofessional , many destined for a career in medicine . these competencies and objectives will serve as a guide and framework for us using a backward design and teaching for understanding ( wiggins and mctighe , 2005 ) approach in the ongoing development of this curriculum . in the evolution of the symbiosis curriculum , we are committed to having students learn how biologists do science by focusing on having students develop critical - thinking skills ( context ) and in becoming intentional learners as they collect and analyze data and model biological systems ( process ) . this takes a nonlinear approach to biological and math material away from concentrating solely on the content material . as an institutional review board ( irb)-approved study , students were tested on biology concepts and mathematics / statistics skills before and after each module . overall , their scores increased from 30.9% for the pretests to 71.33% for the posttests . the gain ( g ) for statistics concept questions was 0.56 , and the g for the biology items was 0.61 ( table 2 ) . assessment of symbiosis i a similar curriculum was taught for the east tennessee state university governor 's school : scientific models and data analysis during summer 2009 . their scores increased from a mean of 34% for the pretests to a mean of 65% for the posttests for the subset of concept questions . in spring 2008 , a comprehensive concept pretest to cover modules 712 in the interdisciplinary course symbiosis ii was developed . these items were identified as either predominantly math , predominantly biology , or as truly integrated conceptually . the final for this course included 33 of the items from the pretest and were used in the analysis . the scores for the biology concept questions increased from 27.3 to 43.6% , the scores for the predominantly math items increased from 26.3 to 65.7% , and the scores for the integrated items increased tremendously from 28.6 to 83.1% for a normalized g of 0.76 . combined scores went from 27.7% for the pretest to 66.2% for the posttest ( table 3 ) . similar results were obtained for cohort 2 in spring 2009 as shown in table 3 . assessment of symbiosis ii the first cohort continued their interdisciplinary biology program with the six modules of symbiosis iii in fall 2008 ; it consisted of lecture units with wet and dry labs to reinforce concepts ( table 4 ) . assessment of symbiosis iii the test items along with the curriculum were developed by the teaching faculty . to substantiate these gains as being significant , we have developed an assessment tool that will allow us to compare symbiosis students with students completing the current , more traditional curriculum . in a project of this scope , assessment is a major consideration to decide if the results of the symbiosis project are an improvement to students ' conceptual learning over the standard curriculum . the general purposes of assessment vary from attempting to measure how well students are learning content material to assessing teaching pedagogy to improve teaching effectiveness . although it is evident that the pretest posttest assessments can demonstrate evidence of course content learning and assess instructional effectiveness , it also measures entering student preparedness for course material . furthermore , the instrument can be used to assess knowledge retention from sequential course prerequisites ( s.j . our multifaceted plan has the unique opportunity to collect a large data set from the standard lecture - based introductory biology courses over a 2-yr period . it is important to note that perhaps the best way to address the shortcomings of a pre-,mid- , and posttest study is to use a control group ( colosi and dunifon , 2006 ) . several cohorts of the etsu biology ( ibms 1110 , 1120 , 1130 ) students , beginning in 2009 , have been used as a comparison group . these courses will be compared with the symbiosis ( ibms 1110 , 1130 , 1120 ) courses beginning in the 2011 school year . although this aspect was not envisioned in our initial objectives , it will be a landmark assessment study in biology curriculum development considering the inclusion of the large comparison group . the purpose of this protocol is to investigate the effect of instruction on biology students ' understanding of integrated biology and mathematical concepts in both the traditional biology curriculum and the implemented symbiosis curriculum . to avoid the danger of assessment pushing teaching and learning in undesirable directions that are counterproductive ( britton and schneider , 2007 ) , the instructor of record does not and will not have access to the assessment items . this program is an etsu irb approved protocol with no known associated risks to the participants , and all members of the interdisciplinary assessment team are collaborative institutional training initiative trained . the assessment test consists of 60 multiple - choice questions , with 20 questions applicable to each of the three semesters . multiple - choice questions are advantageous in a large - scale study of this nature for efficiency reasons , especially because we are doing an item analysis of each question . a pool of several hundred questions written by the interdisciplinary team was reviewed to choose the top 100 most appropriate questions that ran the scope of the curricular material . from this pool of 100 questions , 20 questions relating to each of the three semesters was chosen to produce the 60-item assessment test . these items are unique to the curriculum and also there is no standardized pool of biology concept questions in the research literature , especially at the quantitative level for introductory biology . to find the reliability of this 60-item multiple - choice test ( 55 questions have five alternative answers ; four questions have four alternative answers ; and one question has two alternative answers ) , we determined the probability of scoring 70% or higher by guessing alone . data collection , rausch analyses , computation of scale scores , and statistical tests ( t tests , anovas , analyses of covariance , and normalized gain ) will be used to determine students ' changes in content knowledge . the inherent difficulty with raw scores is that not all of the items are equally difficult . to address this issue , several scaling methods can be used , the most well known being the rausch model . other parameters include pre- and postcourse surveys such as the sure iii , sure follow up survey , cure projects of the grinnell model ( www.grinnell.edu/academic/psychology/faculty/dl/sure&cure ) , in - class clicker technology , and self - assessment journals . in higher education , there are no empirical data showing student gains from integrated mathematics and science curriculum projects . achieving successful curricular change was driven by our willingness to embrace the vision of bio2010 . during the development of the courses , we came to the conclusion that the approach of teaching biology with math has greatly added to the conceptual richness of biology instruction while giving the math a contextual basis for instruction . our ability to distinguish and overcome obstacles and our willingness to be open - minded , along with cooperation , communication , and collaboration , were essential to the success of the program . the new curriculum is a shift from the secure and familiar traditional educational methodologies to a more student - centered approach . the complete six - phase , multiyear project covers a lot of material and will be one of the most complete , controlled comparisons of teaching methodology that has been reported . the assessment protocol will provide the teaching faculty in biology and mathematics with valuable information on current curriculum and teaching practices compared with the efficacy of the symbiosis curriculum , document student achievement of learning outcomes , and identify effective and ineffective practices . as such , the departments will be able to direct the necessary resources and interventions that will ensure the success of the implementation of the symbiosis curriculum .

micrornas are small ( 2022 nt ) , evolutionarily conserved , noncoding single - stranded rnas discovered in the 1990s [ 1 , 2 ] , functioning to target 3 untranslated region ( utr ) of mrnas in antisense sequence specific way and regulate genes posttranscriptionally for degradation or translation suppression . micrornas target 13% of all eukaryotic genes yet regulating ~30% of protein - coding genes . the mirnas are first transcribed by rna polymerase ii in the nucleus as large primary transcript ( pri - mirna ) , either from independent genes or from clustered genes encoding several mirnas and further processed into ~70 nt pre - mirna with hairpin structure by drosha , a rnase iii type endonuclease ( rn3 ) in the nucleus . alternatively , in the nucleus , a small class of mintron without the stem - loop and the flanking single - strand structure as in pri - mirna required for drosha processing , could be generated by passing drosha - dependent pathway . in the cytoplasm , ~20 bp mirna / mirna * duplex are generated by dicer , another rn3 endonuclease . one of the mirna duplex strands is further incorporated into protein - rna complex called rna - induced silencing complex ( risc ) , although in some cases , both arms of the pre - mirna hairpin could generate mature mirnas [ 79 ] . mirnas interact with target mrna by sequence complementarity , and in perfect base pairing usually triggers endonucleolytic mrna cleavage ; however , in most situations , such base pairing is imperfect , resulting in translational suppression . the key component of this risc machinery is ago protein family ( ago 14 ) , but only ago 2 is known to have the catalytic enzyme function [ 11 , 12 ] . besides ago proteins , gw182 protein is also recruited to the risc complex and together localize in cytoplasmic foci called processing bodies ( p bodies or gw bodies ) , where mrna is sequestered from being translated [ 1316 ] . there are different experimental and bioinformatics approaches to predict mirna targets . at a minimum , the precise matching to 3utr of mrna in multiple copies should be within the first 28 bases from the 5 end of the mature mirna , called the seed region [ 1720 ] . to date , over 2000 human mirnas have been annotated in the sanger mirbase ( release 18 , http://www.mirbase.org/cgi-bin/browse.pl?org=hsa ) . the mirna network is highly redundant , since a single mirna may have multiple target mrnas , and in turn , a single mrna could be targeted by many mirnas . various mirnas have been shown to be involved in a myriad of cellular processes including differentiation , metabolism , apoptosis , and development . physiologically , and pathologically , mirnas have been reported to play roles in cancers , inflammatory responses , diabetes , and autoimmunity [ 23 , 24 ] . multiple evidence suggest that micrornas play a significant role in the posttranscriptional genetic regulation in stem and progenitor cells . they are involved in a number of hematological malignancies such as acute lymphoblastic leukemia , acute myeloid leukemia , chronic lymphocytic leukemia , chronic myelogenous leukemia , diffuse large - b - cell lymphoma , and others . therefore , mirna profiling is critical in order to distinguish stem cells of the different origins , developmental stages , and genetic conditions . recent studies have demonstrated a causative role for mirnas in malignant diseases development in the hematopoietic system . for instance , overexpression of mir-155 or mir-29a in the mouse hematopoietic system leads to a myeloproliferative disorder or leukemia , respectively . on the other hand , tumor suppressor mirnas such as mir-15a/16 - 1 are found to be deleted in a subset of lymphomas and have been shown to cause chronic lymphocytic leukemia in mice [ 31 , 32 ] . microrna-125b has been demonstrated to cause pathological myeloid cells expansion in a dose - dependant manner , and mir-155 is known to induce polyclonal expansion followed by b - cell malignancy development . in another study on human umbilical cord blood , two particular mirnas - hsa - mir-520h and hsa - mir-526b*- levels appeared to be elevated . interestingly , abcg2 , an important factor of stem cells maintenance , is a known target of hsa - mir-520h . studies have implicated important functions of mirna on hematopoietic development as well as innate and adaptive immune responses . toll - like receptor ( tlr ) signaling leads to transcriptional activation of a large class of proinflammatory cytokines as well as multiple mirnas . for example , mir-146a and mir-155 have been shown to be upregulated upon exposure to lps in the monocytic leukemia cell line thp-1 . more importantly , two key components of tlr4 signaling pathway , traf6 and irak1 have been verified to be targets for mir-146a . this study for the first time profiled the mirnas alterations in tlr signaling and proposed the mirnas as negative regulators of tlr activation . mir-155 is another well - studied microrna reported to be activated by several tlr pathways [ 36 , 37 ] , and its negative regulatory role during tlr - mediated activation has also been addressed [ 38 , 39 ] . more interestingly , il-10 is shown to inhibit tlr - induced mir-155 . to understand global mirnas ' importance in b and t development , studies were performed in which knocking out dicer at different stages of b and t development resulted in blockage of further differentiation [ 4143 ] . in addition , mir-155 has been found to be one of the most important mirnas in both b and t cells as well as antigen presentation by dendritic cells ( dcs ) and is required for normal germinal center ( gc ) response [ 44 , 45 ] , b - cell class switching , th1/th2 polarization , and treg development both in the thymus and peripheral . considering the importance of mirnas in the immune system raises the question whether or not there is direct link between mirnas abnormalities and immune disorders or autoimmune diseases . interestingly , the discovery in 2002 of gw bodies ( gwbs ) , where mirna - mrna reside for degradation was from serum from an autoimmune patient with motor and sensory neuropathy . subsequently , anti - gwb autoantibodies in the serum have been identified from patients with various autoimmune disorders , indicating an involvement of general mirna pathway and autoantibody production . dysregulated mirna expression has been associated with autoimmunity , for example , mir-146a was underexpressed in pbmc from sle patients when compared with healthy control . the study further showed that mir-146a is a negative regulator of type i interferon ( ifn ) pathway by targeting interferon regulatory factor ( irf ) 5 and signal transducers and activators of transcription ( stat ) 1 , thus the decrease in mir-146a may contribute to the increased type i ifn signaling pathway observed in sle . a recent study in murine models ( mrl - lpr , c57bl/6-lpr , and nzb / nzw f1 ) of sle using a combination of microarray and real - time rt - pcr approaches , dai et al . identified that mir-182 - 96 - 183 cluster , mir-31 , and mir-155 are among those consistently upregulated mirnas across different genetic background strains of mice . in addition to important contribution of mir-155 to physiological immune response , its activity in autoimmune circumstances was also investigated . a murine experimental autoimmune encephalomyelitis ( eae ) model with mir-155 was shown to be resistant to eae pathology . thus , unregulated mir-155 may be a link between inflammation and cancer via inducing a high proliferation rate resulting in increased mutations . the mir-17 - 92 cluster locating in human chromosome 13q31 is known as an onco - mir , and this genomic region is often amplified in lymphomas and other cancer , and the mature mir-17 - 92 expression is highly elevated in malignant cells [ 27 , 5355 ] . results showed that mir-17 - 92 targets phosphatase and tensin homolog ( pten , tumor suppressor ) and bim ( proapoptotic molecule ) mrna directly resulting in lymphoproliferative and autoimmune diseases . in a current study from our lab on microrna abnormalities in nzb / nzw f1 lupus model by using type i and type iii interferons ( ifn- and ifn- ) as exogenous disease accelerators , we identified upregulation of several micrornas correlated to disease severity , yet not with the ifn treatment . mir-15a was one of the most significant elevated micrornas as autoimmunity developed in these mice and the level of splenic mir-15a was correlated to the level of anti - dsdna igg , in addition , the cellular level of mir-15a was also reflected in the plasma ( manuscript accepted for publication ) . multiple myeloma ( mm ) is characterized by a clonal expansion of plasma b cells in the bone marrow or in extramedullary sites which results in high levels of monoclonal immunoglobulins in the serum . cytogenetic abnormalities are present in many mm cases , characterized by either hyperdiploidy with the presence of trisomies of odd chromosomes or nonhyperdiploidy with chromosomal aberrations and translocations involving the igh locus on chromosome 14 . in addition to these advancements in understanding mm pathogenesis , studies on the role of micrornas in recent years have shown them to be key players in mm development not only in the sustenance of malignant cells but also in the initiation of malignancy due to methylation of micrornas that function as tumor suppressors . studies investigating micrornas in mm began in 2007 with the discovery that interleukin 6 ( il-6 ) indirectly induces the transcription of mir-21 through signal transducer and activator of transcription 3 ( stat3 ) transcription in the human myeloma cells line . the same upstream enhancer controls mir-21 and stat3 transcription , and stat3 controls the transcription of survivin , bcl2 , and mcl-1 . a microrna microarray analysis in 20 myeloma samples revealed that mir-335 and mir-342 - 3p were upregulated and may be involved in plasma cell homing and other interactions in the bone marrow . for example , mir-106b-25 cluster , mir-181a / b , and mir-32 target a histone acetyltransferase , p300/cbp - associated factor ( pcaf ) that reversibly acetylates transcriptional regulators including p53 , thus accounting for the low levels of pcaf observed in mm cells . also mir-1792 downregulates bim , a proapoptotic molecule , and mir-19a / b target socs-1 , a silencer of the stat3 , thus enhancing the oncogenicity of mm cells [ 60 , 61 ] . as seen in cll , mir-15a/16 - 1 normally encoded within the dleu2 , a gene frequently deleted in lymphocytic leukemia , mir-15a/16 - 1 activity is central to the antiproliferative activity of dleu2 , as mir-15a/16 - 1 inhibits cyclind1 , cyclind2 , and cdc25a . for example , mir-25 and mir-30d are increased in mm and target the 3utr of the p53 gene . also , mm cells have low levels of mir-192 , mir-194 , and mir-215 which targets mdm2 , a p53 antagonist , thereby lowering p53 levels and increasing oncogenic potential . moreover , the promoter of mir-34b / c , a transcriptional target of p53 was found to be hypermethylated and thus inactivated in multiple myeloma cell lines . hypermethylation of the promoters of various tumor suppressors such as mir-124 - 1 ( a target of cdk6 ) , mir-203 ( a target of cyclic - responsive element - binding protein which increases proliferation ) , and mir-29b ( a target of mcl-1 which antagonizes il-6 ) increase the tumorigenicity of myeloma cells . extranodal marginal zone lymphomas are most associated with mucosal - associated lymphoid tissue ( malt ) and are characterized by clonal proliferation of plasma cells that produce the immunoglobulin a isotype . investigations involving micrornas have found the mir-203 promoter to be hypermethylated in samples of gastric lymphoma , and this microrna targets the c - abl1 oncogene , thus enabling tumor growth and proliferation . in addition , mir-150 and mir-155 were upregulated , while mir-184 , mir-205 and mir-200a / b / c ( which targets cyclin e2 ) were downregulated . in another hyperimmunoglobulin disorder , immunoglobulin a nephropathy ( igan ) is characterized by the deposition of immune complexes in the kidney mesenchyme causing renal injury and usually coincides with mucosal infections . these immune complexes are composed of iga1 molecules that are galactose - deficient , causing a conformational change in the molecule , and autoantibodies ( iga or igg ) form to its exposed epitopes . since mir-155 and mir-146 are involved in b lymphocyte development , their levels were examined in 43 igan biopsy specimens and urine samples . the results showed that mir-146 and mir-155 were high in igan biopsy and urine sediment , suggesting their role in igan pathogenesis . micrornas ( mirnas ) have been well studied in various cancers including leukemias [ 73 , 74 ] . acute myeloid leukemia ( aml ) is a hematopoietic progenitor cell - originated malignant disorder affecting the myeloid lineage , which could be classified into subtypes based on the differentiation stages of the malignant cells found in peripheral blood and in bone marrow . among various symptoms and manifestations identified in association with aml , one of the most common characteristics involved in ~50% of aml patients is a group of cytogenetic abnormalities , which is considered to be contributing to the disease heterogeneity and with prognostic significance . other aml patients without detectable chromosomal abnormalities may display mutations or dysregulations in specific genes , a signature ubiquitously found in cancers [ 7779 ] . microrna signatures in aml have been sought , and many groups of researchers performed large - scale profiling of mirna expression in different populations of aml patients . in the first study where aml patient samples were compared to acute lymphoblastic leukemia ( all ) , both groups with similar chromosomal alterations , 27 mirnas were reported to be different between the two groups . importantly , mir-146a was inversely correlated to overall survival in both aml and all . however , these studies focused on mirna profile distinguishing aml from all , which was not sufficient for understanding the abnormalities of mirnas expression exclusive to aml . another study compared 122 aml samples to cd34 + cells from 10 normal controls . among the 122 aml samples , 60 cases were untreated and 54 relapsed or refractory . by microarray profiling , 26 micrornas were downregulated in aml samples . several of these downregulated mirnas in aml were also underexpressed in mature myeloid cells suggesting that mirnas related to the differentiation patterns in aml ( mir-126 , mir-130a , mir-93 , mir-125a , and mir-146 ) . in correlating cytogenetic abnormalities with mirna expression , 14 downregulated and 8 upregulated mirnas were associated with 11q23 translocation versus all other aml , including the downregulation of mir-196 and mir-15a , and overexpression of mir-21 in t(6 ; 11 ) with worse prognosis . in aml patients ( n = 36 ) achieving complete remission the levels of mir-15a/16 were upregulated . all - trans retinoic acid ( atra ) in vitro treatment in aml cell lines and primary leukemic cells induced mir-15a/16 upregulation , in addition , mir-15a/16 enhanced the effects of atra inducing leukemic cell differentiation.despite the poor overall survival of these aml patients , the study showed several associations between mirna expression and the outcome of patients , especially the overexpression of mir-199a and mir-191 , identified in aml with trisomy 8 and associated with poor outcome . this study was the first to identify the distinct mirnas profile between aml patients and normal control , and the subsets of mirnas related to cytogenetic groups and disease outcome . almost at the same time , a study with 215 heterogeneous aml samples was performed to demonstrate the signatures of mirnas expression in cytogenetic and molecular subtypes . a group of upregulated mirnas were prominent in t(15;17 ) cases . in contrast , t(8;21 ) was characterized by downregulated mirna alterations , for example , tumor suppressor let-7 . in molecular subgroups of aml , nucleophosmin ( npm1 ) mutations , which represent the most common molecular abnormality in aml , are associated with overexpression of homeobox genes ( hox ) . upregulation of mir-10a , mir-10b , mir-196a and mir-196b , was identified in aml carrying npm1 mutations , and these mirnas were located within the hox genes . although mir-196a directly targets hoxb8 mrna , the upregulated mir-196a in this aml subgroups may represent a breakage in the regulation loop between mirnas and hox genes . consistent with other studies , mir-155 was significantly upregulated in amls with internal tandem duplications of flt3(flt3-itd ) , corroborating the oncogenic effect of mir-155 in myeloid cells in addition to such effects in lymphoid lineages [ 84 , 87 , 88 ] . in comparing aml to normal cd34 + cells , upregulation of mir-21 in amls was found , consistent with other studies and further strengthening the importance of mir-21 in aml [ 82 , 84 ] . interestingly , in an analysis of aml subgroups the t(8;21 ) and inv(16 ) were grouped together by mirna profile , supporting the notion that both subgroups belong to core - binding factor ( cbf ) amls , suggesting some common pathways shared by cbf - amls . overexpression of mir-224 , mir-368 , and mir-382 was restricted to the t(15;17 ) samples , while mir-17 - 92 cluster was overexpressed exclusively in mixed - lineage leukemia ( mll ) rearrangements . in addition , in a study of 100 amls , comparing leukemic samples to normal bone marrow , mir-155 and mir-181a were upregulated . mir-181a has been reported to target p27 in aml cell lines , resulting in an abrogation of 1 , 25-dihydroxyvitamin d3 ( 1,25d ) induced differentiation in aml cell lines . a recent study classified aml cases into favorable , moderate , and poor as the predicted outcome according to the karyotype . mir-181a high expression was suggested to be associated with better - risk groups suggesting a potential therapeutic approach involving manipulation of mir-181a level in aml patients . in contrast to elevated mir-181a as favorable prognostic factor , mir-155 upregulation predicts poor prognosis in aml . cll is characterized by the accumulation of malignant b-1 cells ( cd5cd19cd20cd23igm ) in peripheral lymphoid organs , bone marrow , and peripheral blood . it accounts for 30% of all leukemias in the western world , making it the most common lymphoid malignancy with mainly elderly with disease . cll is broadly classified into aggressive ( zap70-unmutated igh ) and indolent ( zap70-mutated igh ) . prominent among them are 11q23 deletions ( atm ; mir-34b / c cluster ) , trisomy 12 ( increased mdm2 ) , 17p deletion ( tp53 ) , and 13q14 deletions ( mir-15a/16 - 1 ) . dysregulation of several micrornas like mir-15a/16 - 1 , mir-34 cluster , mir-155 , mir-29 , and mir-181b has been implicated in the pathogenesis of cll . the most common genetic abnormality in cll patients is the deletion of 13q14 region ( 5060% of cll cases ) that encodes a crucial microrna locus , mir-15a/16 - 1 [ 30 , 96 ] . decreased mir-15a/16 - 1 confers a growth advantage as these micrornas target key cell cycle regulatory and antiapoptotic proteins such as cyclin d1 and bcl2 [ 97 , 98 ] . it is interesting to note that nzb mice ( spontaneously occurring mouse model of cll ) also exhibit a 50% reduction in the level of mir-15a/16 - 1 , that is associated with a point mutation and deletion in the 3 flanking region of mir-16 - 1 . moreover targeted deletion of the mir-15a/16 - 1 locus or a larger surrounding minimal deleted region ( mdr ) led to the development of cll in mice , further confirming the tumor suppressor function of this locus . other micrornas are abnormal in cll including mir-29 and mir-181 , which target tcl1 , a gene that is highly elevated in aggressive cll . mir-29 expression is decreased in aggressive cll , while it is increased in indolent cll as compared to normal volunteers [ 100 , 101 ] . thus , the same microrna can function as both an oncogene and as a tumor suppressor in cll . mir-34a / b / c is decreased in patients with 11q deletions . normally , upon transactivation by tp53 , mir-34 expression would result in decreased zap70 . mir-34a has also been shown to target e2f1 and b - myb oncogenes in cll as well as aml . mir-155 , mir-150 , and mir-21 expression is increased in b - cll cells as compared to normal b cells [ 23 , 104 ] . v - myb is found to be elevated in cll patients , and it stimulates the mir-155 host gene . the oncogenic potential of mir-155 is further supported by the development of b - cell malignancies in e-mmu - mir-155 transgenic mice . using a poorly understood mechanism , micrornas are secreted into body fluids such as serum and urine , and their levels can be used as noninvasive biomarkers for diagnosis and monitoring of cancer and various other diseases [ 106 , 107 ] . in a recent , study it was shown that elevated mirna levels in serum may offer early cll detection and differentiation between zap70 status . the authors further concluded that increased expression of mir-150 , mir-29a , mir-222 , and mir-195 can be used as a highly sensitive diagnostic test for cll . in this paper , a variety of blood disorders were discussed in terms of microrna abnormalities observed . one microrna family of interest stood out as a potential regulator of cell fate ( table 1 and figure 1 ) . recently , mir-15 family members ( mir-15a / b , mir-16 , mir-103 , mir-107 , mir-195 , and mir-497 ) have been grouped together due to their identical agcagc sequence at 5 end seed region ( nucleotides 27 ) [ 109 , 110 ] , which offers this mirna group various overlapping functions in gene - regulatory pathways and disease scenarios , especially in cancers . mir-15/107 gene group could be upregulated by tumor suppressor p53 , altered by various cell stress [ 111115 ] , or inhibited by myc [ 116 , 117 ] . a broad spectrum of mrnas is targeted by mir-15/107 , importantly , mir-15/16 paralogs regulate cell cycle via targeting of cyclin d1 and induce apoptosis via targeting of bcl-2 , and mir-107 also induces cell cycle arrest . the tight involvement of mir-15/107 in cell growth and cell fate control , and their upstream regulators , such as p53 and myc , which by themselves are important players in tumorigenesis [ 120 , 121 ] , revealed critical mechanisms for abnormalities in cancer development , including leukemias . indeed , all members from mir-15/107 group have been identified to be altered in various tumor cells [ 122125 ] . specifically , underexpression of mir-15a/16 as a result of deletion or mutation of mir-15a/16 loci has been linked to the pathogenesis of cll [ 96 , 99 , 118 ] , similarly in aml and mm , where the downregulation of mir-15a/16 was associated with the loss of control for malignant cells differentiation and proliferation [ 62 , 83 ] . in contrast , in sle which is characterized by elevated plasma cell differentiation contributing to increased autoantibody production [ 126 , 127 ] , splenic mir-15a was increased , and this was significantly correlated with autoantibody levels in lupus - like autoimmune mouse model ( manuscript accepted for publication ) , suggesting a role of mir-15a upregulation in cell cycle arrest in order for plasma cell differentiation . future directions may be directed toward stem cell transplantation for many of these blood disorders . cellular transplantation therapy holds a huge potential for a variety of degenerative , genetic , and malignant conditions treatment . hematopoietic stem cell transplantation is the most widely used form of such a therapy , but many patients do not benefit from that because of the lack of a suitable hla - matched donor . in this sense , patient - specific autologous pluripotent stem cells generation would provide a great opportunity to combine gene therapy with autologous cell transplantation to treat different human conditions including hematological disorders such as aml . for this reason , robust protocols for the generation of safe autologous induced pluripotent stem ( ips ) cells are strongly needed . to this end , micrornas represent an attractive tool for both ips generation efficiency enhancement and gene targeting approaches . it is known that expression of embryonic stem ( es ) cell - specific micrornas such as mir-294 promotes ips cells induction from somatic cells . recently , it has even been demonstrated that the expression of mir-302/367 cluster can directly reprogram mouse and human somatic cells to a pluripotent stem cell state in the absence of the commonly used reprogramming factors . alternatively , inhibition of tissue - specific mirnas would also enhance ips generation , which has been confirmed by antisense silencing of a prodifferentiation let-7 mirna . another application of micrornas lies in promoting patient - specific ips differentiation towards the required cell lineage , for example hsc expansion . mir-145 has been shown to induce es cell differentiation by inhibiting the expression of sox2 , oct4 , klf4 , and c - myc , key reprogramming factors , and led to an increase of hsc number in vivo by more than 8 fold [ 132134 ] . nevertheless , hsc expansion from ips cells by means of micrornas needs to be further developed . hopefully , the recently achieved success in the production of ips cells with the use of mirnas will pave the way for successful in vitro expansion of hscs with mirnas .

common blood disorders include hematopoietic cell malignancies or leukemias and plasma cell dyscrasia , all of which have associated microrna abnormalities . in this paper , we discuss several leukemias including acute myeloid leukemia ( aml ) and chronic lymphocytic leukemia ( cll ) and identify altered micrornas and their targets . immune disorders with altered blood levels of antibodies include autoimmune disorders , such as systemic lupus erythematosus ( sle ) with associated anti - self - autoantibodies and immunoglobulin a nephropathy ( igan ) also have related microrna abnormalities . the alterations in micrornas may serve as therapeutic targets in these blood disorders .

1. Introduction 2. MicroRNAs in Hematopoietic Stem Cells 3. MicroRNAs in the Immune System 4. MicroRNA in Autoimmune Diseases 5. MicroRNAs in Hyperimmunoglobulinemias 6. MicroRNAs in Acute Myeloid Leukemia (AML) 7. MicroRNAs in Chronic Lymphocytic 8. Conclusion

micrornas are small ( 2022 nt ) , evolutionarily conserved , noncoding single - stranded rnas discovered in the 1990s [ 1 , 2 ] , functioning to target 3 untranslated region ( utr ) of mrnas in antisense sequence specific way and regulate genes posttranscriptionally for degradation or translation suppression . micrornas target 13% of all eukaryotic genes yet regulating ~30% of protein - coding genes . the mirnas are first transcribed by rna polymerase ii in the nucleus as large primary transcript ( pri - mirna ) , either from independent genes or from clustered genes encoding several mirnas and further processed into ~70 nt pre - mirna with hairpin structure by drosha , a rnase iii type endonuclease ( rn3 ) in the nucleus . in the cytoplasm , ~20 bp mirna / mirna * duplex are generated by dicer , another rn3 endonuclease . mirnas interact with target mrna by sequence complementarity , and in perfect base pairing usually triggers endonucleolytic mrna cleavage ; however , in most situations , such base pairing is imperfect , resulting in translational suppression . the key component of this risc machinery is ago protein family ( ago 14 ) , but only ago 2 is known to have the catalytic enzyme function [ 11 , 12 ] . there are different experimental and bioinformatics approaches to predict mirna targets . at a minimum , the precise matching to 3utr of mrna in multiple copies should be within the first 28 bases from the 5 end of the mature mirna , called the seed region [ 1720 ] . to date , over 2000 human mirnas have been annotated in the sanger mirbase ( release 18 , http://www.mirbase.org/cgi-bin/browse.pl?org=hsa ) . various mirnas have been shown to be involved in a myriad of cellular processes including differentiation , metabolism , apoptosis , and development . physiologically , and pathologically , mirnas have been reported to play roles in cancers , inflammatory responses , diabetes , and autoimmunity [ 23 , 24 ] . they are involved in a number of hematological malignancies such as acute lymphoblastic leukemia , acute myeloid leukemia , chronic lymphocytic leukemia , chronic myelogenous leukemia , diffuse large - b - cell lymphoma , and others . therefore , mirna profiling is critical in order to distinguish stem cells of the different origins , developmental stages , and genetic conditions . recent studies have demonstrated a causative role for mirnas in malignant diseases development in the hematopoietic system . on the other hand , tumor suppressor mirnas such as mir-15a/16 - 1 are found to be deleted in a subset of lymphomas and have been shown to cause chronic lymphocytic leukemia in mice [ 31 , 32 ] . in another study on human umbilical cord blood , two particular mirnas - hsa - mir-520h and hsa - mir-526b*- levels appeared to be elevated . studies have implicated important functions of mirna on hematopoietic development as well as innate and adaptive immune responses . toll - like receptor ( tlr ) signaling leads to transcriptional activation of a large class of proinflammatory cytokines as well as multiple mirnas . for example , mir-146a and mir-155 have been shown to be upregulated upon exposure to lps in the monocytic leukemia cell line thp-1 . this study for the first time profiled the mirnas alterations in tlr signaling and proposed the mirnas as negative regulators of tlr activation . to understand global mirnas ' importance in b and t development , studies were performed in which knocking out dicer at different stages of b and t development resulted in blockage of further differentiation [ 4143 ] . in addition , mir-155 has been found to be one of the most important mirnas in both b and t cells as well as antigen presentation by dendritic cells ( dcs ) and is required for normal germinal center ( gc ) response [ 44 , 45 ] , b - cell class switching , th1/th2 polarization , and treg development both in the thymus and peripheral . considering the importance of mirnas in the immune system raises the question whether or not there is direct link between mirnas abnormalities and immune disorders or autoimmune diseases . interestingly , the discovery in 2002 of gw bodies ( gwbs ) , where mirna - mrna reside for degradation was from serum from an autoimmune patient with motor and sensory neuropathy . subsequently , anti - gwb autoantibodies in the serum have been identified from patients with various autoimmune disorders , indicating an involvement of general mirna pathway and autoantibody production . dysregulated mirna expression has been associated with autoimmunity , for example , mir-146a was underexpressed in pbmc from sle patients when compared with healthy control . a recent study in murine models ( mrl - lpr , c57bl/6-lpr , and nzb / nzw f1 ) of sle using a combination of microarray and real - time rt - pcr approaches , dai et al . identified that mir-182 - 96 - 183 cluster , mir-31 , and mir-155 are among those consistently upregulated mirnas across different genetic background strains of mice . thus , unregulated mir-155 may be a link between inflammation and cancer via inducing a high proliferation rate resulting in increased mutations . results showed that mir-17 - 92 targets phosphatase and tensin homolog ( pten , tumor suppressor ) and bim ( proapoptotic molecule ) mrna directly resulting in lymphoproliferative and autoimmune diseases . in a current study from our lab on microrna abnormalities in nzb / nzw f1 lupus model by using type i and type iii interferons ( ifn- and ifn- ) as exogenous disease accelerators , we identified upregulation of several micrornas correlated to disease severity , yet not with the ifn treatment . mir-15a was one of the most significant elevated micrornas as autoimmunity developed in these mice and the level of splenic mir-15a was correlated to the level of anti - dsdna igg , in addition , the cellular level of mir-15a was also reflected in the plasma ( manuscript accepted for publication ) . multiple myeloma ( mm ) is characterized by a clonal expansion of plasma b cells in the bone marrow or in extramedullary sites which results in high levels of monoclonal immunoglobulins in the serum . studies investigating micrornas in mm began in 2007 with the discovery that interleukin 6 ( il-6 ) indirectly induces the transcription of mir-21 through signal transducer and activator of transcription 3 ( stat3 ) transcription in the human myeloma cells line . the same upstream enhancer controls mir-21 and stat3 transcription , and stat3 controls the transcription of survivin , bcl2 , and mcl-1 . a microrna microarray analysis in 20 myeloma samples revealed that mir-335 and mir-342 - 3p were upregulated and may be involved in plasma cell homing and other interactions in the bone marrow . for example , mir-106b-25 cluster , mir-181a / b , and mir-32 target a histone acetyltransferase , p300/cbp - associated factor ( pcaf ) that reversibly acetylates transcriptional regulators including p53 , thus accounting for the low levels of pcaf observed in mm cells . also mir-1792 downregulates bim , a proapoptotic molecule , and mir-19a / b target socs-1 , a silencer of the stat3 , thus enhancing the oncogenicity of mm cells [ 60 , 61 ] . as seen in cll , mir-15a/16 - 1 normally encoded within the dleu2 , a gene frequently deleted in lymphocytic leukemia , mir-15a/16 - 1 activity is central to the antiproliferative activity of dleu2 , as mir-15a/16 - 1 inhibits cyclind1 , cyclind2 , and cdc25a . for example , mir-25 and mir-30d are increased in mm and target the 3utr of the p53 gene . also , mm cells have low levels of mir-192 , mir-194 , and mir-215 which targets mdm2 , a p53 antagonist , thereby lowering p53 levels and increasing oncogenic potential . moreover , the promoter of mir-34b / c , a transcriptional target of p53 was found to be hypermethylated and thus inactivated in multiple myeloma cell lines . hypermethylation of the promoters of various tumor suppressors such as mir-124 - 1 ( a target of cdk6 ) , mir-203 ( a target of cyclic - responsive element - binding protein which increases proliferation ) , and mir-29b ( a target of mcl-1 which antagonizes il-6 ) increase the tumorigenicity of myeloma cells . extranodal marginal zone lymphomas are most associated with mucosal - associated lymphoid tissue ( malt ) and are characterized by clonal proliferation of plasma cells that produce the immunoglobulin a isotype . investigations involving micrornas have found the mir-203 promoter to be hypermethylated in samples of gastric lymphoma , and this microrna targets the c - abl1 oncogene , thus enabling tumor growth and proliferation . in addition , mir-150 and mir-155 were upregulated , while mir-184 , mir-205 and mir-200a / b / c ( which targets cyclin e2 ) were downregulated . in another hyperimmunoglobulin disorder , immunoglobulin a nephropathy ( igan ) is characterized by the deposition of immune complexes in the kidney mesenchyme causing renal injury and usually coincides with mucosal infections . these immune complexes are composed of iga1 molecules that are galactose - deficient , causing a conformational change in the molecule , and autoantibodies ( iga or igg ) form to its exposed epitopes . since mir-155 and mir-146 are involved in b lymphocyte development , their levels were examined in 43 igan biopsy specimens and urine samples . the results showed that mir-146 and mir-155 were high in igan biopsy and urine sediment , suggesting their role in igan pathogenesis . acute myeloid leukemia ( aml ) is a hematopoietic progenitor cell - originated malignant disorder affecting the myeloid lineage , which could be classified into subtypes based on the differentiation stages of the malignant cells found in peripheral blood and in bone marrow . other aml patients without detectable chromosomal abnormalities may display mutations or dysregulations in specific genes , a signature ubiquitously found in cancers [ 7779 ] . microrna signatures in aml have been sought , and many groups of researchers performed large - scale profiling of mirna expression in different populations of aml patients . in the first study where aml patient samples were compared to acute lymphoblastic leukemia ( all ) , both groups with similar chromosomal alterations , 27 mirnas were reported to be different between the two groups . importantly , mir-146a was inversely correlated to overall survival in both aml and all . by microarray profiling , 26 micrornas were downregulated in aml samples . in correlating cytogenetic abnormalities with mirna expression , 14 downregulated and 8 upregulated mirnas were associated with 11q23 translocation versus all other aml , including the downregulation of mir-196 and mir-15a , and overexpression of mir-21 in t(6 ; 11 ) with worse prognosis . in aml patients ( n = 36 ) achieving complete remission the levels of mir-15a/16 were upregulated . this study was the first to identify the distinct mirnas profile between aml patients and normal control , and the subsets of mirnas related to cytogenetic groups and disease outcome . almost at the same time , a study with 215 heterogeneous aml samples was performed to demonstrate the signatures of mirnas expression in cytogenetic and molecular subtypes . a group of upregulated mirnas were prominent in t(15;17 ) cases . in molecular subgroups of aml , nucleophosmin ( npm1 ) mutations , which represent the most common molecular abnormality in aml , are associated with overexpression of homeobox genes ( hox ) . upregulation of mir-10a , mir-10b , mir-196a and mir-196b , was identified in aml carrying npm1 mutations , and these mirnas were located within the hox genes . although mir-196a directly targets hoxb8 mrna , the upregulated mir-196a in this aml subgroups may represent a breakage in the regulation loop between mirnas and hox genes . consistent with other studies , mir-155 was significantly upregulated in amls with internal tandem duplications of flt3(flt3-itd ) , corroborating the oncogenic effect of mir-155 in myeloid cells in addition to such effects in lymphoid lineages [ 84 , 87 , 88 ] . in comparing aml to normal cd34 + cells , upregulation of mir-21 in amls was found , consistent with other studies and further strengthening the importance of mir-21 in aml [ 82 , 84 ] . interestingly , in an analysis of aml subgroups the t(8;21 ) and inv(16 ) were grouped together by mirna profile , supporting the notion that both subgroups belong to core - binding factor ( cbf ) amls , suggesting some common pathways shared by cbf - amls . overexpression of mir-224 , mir-368 , and mir-382 was restricted to the t(15;17 ) samples , while mir-17 - 92 cluster was overexpressed exclusively in mixed - lineage leukemia ( mll ) rearrangements . a recent study classified aml cases into favorable , moderate , and poor as the predicted outcome according to the karyotype . mir-181a high expression was suggested to be associated with better - risk groups suggesting a potential therapeutic approach involving manipulation of mir-181a level in aml patients . cll is characterized by the accumulation of malignant b-1 cells ( cd5cd19cd20cd23igm ) in peripheral lymphoid organs , bone marrow , and peripheral blood . cll is broadly classified into aggressive ( zap70-unmutated igh ) and indolent ( zap70-mutated igh ) . dysregulation of several micrornas like mir-15a/16 - 1 , mir-34 cluster , mir-155 , mir-29 , and mir-181b has been implicated in the pathogenesis of cll . decreased mir-15a/16 - 1 confers a growth advantage as these micrornas target key cell cycle regulatory and antiapoptotic proteins such as cyclin d1 and bcl2 [ 97 , 98 ] . it is interesting to note that nzb mice ( spontaneously occurring mouse model of cll ) also exhibit a 50% reduction in the level of mir-15a/16 - 1 , that is associated with a point mutation and deletion in the 3 flanking region of mir-16 - 1 . other micrornas are abnormal in cll including mir-29 and mir-181 , which target tcl1 , a gene that is highly elevated in aggressive cll . mir-29 expression is decreased in aggressive cll , while it is increased in indolent cll as compared to normal volunteers [ 100 , 101 ] . mir-34a / b / c is decreased in patients with 11q deletions . normally , upon transactivation by tp53 , mir-34 expression would result in decreased zap70 . the oncogenic potential of mir-155 is further supported by the development of b - cell malignancies in e-mmu - mir-155 transgenic mice . using a poorly understood mechanism , micrornas are secreted into body fluids such as serum and urine , and their levels can be used as noninvasive biomarkers for diagnosis and monitoring of cancer and various other diseases [ 106 , 107 ] . in this paper , a variety of blood disorders were discussed in terms of microrna abnormalities observed . one microrna family of interest stood out as a potential regulator of cell fate ( table 1 and figure 1 ) . the tight involvement of mir-15/107 in cell growth and cell fate control , and their upstream regulators , such as p53 and myc , which by themselves are important players in tumorigenesis [ 120 , 121 ] , revealed critical mechanisms for abnormalities in cancer development , including leukemias . indeed , all members from mir-15/107 group have been identified to be altered in various tumor cells [ 122125 ] . specifically , underexpression of mir-15a/16 as a result of deletion or mutation of mir-15a/16 loci has been linked to the pathogenesis of cll [ 96 , 99 , 118 ] , similarly in aml and mm , where the downregulation of mir-15a/16 was associated with the loss of control for malignant cells differentiation and proliferation [ 62 , 83 ] . in contrast , in sle which is characterized by elevated plasma cell differentiation contributing to increased autoantibody production [ 126 , 127 ] , splenic mir-15a was increased , and this was significantly correlated with autoantibody levels in lupus - like autoimmune mouse model ( manuscript accepted for publication ) , suggesting a role of mir-15a upregulation in cell cycle arrest in order for plasma cell differentiation . future directions may be directed toward stem cell transplantation for many of these blood disorders . in this sense , patient - specific autologous pluripotent stem cells generation would provide a great opportunity to combine gene therapy with autologous cell transplantation to treat different human conditions including hematological disorders such as aml . to this end , micrornas represent an attractive tool for both ips generation efficiency enhancement and gene targeting approaches . it is known that expression of embryonic stem ( es ) cell - specific micrornas such as mir-294 promotes ips cells induction from somatic cells . recently , it has even been demonstrated that the expression of mir-302/367 cluster can directly reprogram mouse and human somatic cells to a pluripotent stem cell state in the absence of the commonly used reprogramming factors . alternatively , inhibition of tissue - specific mirnas would also enhance ips generation , which has been confirmed by antisense silencing of a prodifferentiation let-7 mirna . another application of micrornas lies in promoting patient - specific ips differentiation towards the required cell lineage , for example hsc expansion . nevertheless , hsc expansion from ips cells by means of micrornas needs to be further developed . hopefully , the recently achieved success in the production of ips cells with the use of mirnas will pave the way for successful in vitro expansion of hscs with mirnas .

antibodies against phosphotyrosine , akt , phospho - akt ( ser ) , stress - activated protein kinase / jnk , phospho - stress - activated protein kinase / jnk ( thr / tyr ) , and phospho - gsk ( glycogen synthase kinase)-3 ( ser ) were purchased from cell signaling technology ( beverly , ma ) . antibodies against gsk-3 and phospho - c - jun were from santa cruz biotechnology , inc . insulin from porcine pancreas , sodium palmitate , sodium oleate , myriocin , n - acetyl - l - cysteine , rotenone , thenoyltrifluoroacetone , cyanide m - chlorophenylhydrazone , oxypurinol , etomoxir , and tunicamycin was obtained from sigma . dl--tocopherol and 2,7-dichlorofluorescin diacetate ( h2dcfda ) were from wako ( osaka , japan ) . a , h4iiec3 cells were incubated in the presence or absence of ffas ( palmitate ( pal ) or oleate ( ole ) ) and the jnk inhibitor sp600125 ( sp ) for 16 h. total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . b , the values from densitometry of four ( p - jnk ) independent experiments were normalized to the level of total jnk ( p - c - jun was not normalized ; n = 4 ) and expressed as the mean -fold increase over control s.e . * * , p < 0.01 versus control . , p < 0.01 versus palmitate treatment . a , h4iiec3 cells were incubated in the presence or absence of ffas ( palmitate ( pal ) or oleate ( ole ) ) and the jnk inhibitor sp600125 ( sp ) for 16 h. total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . b , the values from densitometry of four ( p - jnk ) independent experiments were normalized to the level of total jnk ( p - c - jun was not normalized ; n = 4 ) and expressed as the mean -fold increase over control s.e . * * , p < 0.01 versus control . , p < 0.01 versus palmitate treatment . cell culture and fatty acid treatment studies were performed in the rat hepatoma cell line h4iiec3 , purchased from the american type culture collection ( manassas , va ) . cells were cultured in dulbecco 's modified eagle 's medium ( invitrogen ) supplemented with 10% fetal bovine serum ( invitrogen ) , penicillin ( 100 units / ml ) , and streptomycin ( 0.1 mg / ml ; invitrogen ) . the cells were cultured at 37 c in a humidified atmosphere containing 5% co2 , with medium changes three times a week . all studies were conducted using 8090% confluent cells , which were treated with the indicated concentrations of ffas in the presence of 2% ffa - free bovine serum albumin ( sigma ) . hepatocytes , grown to 8090% confluence in 6-well plates , were treated with the indicated reagents for 16 h in dulbecco 's modified eagle 's medium . after treatment , the cells were stimulated with insulin ( 1 ng / ml ) for 15 min . then the cells were washed with ice - cold phosphate - buffered saline and lysed in buffer containing 20 mm tris - hcl ( ph 7.5 ) , 5 mm edta , 1% nonidet p-40 , 2 mm na3vo4 , 100 mm naf , and a protease inhibitor mixture ( sigma ) . after sonication with a bioruptor ( cosmo bio , tokyo , japan ) , the lysates were centrifuged to remove insoluble materials . the supernatants ( 10 g / lane ) were separated by sds - page and transferred onto polyvinylidene difluoride membranes ( millipore , billerica , ma ) . for detection of phosphotyrosine insulin receptor and phosphotyrosine irs-2 , the supernatants ( 400 gof protein ) were immunoprecipitated with a phosphotyrosine antibody and protein g beads for 2 h at 4 c before sds - page . the membranes were blocked in a buffer containing 5% nonfat milk , 50 mm tris ( ph 7.6 ) , 150 mm nacl , and 0.1% tween 20 ( tbs - t ) for 1 h at room temperature . they were then incubated with specific primary antibodies and subsequently with horseradish peroxidase - linked secondary antibodies . signals were detected with a chemiluminescence detection system ( ecl plus western blotting detection reagents ; ge healthcare ) . densitometric analysis was conducted directly on the blotted membrane , using a ccd camera system ( las-3000 mini ; fujifilm , tokyo , japan ) and scion image software . quantitative real time pcr total rna was extracted from cultured h4iiec3 hepatocytes using an rneasy mini kit ( qiagen , germantown , md ) , according to the manufacturer 's protocol . the cdna was synthesized from total rna ( 100 ng ) using random hexamer primers , n6 , and a high capacity cdna reverse transcription kit ( applied biosystems , foster city , ca ) . quantitative real time pcr was performed with an abi prism 7900ht ( applied biosystems ) . the set of specific primers and taqman probes in the present study was obtained from applied biosystems . the pcr conditions were one cycle at 50 c for 2 min and 95 c for 10 min , followed by 40 cycles at 95 c for 15 s and 60 c for 1 min . total rna was extracted from h4iiec3 hepatocytes , and cdna was synthesized as described above . the cdna was amplified with a pair of primers ( reverse 5-cca tgg gaa gat gtt ctg gg-3 and forward 5-aca cgc ttg ggg atg aat gc-3 ) corresponding to the rat xbp-1 cdna . the pcr conditions were initial denaturation at 94 c for 3 min , followed by 30 cycles of amplification ( 94 c for 30 s , 58 c for 30 s , 72 c for 30 s ) and a final extension at 72 c for 3 min . figure 3.effect of a jnk inhibitor on palmitate - induced alterations in insulin - stimulated phosphorylation of akt and gsk-3 in h4iiec3 hepatocytes . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and the jnk inhibitor sp600125 ( sp ) for 16 h prior to stimulation with insulin ( 1 ng / ml , 15 min ) . total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . b , the values from densitometry of four ( p - akt or p - gsk-3 ) independent experiments were normalized to the level of total akt or gsk-3 protein , respectively , and expressed as the mean -fold increase over control s.e . * * , p < 0.01 versus palmitate treatment . effect of a jnk inhibitor on palmitate - induced alterations in insulin - stimulated phosphorylation of akt and gsk-3 in h4iiec3 hepatocytes . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and the jnk inhibitor sp600125 ( sp ) for 16 h prior to stimulation with insulin ( 1 ng / ml , 15 min ) . total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . b , the values from densitometry of four ( p - akt or p - gsk-3 ) independent experiments were normalized to the level of total akt or gsk-3 protein , respectively , and expressed as the mean -fold increase over control s.e . * * , p < 0.01 versus palmitate treatment . measurement of intracellular ros the intracellular formation of ros was detected using the fluorescent probe h2dcfda , according to a published method ( 13 ) . briefly , h4iiec3 hepatocytes , grown to 7080% confluence in 96-well plates , were treated with the indicated reagents in dulbecco 's modified eagle 's medium for 8 h. after treatment , the cells were washed with phosphate - buffered saline , loaded with 10 m h2dcfda , and incubated for 30 min at 37 c . the fluorescence was analyzed using a plate reader ( fluoroskan ascent fl , thermolab systems , franklin , ma ) . the cellular concentration of proteins containing carbonyl groups ( those that react with 2,4-di - nitrophenylhydrazine to form the corresponding hydrazone ) was determined spectrophotometrically using a protein carbonyl assay kit ( cayman chemical , ann arbor , mi ) according to the manufacturer 's instructions and as described previously ( 14 ) . differences between two groups were assessed using unpaired , two - tailed t tests . data involving more than two groups were assessed by one - way analysis of variance . all calculations were performed with spss ( version 12.0 for windows ; spss , chicago , il ) . two long chain fatty acids were chosen for the study : palmitate , a c16:0 saturated fatty acid , and oleate , a c18:1 monounsaturated fatty acid . to examine whether ffas impaired insulin signal transduction in h4iiec3 hepatocytes , we assessed the effect of ffas on insulin - stimulated tyrosine phosphorylation of irs-2 and serine phosphorylation of akt and gsk-3 ( fig . 1 ) . incubation with 0.25 mm palmitate inhibited insulin - stimulated tyrosine phosphorylation of irs-2 by 40% in h4iiec3 cells . downstream of irs-2 , insulin - stimulated serine phosphorylation of akt and gsk-3 were also inhibited by 0.25 mm palmitate treatment , by 80 and 70% , respectively , indicating an insulin - resistant state . however , the protein levels of total irs-2 , akt , and gsk-3 were unaffected by palmitate . furthermore , we confirmed that palmitate , but not oleate , impaired insulin - stimulated akt serine phosphorylation in the human hepatoma cell line hepg2 ( supplemental fig . 1 ) . jnk activation by palmitate contributes to palmitate - induced insulin resistance jnk , a stress - activated protein kinase , has been reported to phosphorylate irs-1 and -2 at serine residues ( 15 , 16 ) . serine phosphorylation of irss impairs irs tyrosine phosphorylation , leading to a reduction in insulin receptor - mediated signaling . many studies have verified the role of jnk in fat - induced insulin resistance in several experimental systems ( 7 , 17 , 18 ) . thus , we next examined the effect of ffas on jnk activation and its involvement in insulin signaling . palmitate , but not oleate , dramatically increased phosphorylated jnk and c - jun ( fig . , sp600125 ( 19 ) , reversed the palmitate - induced phosphorylation of c - jun ( fig . 2 ) , suggesting that palmitate activated jnk . to test whether palmitate - induced jnk activation mediated cellular insulin resistance , we inhibited the jnk pathway with sp600125 . sp600125 dose - dependently improved insulin - stimulated serine phosphorylation of akt and gsk-3 in h4iiec3 hepatocytes exposed to palmitate ( fig . these results suggest that jnk activation by palmitate contributed to palmitate - induced insulin resistance . h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) or oleate ( ole ) for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . ( n = 4 ) . * * , p < 0.01 versus control . , p < 0.01 versus 0.25 mm palmitate treatment . h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) or oleate ( ole ) for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . pathways for srebp-1c and er stress are not involved in palmitate - induced jnk activation and insulin resistance in h4iiec3 hepatocytes the srebp-1c pathway has been reported to play a role in diet - induced insulin resistance in vivo . ( 6 ) found that high sucrose diet - induced hyperglycemia and hyperinsulinemia up - regulated hepatic expression of srebp-1c , leading to down - regulation of irs-2 at the transcriptional level . however , in the present study , palmitate dramatically down - regulated the expression of srebp-1c in h4iiec3 hepatocytes ( supplemental fig . thus , palmitate itself did not appear to cause insulin resistance in hepatocytes via the srebp-1c pathway . figure 5.effects of antioxidants on palmitate - induced intracellular ros production in h4iiec3 hepatocytes . h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and antioxidants for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . p < 0.05 versus palmitate treatment alone . * * , p < 0.01 versus palmitate treatment alone . h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and antioxidants for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . p < 0.05 versus palmitate treatment alone . * * , p < 0.01 versus palmitate treatment alone . er stress is induced in insulin - resistant states , such as obesity and type 2 diabetes , and in turn , this stress has been shown to lead to the inhibition of insulin signaling , through overactivation of jnk ( 9 ) . since excessive ffas have been shown to trigger er stress in pancreatic -cells ( 20 ) , we examined whether palmitate caused er stress in h4iiec3 hepatocytes . er stress induces the spliced form of xbp-1 ( xbp-1s ) , which up - regulates the transcription of molecular chaperones , including grp78 ( 78-kda glucose - regulated / binding immunoglobulin protein ) palmitate at 0.25 mm did not alter the expression level of grp78 mrna or the splicing pattern of xbp-1 , unlike tunicamycin , an agent commonly used to induce er stress ( supplemental fig . next , we compared the impact of palmitate and tunicamycin on insulin - stimulated signal transduction and jnk activation ( supplemental fig . the inhibitory effect of tunicamycin on insulin - stimulated serine phosphorylation of akt was mild and not significant compared with that of palmitate . additionally , the increment in phosphorylated jnk by tunicamycin was lower and not significant compared with that of palmitate . these results suggest that er stress played a minor role in palmitate - induced jnk activation and cellular insulin resistance in h4iiec3 hepatocytes . in addition to er stress , increased cellular ros levels are known to stimulate threonine phosphorylation of jnk ( 22 ) . indeed , ros levels are increased in clinical conditions associated with insulin resistance , such as sepsis , burn injuries , obesity , and type 2 diabetes ( 23 ) . furthermore , ffas have been reported to generate ros in various cells , such as pancreatic islet cells ( 24 ) , cardiac myocytes ( 25 ) , and adipocytes ( 23 ) . figure 6.effects of antioxidants on palmitate - induced alterations in insulin - stimulated serine phosphorylation of akt in h4iiec3 hepatocytes . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and antioxidants for 16 h prior to stimulation with insulin ( 1 ng / ml , 15 min ) . total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . b , the values from densitometry of four ( nac ) or five ( -tocopherol ) independent experiments were normalized to the level of total akt protein and expressed as the mean -fold increase over control s.e . * , p < 0.05 versus palmitate treatment . effects of antioxidants on palmitate - induced alterations in insulin - stimulated serine phosphorylation of akt in h4iiec3 hepatocytes . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and antioxidants for 16 h prior to stimulation with insulin ( 1 ng / ml , 15 min ) . total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . b , the values from densitometry of four ( nac ) or five ( -tocopherol ) independent experiments were normalized to the level of total akt protein and expressed as the mean -fold increase over control s.e . * , p < 0.05 versus palmitate treatment . thus , we hypothesized that palmitate increased intracellular ros production and thereby activated jnk , leading to the impaired insulin signaling . to evaluate this , h4iiec3 hepatocytes were incubated with h2dcfda , a fluorescent probe , to visualize intracellular ros , with or without palmitate . h2dcfda - associated fluorescence was elevated by 58% after incubation with 0.25 mm palmitate for 8 h , and palmitate induced more ros production than oleate ( fig . consistent with this , the amount of protein carbonyls , a marker of oxidative stress , significantly increased in palmitate - treated hepatocytes ( 4.6 0.5 nmol / mg protein ) , compared with control cells ( 3.1 0.4 nmol / mg protein ) . these results suggest that ffas , especially palmitate , can cause ros production and oxidative stress in h4iiec3 hepatocytes . we next sought to test whether palmitate - induced ros overproduction had a causal role in insulin resistance by assessing whether two antioxidant reagents , n - acetyl - l - cysteine ( nac ) and -tocopherol , could also act as insulin sensitizers . nac and -tocopherol dose - dependently suppressed palmitate - induced intracellular ros production ; nac at 10 mm and -tocopherol at 0.4 mm suppressed ros production by 50 and 60% , respectively ( fig . 5 ) . in parallel with decreased ros levels , the antioxidants recovered the insulin - stimulated akt phosphorylation impaired by palmitate ; nac at 10 mm and -tocopherol at 0.4 mm recovered the phosphorylation by 40 and 35% , respectively ( fig . 6 ) . furthermore , these antioxidants suppressed palmitate - induced jnk phosphorylation ; nac at 10 mm and -tocopherol at 0.4 mm suppressed it by 80 and 55% , respectively ( fig . these results suggest that palmitate increased ros levels in h4iiec3 hepatocytes and thereby activated jnk , resulting in insulin resistance . palmitate induces ros overproduction in mitochondria to define the source of ros induced by palmitate in h4iiec3 hepatocytes , we examined the cellular pathway involved in ros production , including nadph oxidase , xanthine oxidase , and mitochondria - mediated pathways . palmitate - induced ros production was markedly suppressed by rotenone , an inhibitor of mitochondrial respiratory chain complex i ; thenoyltrifluoroacetone , an inhibitor of mitochondrial respiratory chain complex ii ; and carbonyl cyanide m - chlorophenylhydrazone , an uncoupler of oxidative phosphorylation ( fig . in contrast , ros production in palmitate - treated h4iiec3 cells was not suppressed by apocynin , an inhibitor of nadph oxidase , or oxypurinol , an inhibitor of xanthine oxidase . these results suggest that the mitochondrial respiratory chain is involved in palmitate - induced ros overproduction in h4iiec3 hepatocytes . ffas are metabolized in the mitochondrial fatty acid -oxidation pathway , which supplies the mitochondrial respiratory chain with electrons . large amounts of electrons entering the respiratory chain may cause abnormal reduction of oxygen , leading to ros production . thus , we next examined whether palmitate - induced ros production was dependent on mitochondrial fatty acid -oxidation . cpt-1a ( carnitine palmitoyltransferase-1a ) is the rate - limiting enzyme in mitochondrial fatty acid -oxidation . as expected , etomoxir , a cpt-1 inhibitor , decreased palmitate - induced ros production , by 80% ( fig . palmitate , but not oleate , significantly increased expression of the cpt-1a gene ( fig . this up - regulation may contribute to palmitate - induced ros overproduction , because the accelerated -oxidation should cause excessive electron flux in the respiratory chain . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and antioxidants for 16 h. total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . b , the values from densitometry of four ( nac or-tocopherol ) independent experiments were normalized to the level of total jnk protein and expressed as the mean -fold increase over control s.e . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and antioxidants for 16 h. total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . b , the values from densitometry of four ( nac or-tocopherol ) independent experiments were normalized to the level of total jnk protein and expressed as the mean -fold increase over control s.e . * * , p < 0.01 versus palmitate treatment alone . in the present study , we investigated the direct action of fatty acids on insulin signaling in hepatocytes . the saturated fatty acid palmitate , but not the unsaturated fatty acid oleate , impaired insulin - induced tyrosine phosphorylation of irs-2 , serine phosphorylation of akt , and serine phosphorylation of gsk-3 , all of which are indicative of insulin resistance in cultured h4iiec3 hepatocytes ( fig . , the expression of the srebp-1c gene was down - regulated by adding palmitate to cultured h4iiec3 hepatocytes , which is likely a result of a negative feedback loop for fatty acid synthesis , and irs-2 protein levels were unaffected . ffa - induced insulin resistance has been reported in other insulin - sensitive cells , such as adipocytes ( 18 ) and skeletal muscle cells ( 26 ) . these studies , together with the present results , suggest that ffa inhibits insulin signaling at the level of tyrosine phosphorylation of irss , regardless of cell type . similar to the findings in 3t3-l1 adipocytes ( 18 ) and primary mouse hepatocytes and pancreatic -cells ( 16 ) , the activation of jnk , a known suppressor of the tyrosine phosphorylation of irss , was involved in ffa - induced tyrosine phosphorylation of irs-2 in cultured h4iiec3 hepatocytes . because a jnk inhibitor , sp600125 , largely restored palmitate - induced impairment of the insulin signaling pathway , jnk activation seems to play a major role in the development of palmitate - induced insulin resistance in h4iiec3 hepatocytes . our results support in vivo findings that jnk is activated in the liver of an animal model of obesity and diabetes in which ffa influx into the liver is elevated ( 9 , 27 ) . the overexpression of jnk in mouse liver resulted in hepatic insulin resistance at the level of irs tyrosine phosphorylation , and the overexpression of a dominant negative mutant of jnk in the liver accelerated hepatic insulin signaling ( 17 ) . given that jnk is activated by many types of cellular stresses ( 28 ) , we next searched for a link between palmitate treatment and jnk activation in h4iiec3 hepatocytes . er stress was unlikely to mediate palmitate - induced insulin resistance in h4iiec3 hepatocytes , because palmitate caused insulin resistance independent of er stress , whereas tunicamycin caused er stress without affecting insulin action . instead , we found that palmitate - induced ros generation mediated insulin resistance . ros are one of many factors suggested to have a possible role in insulin resistance ( 29 , 30 ) . ros include reactive products , such as superoxide anion , hydrogen peroxide , and hydroxyl radical , which are formed as by - products of mitochondrial oxidative phosphorylation ( oxphos ) . thus , as a rule , increased mitochondrial oxphos flux leads to increased formation of ros ( 31 , 32 ) . ros can also be produced during -oxidation of fatty acids , especially as a by - product of peroxisomal acyl - coa oxidase activity ( 32 ) . additionally , ros can be produced by dedicated enzymes , such as nadph oxidase ( 33 ) , present in phagocytic cells , where ros are an important part of cellular defense mechanisms . using specific inhibitors of subcellular ros , we identified mitochondrial oxphos as an important source of palmitate - induced ros generation in h4iiec3 hepatocytes . a final metabolite of fatty acids , acetyl - coa , is metabolized in the trichloroacetic acid cycle . in the processes of fatty acid -oxidation and the trichloroacetic acid cycle , nadh and fadh2 figure 8.effects of ros - producing pathway inhibitors on palmitate - induced ros production in h4iiec3 hepatocytes . h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and each ros - producing pathway inhibitor for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . ( n = 4 ) . * , p < 0.05 versus palmitate treatment alone . * * , p < 0.01 versus palmitate treatment alone . rot , rotenone ; apo , apocynin ; oxy , oxypurinol ; ttfa , thenoyltrifluoroacetone ; cccp , carbonyl cyanide m - chlorophenylhydrazone . effects of ros - producing pathway inhibitors on palmitate - induced ros production in h4iiec3 hepatocytes . h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and each ros - producing pathway inhibitor for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . ( n = 4 ) . * , p < 0.05 versus palmitate treatment alone . * * , p < 0.01 versus palmitate treatment alone . rot , rotenone ; apo , apocynin ; oxy , oxypurinol ; ttfa , thenoyltrifluoroacetone ; cccp , carbonyl cyanide m - chlorophenylhydrazone . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and the cpt-1 inhibitor etomoxir ( eto ) for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . b , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) or oleate ( ole ) for 16 h. total rna was extracted and subjected to reverse transcription . using the cdna as a template , the amounts of cpt-1 mrna were detected by real time pcr . the values were normalized to the level of 18 s ribosomal rna and expressed as mean -fold increase over control ( n = 3 ) . * , p < 0.05 versus control . * * , p < 0.01 versus palmitate treatment alone . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and the cpt-1 inhibitor etomoxir ( eto ) for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . b , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) or oleate ( ole ) for 16 h. total rna was extracted and subjected to reverse transcription . using the cdna as a template , the amounts of cpt-1 mrna were detected by real time pcr . the values were normalized to the level of 18 s ribosomal rna and expressed as mean -fold increase over control p < 0.05 versus control . * * , p < 0.01 versus palmitate treatment alone . nac , a scavenger of ros , dose - dependently restored glutathione in palmitate - treated cells ( supplemental fig . however , glutathione restoration by nac was unable to completely rescue palmitate - induced insulin resistance . furthermore , the combination of nac and -tocopherol did not completely reverse jnk activation ( supplemental fig . 6 , a and b ) and only partly rescued palmitate - induced insulin resistance ( supplemental fig . therefore , other mechanisms may also be involved in insulin resistance caused by palmitate . de novo ceramide synthesis is a potential pathway contributing to palmitate - induced jnk activation . ceramide derived from saturated fatty acids has been reported to activate jnk and inhibit insulin - induced akt phosphorylation in myocytes ( 3436 ) . in our investigation , palmitate increased the intracellular content of ceramide in h4iiec3 hepatocytes ( supplemental fig . unfortunately , even at the maximum myriosin concentration , the intracellular accumulation of ceramide was not blocked by myriosin , a potent inhibitor of serine palmitoyltransferase at the first step in ceramide biosynthesis ( supplemental fig . 8) . furthermore , ceramide accumulation was not blocked when myriosin was used in combination with fumonisin b1 , an inhibitor of ceramide synthase ( data not shown ) . therefore , we can not rule out the possibility that intracellular ceramide contributes to palmitate - induced insulin resistance in h4iiec3 hepatocytes . further studies are required to assess the role of the ceramide pathway in palmitate - induced insulin resistance in hepatocytes . proposed model for palmitate - induced hepatic insulin resistance . in the present study , etomoxir , an inhibitor of cpt-1 , decreased palmitate - induced intracellular ros production . additionally , palmitate , but not oleate , significantly increased the expression of the cpt-1a gene , which may account for the observed differences in insulin action between palmitate and oleate . recently , it was reported that fatty acid composition may be a determinant in insulin sensitivity ( 37 , 38 ) . in this regard , we investigated the effect of oleate on insulin signaling in palmitate - treated hepatocytes . surprisingly , oleate dose - dependently reversed palmitate - induced ros generation and jnk phosphorylation and rescued palmitate - induced phosphorylation of akt.3 further investigations aimed at elucidating the molecular basis underlying the differential roles and interactions of ffas are required . in conclusion , this study identified mitochondrial ros generation as a critical factor in palmitate - induced hepatic insulin resistance . palmitate may induce cpt-1 expression , accelerate metabolism , supply excess electrons for mitochondrial oxphos , and generate ros . ros then desensitize the insulin signaling pathway by activating jnk , impairing tyrosine phosphorylation of irs-2 , and causing hepatic insulin resistance ( fig . the results suggest that an initial event in high fat / sucrose diet - induced or obesity - induced insulin resistance in the liver is mitochondrial ros generation , which could potentially be a therapeutic target . in addition to previously suggested jnk inhibitors or antioxidants , mitochondrial uncouplers , such as cyanide m - chlorophenylhydrazone , may provide a candidate therapeutic strategy for this pathway by preventing ros generation .

visceral adiposity in obesity causes excessive free fatty acid ( ffa ) flux into the liver via the portal vein and may cause fatty liver disease and hepatic insulin resistance . however , because animal models of insulin resistance induced by lipid infusion or a high fat diet are complex and may be accompanied by alterations not restricted to the liver , it is difficult to determine the contribution of ffas to hepatic insulin resistance . therefore , we treated h4iiec3 cells , a rat hepatocyte cell line , with a monounsaturated fatty acid ( oleate ) and a saturated fatty acid ( palmitate ) to investigate the direct and initial effects of ffas on hepatocytes . we show that palmitate , but not oleate , inhibited insulin - stimulated tyrosine phosphorylation of insulin receptor substrate 2 and serine phosphorylation of akt , through c - jun nh2-terminal kinase ( jnk ) activation . among the well established stimuli for jnk activation , reactive oxygen species ( ros ) played a causal role in palmitate - induced jnk activation . in addition , etomoxir , an inhibitor of carnitine palmitoyltransferase-1 , which is the rate - limiting enzyme in mitochondrial fatty acid -oxidation , as well as inhibitors of the mitochondrial respiratory chain complex ( thenoyltrifluoroacetone and carbonyl cyanide m - chlorophenylhydrazone ) decreased palmitate - induced ros production . together , our findings in hepatocytes indicate that palmitate inhibited insulin signal transduction through jnk activation and that accelerated -oxidation of palmitate caused excess electron flux in the mitochondrial respiratory chain , resulting in increased ros generation . thus , mitochondria - derived ros induced by palmitate may be major contributors to jnk activation and cellular insulin resistance .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

insulin from porcine pancreas , sodium palmitate , sodium oleate , myriocin , n - acetyl - l - cysteine , rotenone , thenoyltrifluoroacetone , cyanide m - chlorophenylhydrazone , oxypurinol , etomoxir , and tunicamycin was obtained from sigma . a , h4iiec3 cells were incubated in the presence or absence of ffas ( palmitate ( pal ) or oleate ( ole ) ) and the jnk inhibitor sp600125 ( sp ) for 16 h. total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . b , the values from densitometry of four ( p - jnk ) independent experiments were normalized to the level of total jnk ( p - c - jun was not normalized ; n = 4 ) and expressed as the mean -fold increase over control s.e . a , h4iiec3 cells were incubated in the presence or absence of ffas ( palmitate ( pal ) or oleate ( ole ) ) and the jnk inhibitor sp600125 ( sp ) for 16 h. total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . b , the values from densitometry of four ( p - jnk ) independent experiments were normalized to the level of total jnk ( p - c - jun was not normalized ; n = 4 ) and expressed as the mean -fold increase over control s.e . all studies were conducted using 8090% confluent cells , which were treated with the indicated concentrations of ffas in the presence of 2% ffa - free bovine serum albumin ( sigma ) . figure 3.effect of a jnk inhibitor on palmitate - induced alterations in insulin - stimulated phosphorylation of akt and gsk-3 in h4iiec3 hepatocytes . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and the jnk inhibitor sp600125 ( sp ) for 16 h prior to stimulation with insulin ( 1 ng / ml , 15 min ) . effect of a jnk inhibitor on palmitate - induced alterations in insulin - stimulated phosphorylation of akt and gsk-3 in h4iiec3 hepatocytes . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and the jnk inhibitor sp600125 ( sp ) for 16 h prior to stimulation with insulin ( 1 ng / ml , 15 min ) . two long chain fatty acids were chosen for the study : palmitate , a c16:0 saturated fatty acid , and oleate , a c18:1 monounsaturated fatty acid . to examine whether ffas impaired insulin signal transduction in h4iiec3 hepatocytes , we assessed the effect of ffas on insulin - stimulated tyrosine phosphorylation of irs-2 and serine phosphorylation of akt and gsk-3 ( fig . incubation with 0.25 mm palmitate inhibited insulin - stimulated tyrosine phosphorylation of irs-2 by 40% in h4iiec3 cells . downstream of irs-2 , insulin - stimulated serine phosphorylation of akt and gsk-3 were also inhibited by 0.25 mm palmitate treatment , by 80 and 70% , respectively , indicating an insulin - resistant state . furthermore , we confirmed that palmitate , but not oleate , impaired insulin - stimulated akt serine phosphorylation in the human hepatoma cell line hepg2 ( supplemental fig . jnk activation by palmitate contributes to palmitate - induced insulin resistance jnk , a stress - activated protein kinase , has been reported to phosphorylate irs-1 and -2 at serine residues ( 15 , 16 ) . serine phosphorylation of irss impairs irs tyrosine phosphorylation , leading to a reduction in insulin receptor - mediated signaling . thus , we next examined the effect of ffas on jnk activation and its involvement in insulin signaling . palmitate , but not oleate , dramatically increased phosphorylated jnk and c - jun ( fig . , sp600125 ( 19 ) , reversed the palmitate - induced phosphorylation of c - jun ( fig . to test whether palmitate - induced jnk activation mediated cellular insulin resistance , we inhibited the jnk pathway with sp600125 . sp600125 dose - dependently improved insulin - stimulated serine phosphorylation of akt and gsk-3 in h4iiec3 hepatocytes exposed to palmitate ( fig . these results suggest that jnk activation by palmitate contributed to palmitate - induced insulin resistance . h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) or oleate ( ole ) for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . pathways for srebp-1c and er stress are not involved in palmitate - induced jnk activation and insulin resistance in h4iiec3 hepatocytes the srebp-1c pathway has been reported to play a role in diet - induced insulin resistance in vivo . thus , palmitate itself did not appear to cause insulin resistance in hepatocytes via the srebp-1c pathway . figure 5.effects of antioxidants on palmitate - induced intracellular ros production in h4iiec3 hepatocytes . h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and antioxidants for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and antioxidants for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . er stress is induced in insulin - resistant states , such as obesity and type 2 diabetes , and in turn , this stress has been shown to lead to the inhibition of insulin signaling , through overactivation of jnk ( 9 ) . next , we compared the impact of palmitate and tunicamycin on insulin - stimulated signal transduction and jnk activation ( supplemental fig . the inhibitory effect of tunicamycin on insulin - stimulated serine phosphorylation of akt was mild and not significant compared with that of palmitate . these results suggest that er stress played a minor role in palmitate - induced jnk activation and cellular insulin resistance in h4iiec3 hepatocytes . figure 6.effects of antioxidants on palmitate - induced alterations in insulin - stimulated serine phosphorylation of akt in h4iiec3 hepatocytes . effects of antioxidants on palmitate - induced alterations in insulin - stimulated serine phosphorylation of akt in h4iiec3 hepatocytes . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and antioxidants for 16 h prior to stimulation with insulin ( 1 ng / ml , 15 min ) . thus , we hypothesized that palmitate increased intracellular ros production and thereby activated jnk , leading to the impaired insulin signaling . we next sought to test whether palmitate - induced ros overproduction had a causal role in insulin resistance by assessing whether two antioxidant reagents , n - acetyl - l - cysteine ( nac ) and -tocopherol , could also act as insulin sensitizers . these results suggest that palmitate increased ros levels in h4iiec3 hepatocytes and thereby activated jnk , resulting in insulin resistance . palmitate induces ros overproduction in mitochondria to define the source of ros induced by palmitate in h4iiec3 hepatocytes , we examined the cellular pathway involved in ros production , including nadph oxidase , xanthine oxidase , and mitochondria - mediated pathways . palmitate - induced ros production was markedly suppressed by rotenone , an inhibitor of mitochondrial respiratory chain complex i ; thenoyltrifluoroacetone , an inhibitor of mitochondrial respiratory chain complex ii ; and carbonyl cyanide m - chlorophenylhydrazone , an uncoupler of oxidative phosphorylation ( fig . in contrast , ros production in palmitate - treated h4iiec3 cells was not suppressed by apocynin , an inhibitor of nadph oxidase , or oxypurinol , an inhibitor of xanthine oxidase . these results suggest that the mitochondrial respiratory chain is involved in palmitate - induced ros overproduction in h4iiec3 hepatocytes . ffas are metabolized in the mitochondrial fatty acid -oxidation pathway , which supplies the mitochondrial respiratory chain with electrons . thus , we next examined whether palmitate - induced ros production was dependent on mitochondrial fatty acid -oxidation . cpt-1a ( carnitine palmitoyltransferase-1a ) is the rate - limiting enzyme in mitochondrial fatty acid -oxidation . as expected , etomoxir , a cpt-1 inhibitor , decreased palmitate - induced ros production , by 80% ( fig . palmitate , but not oleate , significantly increased expression of the cpt-1a gene ( fig . this up - regulation may contribute to palmitate - induced ros overproduction , because the accelerated -oxidation should cause excessive electron flux in the respiratory chain . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and antioxidants for 16 h. total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and antioxidants for 16 h. total cell lysates were resolved by sds - page , transferred to a pvdf membrane , and immunoblotted with the indicated antibodies . the saturated fatty acid palmitate , but not the unsaturated fatty acid oleate , impaired insulin - induced tyrosine phosphorylation of irs-2 , serine phosphorylation of akt , and serine phosphorylation of gsk-3 , all of which are indicative of insulin resistance in cultured h4iiec3 hepatocytes ( fig . , the expression of the srebp-1c gene was down - regulated by adding palmitate to cultured h4iiec3 hepatocytes , which is likely a result of a negative feedback loop for fatty acid synthesis , and irs-2 protein levels were unaffected . ffa - induced insulin resistance has been reported in other insulin - sensitive cells , such as adipocytes ( 18 ) and skeletal muscle cells ( 26 ) . similar to the findings in 3t3-l1 adipocytes ( 18 ) and primary mouse hepatocytes and pancreatic -cells ( 16 ) , the activation of jnk , a known suppressor of the tyrosine phosphorylation of irss , was involved in ffa - induced tyrosine phosphorylation of irs-2 in cultured h4iiec3 hepatocytes . because a jnk inhibitor , sp600125 , largely restored palmitate - induced impairment of the insulin signaling pathway , jnk activation seems to play a major role in the development of palmitate - induced insulin resistance in h4iiec3 hepatocytes . the overexpression of jnk in mouse liver resulted in hepatic insulin resistance at the level of irs tyrosine phosphorylation , and the overexpression of a dominant negative mutant of jnk in the liver accelerated hepatic insulin signaling ( 17 ) . er stress was unlikely to mediate palmitate - induced insulin resistance in h4iiec3 hepatocytes , because palmitate caused insulin resistance independent of er stress , whereas tunicamycin caused er stress without affecting insulin action . instead , we found that palmitate - induced ros generation mediated insulin resistance . using specific inhibitors of subcellular ros , we identified mitochondrial oxphos as an important source of palmitate - induced ros generation in h4iiec3 hepatocytes . in the processes of fatty acid -oxidation and the trichloroacetic acid cycle , nadh and fadh2 figure 8.effects of ros - producing pathway inhibitors on palmitate - induced ros production in h4iiec3 hepatocytes . h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and each ros - producing pathway inhibitor for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . rot , rotenone ; apo , apocynin ; oxy , oxypurinol ; ttfa , thenoyltrifluoroacetone ; cccp , carbonyl cyanide m - chlorophenylhydrazone . effects of ros - producing pathway inhibitors on palmitate - induced ros production in h4iiec3 hepatocytes . h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and each ros - producing pathway inhibitor for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . rot , rotenone ; apo , apocynin ; oxy , oxypurinol ; ttfa , thenoyltrifluoroacetone ; cccp , carbonyl cyanide m - chlorophenylhydrazone . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and the cpt-1 inhibitor etomoxir ( eto ) for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . a , h4iiec3 cells were incubated in the presence or absence of palmitate ( pal ) and the cpt-1 inhibitor etomoxir ( eto ) for 8 h. intracellular ros production was quantified using the fluorescent probe h2dcfda . however , glutathione restoration by nac was unable to completely rescue palmitate - induced insulin resistance . 6 , a and b ) and only partly rescued palmitate - induced insulin resistance ( supplemental fig . de novo ceramide synthesis is a potential pathway contributing to palmitate - induced jnk activation . therefore , we can not rule out the possibility that intracellular ceramide contributes to palmitate - induced insulin resistance in h4iiec3 hepatocytes . further studies are required to assess the role of the ceramide pathway in palmitate - induced insulin resistance in hepatocytes . in the present study , etomoxir , an inhibitor of cpt-1 , decreased palmitate - induced intracellular ros production . additionally , palmitate , but not oleate , significantly increased the expression of the cpt-1a gene , which may account for the observed differences in insulin action between palmitate and oleate . surprisingly , oleate dose - dependently reversed palmitate - induced ros generation and jnk phosphorylation and rescued palmitate - induced phosphorylation of akt.3 further investigations aimed at elucidating the molecular basis underlying the differential roles and interactions of ffas are required . in conclusion , this study identified mitochondrial ros generation as a critical factor in palmitate - induced hepatic insulin resistance . ros then desensitize the insulin signaling pathway by activating jnk , impairing tyrosine phosphorylation of irs-2 , and causing hepatic insulin resistance ( fig . the results suggest that an initial event in high fat / sucrose diet - induced or obesity - induced insulin resistance in the liver is mitochondrial ros generation , which could potentially be a therapeutic target . in addition to previously suggested jnk inhibitors or antioxidants , mitochondrial uncouplers , such as cyanide m - chlorophenylhydrazone , may provide a candidate therapeutic strategy for this pathway by preventing ros generation .

biogeochemical processes occurring in soil environments such as decomposition and mineralization of organic matter ( om ) significantly affect nutrient cycling , subsequently influencing the climate and the biosphere . moreover , soil is an important habitat for soil microbes and animals , and serves as physical and cultural environment for humankind ( blum , busing and montanarella 2004 ) . soil microbes are major drivers of biogeochemical cycles and are a considerable pool of belowground terrestrial biomass . every gram of soil harbors thousands of bacterial , archaeal and eukaryotic taxa , and this taxonomic diversity is mirrored by the diversity of their physiologies , life styles ( i.e. oligotrophy - copiotrophy ) and associated functional classes of proteins ( fierer et al . microbial diversity is highly variable in terrestrial ecosystems , depending on many factors , such as plant cover , animal activity , soil moisture , temperature , aeration , porosity , nutrient availability , ph and salinity ( kennedy et al . 2004 ; maron , mougel and ranjard 2011 ; van horn et al . when comparing a broad range of soil ecosystem types acidobacteria and verrucomicrobia turned out to be the most abundant taxonomic groups followed by actinobacteria , bacteroidetes , planctomycetes and archaea ( barbern et al . these groups vary across different biomes e.g. actinobacteria , bacteroidetes and cyanobacteria phyla dominate in desert soils ( fierer et al . 2012 ) , while arctic permafrost peatland soils were dominated by actinobacteria , verrucomicrobia and bacteroidetes ( tveit et al . this phylogenetic information enables the determination of changes in ecological life styles in response to treatments , as has been shown for n gradients ( fierer et al . a variety of genes expressed for plant degradation were comparable among climatic zones , including arctic permafrost peatland soil and temperate and subtropical soils ( tveit et al . 2013 ) , displaying similar metabolic potential . however , n - fertilization resulted in increased gene abundances for dna / rna replication , electron transport and protein metabolism ( fierer et al . 2012 ) , while desert microbial communities are characterized by a high abundance of genes associated with osmoregulation and dormancy , and genes associated with nutrient cycling and catabolism of plant - derived organic compounds are less abundant ( fierer et al . 2012 ) . however , to which extent these genes are actually expressed and hence become physiologically active has yet to be determined . notably , changes in microbial composition might be of minor relevance for soil ecosystem functions , due to functional redundancy ( souza et al . metagenome information thus represents only the functional potential and giving no indication of the relative activity of the phyla present . therefore , to assess function and potentially link biodiversity and ecosystem functioning , it is of upmost importance to not only measure gene abundance , but also the actual expression and activity of functional proteins ( prosser 2015 ; delgado - baquerizo et al . provided the number of studies that have successfully applied metaproteomics on soil and leaf litter environments continues to grow , including e.g. metaproteome analysis of permafrost soil ( hultman et al . 2015a ) , soil restoration and ecosystem processes ( bastida et al . 2015b ) and a recent study that focused on the active microbial players in short - term degradation of plant - derived n ( starke et al . the latter is a novel protein stable isotope probing ( sip ) approach applying isotopic - n labeled plant material in a metaproteomics experiment , to track n from plants into microbes . a bacterial dominated short - term assimilation of plant - derived n was shown , and oligotrophic and copitrophic life styles of soil organisms in terms of temporal leaf litter n utilization patterns illustrate a new cutting edge approach to determine ecological attributes of soil microbes ( starke et al . 2016 ) . due to its large potential for providing a link between functional and phylogenetic information of soil microbial communities , as exemplified by the aforementioned studies , there has been growing interest in the application of metaproteomics in soil ecology to study microbially driven ecosystem functions ( e.g. methanogensis in permafrost soils ; hultman et al . however , soil metaproteomics still faces several challenges , including the heterogeneity of soil matrices , high microbial diversity , the ecosystem - specific dominance of few microbial species and limited metagenomic information and data handling ( keller and hettich 2009 ; schneider and riedel 2010 ; siggins , gunnigle and abram 2012 ; becher et al . protein extraction of soils is often difficult due to the presence of other organic compounds , such as complex carbohydrates , lipids and phenolic compounds ( e.g. lignin ) , and humic substances ( hs ) as well as inorganic compounds from the soil matrix , such as silt and clay minerals . coextraction of hs , which are contained in litter and soil , as well as the presence of large reactive surfaces of soil minerals ( e.g. clay ) not only complicate protein extraction but also interfere with the separation of peptides ( bastida et al . 2014 ) and quantification ( criquet , farnet and ferre 2002 ; ogunseitan 2006 ) due to protein modifications . these limitations for extraction are due to the fact that proteins can be adsorbed , linked anchored or embedded on / to / in solid particles such as clay , clay minerals , and soil om organo - mineral complexes ( nielsen , calamai and pietramellara 2006 ; tomaszewski , schwarzenbach and sander 2011 ) , which thereby reduce extraction efficiency ( sander , tomaszewski and schwarzenbach 2011 ) . adsorption of proteins to clays is a rapid process , which is only partly reversible ( nielsen , calamai and pietramellara 2006 ) , and is based on the large specific surface area of clay minerals ( giagnoni et al . 2011 ) . while it was shown that even whole cells can be sorbed to mineral surfaces ( i.e. clays ) , which depend on the ph , the charge of the clay mineral and the mg concentration ( jiang et al . 2007 ) . however , the adhesion of cells to soil particles is governed by their surface charges and global hydrophobic and hydrophilic characteristics ( doyle 2000 ) . hs and proteins are bound reversibly by a cation exchange process , which depends on the cation exchange capacity ( cec ) of the soil , the amino acid composition and the isoelectric point of the target proteins . moreover , protein polarity may affect sorption in aqueous solution through hydrophobic interactions ( norde , tan and koopal 2008 ) , though hydrophobic surfaces may reduce proteins sorption in soils ( keiblinger et al . reduced protein availability through clay - enzyme complexes has been shown for artificial soil mixtures with high cec or clay content by lower numbers of protein spots ( giagnoni et al . 2011 ) . to this end , the choice of purification methods or the extraction buffer and additives to it depends not only on the soil type but also on the goal of the investigation . ( i ) sample handling ( including obtaining a representative sample , homogenization , pooling and storage conditions , fig . 1b ) , ( iii ) processing of soil protein extracts ( including removal of interfering substances , pre - fractionation of proteins or peptides and mass spectrometry ( ms ) analysis ( fig . 1c ) , ( iv ) data analysis ( including spectra handling and database assembly for peptide and protein identification , fig . all steps are crucial for obtaining , holding and sharing high - quality soil metaproteome data , and some of these steps have recently been reviewed in detail ( keller and hettich 2009 ; schneider and riedel 2010 ; siggins , gunnigle and abram 2012 ; becher et al . we focus on differences in sample preparation and published protocols ( table 1 ) and try to synthesize knowledge to provide a step - by - step guideline of how to best proceed in soil and leaf litter protein extraction ( fig . , the current work presents recent advances in data analysis and data interpretation using novel bioinformatic tools ( fig . the wider objective of the present work is to ( i ) highlight the need for standardized methodology , which would ensure better comparability of future soil metaproteomic analyses , and to ( ii ) provide the basis for future meta - analysis by including additional environmental parameters and different ecosystem properties into metaproteome datasets . from sampling to data . researchers are confronted with various sampling methods and procedures that have to be carefully selected and combined for ( a ) sample preparation , including soil sampling homogenization and storage , ( b ) protein isolation and ( c ) shotgun proteomics ( from top to down ) . researchers can select or combine various methods for ( a ) data analysis , and ( b ) data interpretation ( from top to down ) . consecutive steps are connected by lines ( dashed lines represent workflows not suitable for high - throughput analyses ) . as soils of the globe are multifaceted , they are classified into groups based on their soil morphology , behavior or genesis in soil science . due to their varying characteristics in multiple scales , a case - by - case evaluation of sample handling as well as protein extraction strategies ( see also fig . 1 ) are necessary for proper metaproteomics experiments , to ensure that the material extracted from the particular soil and/or site is representative for the entire soil community . small differences in sample handling and preparation can introduce variability and may thereby dramatically alter the recovered species abundance and diversity to the measured data ( rubin et al . to minimize artificially introduced variability , sample handling and preparation should involve as few steps as possible . in the following paragraphs , we will guide the reader step by step from the soil sampling to the analysis of metaproteomic data . we believe that meta - omics studies of soil ecosystems should also provide contextual data such as soil ph , organic carbon ( corg ) , n - content , sampling depth , soil texture and cec ( for soils ) ( table 1 ) . as for instance these parameters might help to evaluate the potential of extracellular enzymes and , moreover , intracellular proteins that are released from the inner cells during extraction attaching to hs and mineral surfaces ( for more details , see also section sample matrix interference of hs and physico - chemical parameters ) . in addition , information on the study site including latitude and longitude , altitude , climate including mean annual temperature and precipitation , nutrient concentrations and bedrock material should be provided . usually basic soil / environmental parameters obtained in a study are highly dependent on the hypotheses and the experimental design . however , for choosing an appropriate protein extraction , protocol knowledge on the before mentioned parameters ( partly displayed in table 1 ) is needed . as with any technique , metaproteomics per se hence , we need to implement additional data i.e. chemical background , soil history , microbial biomass and enzyme activities , to provide the basis to unravel the major biotic and abiotic drivers of the active abundant communities , not only for individual experiments but also for future , cross - biome meta - analysis . the spatial and temporal heterogeneity of the soil matrix need to be considered by obtaining a representative sample of the natural situation for metaproteome analysis . so far , analysis of replicates in soil metaproteomic studies has been hampered by large costs and time - consuming analysis , resulting in numerous studies based on only one or few replicates ( myrold , zeglin and jansson 2014 ) . as analysis costs per sample will drop , future studies should employ well - established soil sampling strategies and a larger number of biological and technical replicates . however , without giving any further details , such strategies might include sampling time , sample amount , sampling device , stratified sampling ( horizontal and vertical distribution ) , composite samples ( pooling ) when appropriate ( pettitt and mcbratney 1993 ) and/or apply a replicated sampling design ( for details , see boeddinghaus et al . the individual sampling design is , however , dependent on the ecosystem type and the research objective . soils are also strongly stratified horizontally with one or more organic horizons on top of mineral horizons , depending on soil type . these layers generally harbor the highest abundance of microbes and are also more prone to fluctuations in temperature and moisture compared to subsoil . most metaproteomic studies thus focus on top soil horizons ( 015 cm ; see table 1 ) . apart from the spatial variability , it is necessary to evaluate the seasonal impact or temporal variation , as environmental conditions such as aeration , nutrient diffusion and redox potential can vary strongly over time . while field conditions by definition include seasonal variation in a specific environment , these fluctuations can be reduced or controlled by changing only a few parameters in laboratory studies as has been demonstrated for soil ( bastida et al . 2012b ; starke et al . given the spatio - temporal variability of climatic and pedologic characteristics in the field scale that shape the active soil microbial community , we highlight the importance of measuring these covariables in metaproteomic studies as already mentioned before . samples for soil metaproteome analysis are routinely sieved ( < 2 mm , see table 1 ) to homogenize the sample and minimize contamination with plant and animal protein ( fig . high clay and/or moisture content , however , can inhibit effective sieving in which case removal of visible organic debris and sample homogenization has to be done manually . several studies investigated the effect of storage conditions ( mainly freezing and drying ) on microbial parameters ( lee et al . 2007 ; results suggest that responses to storage are strongly soil dependent ( bandick and dick 1999 ) and seems to become more critical with increasing organic matter ( om ) content ( lee et al . 2007 ; 1a ) are summarized in table 1 , including air - drying , freeze - drying , freezing as well as deep freezing at 80c and storage in rna later . unfortunately , om content and texture of the soils processed are not always given ( table 1 ) hampering systematic investigations of storage conditions on soil metaproteomes . processing fresh samples whenever possible or storage at 80c is recommended to minimize the activity of naturally occurring proteases to avoid detrimental effects on protein abundance of environmental samples . ( 2015 ) , who suggest active gene expression and translation even in permafrost soil where proteins can be preserved for long periods under subzero conditions . however , a detailed comparison of the influence of storage conditions in terms of temperature and time on the stability and activity of soil proteins is urgently needed . an optimal protein extraction protocol contains at least three important steps : ( i ) quantitative extraction of proteins from the environmental matrix ( including steps for cell lysis , choice of buffer for solubilization and chemical reduction ) , ( ii ) protein purification ( i.e. to remove lysed cellular debris , residual sample matrix , interfering chemical substances ) and ( iii ) protein concentration ( fig . although a universal extraction protocol that provides good protein yields from wide range of soils would be desirable , this goal might be therefore , several protein extraction methods have been developed for specific research questions ( wang et al . some of these have been optimized and compared regarding their efficiency by our group ( keiblinger et al . several studies aimed at extracting the entire protein complement of an environmental sample by employing different strategies such as ( i ) indirect extraction , where microbes become enriched prior to extraction ( see table 1 , i.e # 9 , 10 ) , ( ii ) separation by means of density gradient centrifugation ( dgc ) prior to protein extraction ( to separate microorganisms from the environmental matrix , table 1 , i.e. # 2 , 7 ) and ( iii ) direct extraction ( lysis in the environmental matrix , table 1 , i.e. # 1 , 3 , 4 ) . the first two options reduce or eliminate problems that derive from interfering substances such as hs or mineral surfaces ( bastida et al . 2013 ) , which can reduce extraction efficiency ( sander , tomaszewski and schwarzenbach 2011 ) but are confined by ( i ) focusing only on the cultivable fraction or ( ii ) strongly biased extractions ( bastida et al . however , direct extraction might lead to a more comprehensive protein recovery from bacteria , fungi , protozoa and multicellular organisms ( wohlbrand , trautwein and rabus 2013 ) . 1b ) , which is obtained via ( i ) physical / mechanical lysis including heat , pressure ( french press , sonication or bead milling using glass beads ) ( mueller and pan 2013 ) , snap - freezing and grinding in liquid nitrogen with mortar and pistil ; freeze - thaw cycles , ( ii ) chemical lysis ( using detergents and stabilizing agents ; mueller and pan 2013 ) ; or ( iii ) enzymatic lysis that involves lysozyme cleavage of gycosidic bondages . for the choice of cell lysis method , physical cell rupture is usually more effective for gram - negative bacteria , due to their thinner peptidoglycan layer compared to gram - positive bacteria ( bakken and frostegrd 2006 ) . fungal lysis in soils samples can be obtained by bead beating or grinding in liquid n2 resulting in similar recoveries ( van elsas et al . however , grinding is laborious ; it might be also inefficient for sandy soils , as it is not possible to pulverize them with mortar and pistil . to this end , grinding seems to be most applicable for plant material , leaf litter and soils with high humic and low sand content or compost . among physical procedures , sonication is a commonly used method for protein extraction from soils , as it favors the solubilization of stabilized proteins , and also breaks soil aggregates ( nannipieri 2006 ; ogunseitan 2006 ) . chemical methods use lysis buffers for cell disruption they include either ionic detergents or non - ionic detergents . among ioinic detergents , anionic such as sodium dodecyl sulfate ( sds ) or cationic such as ethylenediaminetetraacetic acid ( edta ) or zwitter ionic reagents such as chaps ( 3-((3-cholamidopropyl ) dimethylammonio)-1-propanesulfonate ) are applied to dissolve cell membranes to release proteins . on the other hand , non - ionic detergents ( i.e. triton x-100 , nonylphenoxypolyethoxyethanol ( np-40 ) ) offer the advantage that proteins are not denatured , by still solubilizing membrane proteins . although detergents such as edta also inhibit polyphenol oxidases and metalloproteases , by building complexes with metal ions , -mercaptoethanol is often added to soil protein extraction buffers as a reducing agent , as it prevents oxidation of proteins . alternatively , enzymes can either be used alone or in combination with chemicals and/or physical means to lyse cells ( gianfreda and rao 2014 ) . a combination of mild mechanical methods ( i.e. sonication ) in detergents ( i.e. sds ) with other additives , such as enzymes and/or protease inhibitors cocktails , is a good strategy for direct cell lysis in soil samples , depending on the target cells and soil type and further downstream processing . basic knowledge of soil and environmental characteristics might aid the choice of an extraction procedure appropriate for the research question . thus , it will be at least possible to evaluate which challenges during protein extraction can be expected ( such as high humus content or clay - rich soils with high cec ) and to adopt existing protocols that provided promising results on similar soils , in comparable habitats . however , these parameters should not be taken individually , as clay and om are often well related with hs because clays retard the decomposition of om ( nannipieri 2006 ) . together with the aforementioned cell lysis , the extraction buffer should often meet the requirements for the removal of hs and/or to target stabilized proteins . specifically , salt solutions ( i.e. cacl2 ) of inorganic divalent cations ( 10100 mm ) have been used to release naturally immobilized proteins from hs by desorption ( criquet , farnet and ferre 2002 ) from hs . the extraction buffer often contains polyvinylpolypyrrolidone ( pvpp ) and hexadecyltrimethylammonium bromide ( ctab ) because they form complexes with humic acids . stabilized enzymes are efficiently extracted with buffers at slightly alkaline conditions ( nannipieri 2006 ) . this illustrates the importance of the ph of the soil and the extraction buffer , as it governs sorption of proteins to minerals and removal of interfering substances , and it also influences protein structure ( bastida et al . the ph of the extraction buffer has a strong influence on cell extraction , and considerably increases with ph in the range from 5 to 8 ( bakken and frostegrd 2006 ) . therefore , for direct extraction of soils , a ph of 7 or somewhat higher should result in sufficient amount of cells . to achieve naoh ( benndorf et al . 2007 ) or alkaline pyrophosphate ( masciandaro et al . alternatively , a subsequent phenolic extraction protocol has been used ( wang et al . 2006 ; benndorf et al . 2007 ; chen , rillig and wang 2009 ; keiblinger et al . this phenol including extraction preferentially dissolves nucleic acids , carbohydrates and cell debris in the aqueous phase , while proteins and lipids are contained in the phenolic phase . the application to samples that contain interfering compounds resulted in more protein bands or spots on the gels and less proteolysis , and also downstream processing including bioinformatic analysis resulted better results for phenol - extracted proteins for plant tissue ( pavokovi , krinik and krsnik - rasol 2012 ) . the major drawbacks of phenol - based extractions are the corrosivity and toxicity of the chemical , and the time intensive extraction with the phase separation . to ease the phase separation , the addition of sucrose pushes the phenol phase to the top and facilitates recovery ( faurobert , pelpoir and chab 2007 ) . the former shows already that a combination of strategies can be useful for sufficient protein yields from soils . similarly , nicora et al . , ( 2013 ) suggested to combine the use of desorption buffers and positive polar amino acids that bind to the sorption sites of the soil prior to cell lysis . this strategy might be useful for silty and clayey soil , soils that are characterized by a high cec . beside the choice of extraction buffer , the potential steps for getting rid of hs are based on physico - chemical separation principles . these strategies can be easily applied with various protein extraction buffers either before ( using pvpp during grinding in liquid nitrogen ; keiblinger et al . proteins and hs can be fractionated by size , using gel filtration raisins ( sepharose 4b , sephadex or sephacryl ) or ultrafiltration with spin filters ( 10 kmwco cut off ) , fig . columns packed with pvpp ( kabir et al . 2003 ; masciandaro et al . 2008 ) as well as commercial ones are used to separate hs from proteins by the aid of different binding abilities to a polymeric matrix . the precipitation of hs by alnh4(so4)2 has to our knowledge not been used for the extraction of proteins from soil so far , but might be a potential solution ( braid , daniels and kitts 2003 ) . elimination of coextractants consequently may also reduce target proteins ; to this end , recoveries of extraction should be monitored during all extractions by adding a standard protein spike to evaluate the extraction efficiency . there are several factors that might affect protein yields during extraction ( i.e. cell lysis , ph and detergents of the extraction buffer , denaturation agents and application of phenol and precipitation method , table 1 ) . table 1 lists different extraction protocols for forest soils , agricultural soils and rhizosphere soils together with soil physicochemical parameters such as ph , cec , organic c , n content , soil texture , extraction strategy applied , extracted protein concentration or number of proteins ( spots ) or ( if applicable ) assigned proteins . owing to the complexity of the soil matrix , the reader would not be surprised that a unified extraction protocol for soils can not be recommended at the moment . although some suggestions are given above , based on the strong variation of conditions for sample handling and extraction , and further downstream processing as well as samples from strongly differing biomes given in table 1 , it is not even possible for agricultural and forest soils . the entire proteome of a microorganism consists of all its extracellular , cytoplasmic and membrane proteins . many extracellular proteins have successfully been recovered from cultures grown on leaf litter ( schneider et al . 2010 ) . while studies on leaf litter ( keiblinger et al . 2012a 2012 ) aimed at capturing the entire metaproteome , these analyses include information on the extracellular fraction recovered by the extraction with sds buffer ( extraction conditions recently reviewed by becher et al . in contrast to leaf litter , the complexity of the soil matrix ( vos et al . extracellular enzymes are often reached by indirect extraction or prior washing , as soil washing releases cells from the soil matrix . however , this step introduces another level of uncertainty as stabilized enzymes are not reached . in general , it should be mentioned that alkaline conditions are unfavorable for extraction of extracellular enzymes as cell lysis can occur , thereby including untargeted intracellular proteins . extracellular proteins have been isolated from a greenhouse soil and forest soils , using extraction buffers containing phosphate ( murase et al . 2008 ) at ph 6 ( see also table 1 ) . in a recent study , bastida , hernandez and garcia ( 2014 ) found that chourey 's method ( 2010 ) was better suited than singleton 's ( 2003 ) to recover more extracellular proteins in metaproteomics from forest and agricultural soils . after extraction , it is often necessary to concentrate proteins ( fig . 1b ) as amplification of low - abundant proteins is not possible ( in contrast , e.g. dna amplification via pcr ) . to this end , proteins can either be concentrated by reducing the sample volume ( through freeze drying , heating , ultrafiltration or by vacuum centrifugation ; criquet , farnet and ferre 2002 ) by dialysis or desalting methods ( ogunseitan 2006 ) ; however , most commonly in soil metaproteomics is precipitation ( chourey et al . while reducing sample volume can also increase the concentration of interference compounds ( i.e. humics ) , precipitation includes purification from undesirable substances . for soil protein extracts , most often trichloroacetic acid ( tca ) or methanol ammonium acetate precipitation ( table 1 ) tca precipitation is achieved by changing the ph , and reducing the solubility of proteins in solution . in contrast , methanol ammonium acetate precipitation in methanol combines salt - induced precipitation and organic solvents . although adding a 4-fold amount of methanol efficiently precipitates most proteins , adding an organic base , ammonium acetate , increases yields for acidic solutions . while tca is known to be an efficient precipitation agent for soil proteins extracted with sds buffers , it has several disadvantages . among them are ( i ) a potential loss of large proteins ( carpentier et al . 2005 ) ; ( ii ) the coprecipitation of interfering substances such as dna and protein - dna aggregates ( pavokovi , krinik and krsnik - rasol 2012 ) ; ( iii ) protein pellets need to be washed with acetone or a base to remove the remaining acid from the proteins ; ( iv ) the risk that proteins are non - functional afterwards , which is problematic with 2de ; ( v ) and finally tca precipitated proteins are difficult to re - solubilized , here preferentially small proteins are redissolved ( carpentier et al . ammonium acetate precipitation is often used in combination with phenol - based extraction procedures ( carpentier et al . ; pavokovi , krinik and krsnik - rasol 2012 ) , and might be more suitable for soils with large amounts of hs . as mentioned above , rehydration of precipitated proteins is sometimes problematic as the protein pellets do not dissolve well . for this a variety of buffers ( i.e. guanidine buffer , sds sample buffer ) can be applied ; for more details , see table 1 . rehydration buffers containing chaotropes ( typically urea and thiourea ) might improve protein yields ( weiss and grg 2008 ) . prior to further processing , the evaluation of the protein concentration is helpful . as most colorimetric assays such as bradford ( whiffen , midgley and mcgee 2007 ) interfere with hs , roberts and jones ( 2008 ) suggested that total protein concentrations should be determined by acid hydrolysis followed by amino acid measurements . this strategy has been successfully applied recently in soil metaproteomics ( bastida , hernandez and garcia 2014 ) . the complexity of environmental samples still outstrips the capabilities of state - of - the - art ms approaches . thus , separation of proteins / peptides is mandatory to reduce sample complexity before ms analysis ( fig . in early ( soil ) metaproteomic studies , 2d gel - based protein separation methods were successfully employed ( klaassens , de vos and vaughan 2007 ; benndorf et al . 2007 ; wilmes , wexler and bond 2008 ) however , this technology has major drawbacks , particularly regarding the analysis of proteins with extreme molecular weights , isoelectric points or hydrophobicity values . these restrictions were relaxed by 1d gel - based or gel - free fractionation methods . gel - free approaches include different protein extraction procedures , followed by in - solution digestion to peptides . peptides are further separated by reversed - phase rp - lc or a chromatographic separation in two dimensions using strong cation exchange chromatography in combination with rp - lc . proteins separated by gels can be enzymatically digested in - gel while gel - free approaches take advantage of in - solution or on - filter protein digestion ( fig . . identification rate of particularly low - abundant proteins after 1d gel - based fractionation can be improved by normalizing the size of fractions ( gel pieces ) to the contained protein amount ( yin et al . weston , bauer and hummon ( 2013 ) showed that filter - based digestion resulted in an 18% higher protein identification rate compared to in - solution digestion , which might be due to an additional denaturating protein solubilization step . the advantage of a gel - based fractionation is the combination of protein denaturation and separation , while it is more time consuming than gel - free fractionation that benefits from reduced processing time , and therefore has a greater high - throughput potential . one of the most frequently used strategies in such proteomic experiments is tandem ms of peptides after enzymatic protein digestion . ms analysis is followed by subsequent correlation of resulting spectra with those of theoretic peptides from a given protein database ( protein db or target db ) ( eng , mccormack and yates 1994 ; yates et al . , this approach evolved to the preferred strategy for protein identification , quantification and detection of chemical peptide modifications in large - scale soil metaproteomic studies ( aebersold and mann 2003 ) . however , this approach does not allow direct protein identifications but is based on two matching steps : ( i ) matching the experimental spectra to theoretical spectra obtained from a given protein db after in silico digestion and ( ii ) inferring the original proteins based on the resulting peptide - to - spectrum matches ( psms ) . thus , only protein sequences represented in the target db can be identified ( fig . alternatively , spectral libraries can be used to correlate experimental spectra directly with identified reference spectra ( fig . these reference spectra have to meet high - quality criteria and , thus , their generation is costly and not practicable in dimensions demanded by metaproteomics . however , high - quality spectra can be used as a reference even if they are identified not yet . tools such as scanranker support selection of unidentified high - quality spectra by automatic routines ( ma et al . 2011 ) whose occurrence can be then followed across different samples and ecosystems ( muth et al . promising spectra can be then submitted to de novo sequencing ( hughes , ma and lajoie 2010 ) . as mentioned before , correlating experimental spectra with theoretic spectra of peptides from a given protein db is the most frequently used proteomics approach . quality and performance of spectra correlation crucially depend on the size of the search space that is defined by both ( i ) the number of recorded spectra to compare and ( ii ) the number of theoretic or reference spectra compared to . an increased search space inevitably leads to an increase in ( i ) computational costs , ( ii ) potential of false positives ( or false negatives ) and ( iii ) frequency of psms matching to two or more proteins . to reduce the number of spectra submitted to 2a ) ( flikka et al . 2006 ; ding , shi and wu 2009 ; zou et al . 2009 ; redundant spectra can be clustered into metaspectra to further reduce the number of spectra to correlate that positively affects not only false discovery rates ( fdr ) but also analysis speed ( flikka et al . thus , protein db selection plays a pivotal role in metaproteomics ( tanca et al . a customized protein db is ideally assembled based on a matched full metagenome from the same sample as analyzed by metaproteomics . by this , optimal identification rates can be achieved as previously shown ( morris et al . alternatively , the taxonomic sample composition revealed by 16s and/or 18s rna sequencing data can be used to deduce the pseudo - metagenome . using a six - frame translation of the metagenome sequence produces more complex protein dbs , but can be helpful to increase the metaproteome coverage . finally , unmatched metagenome data can be also successfully used for protein db assembly as previously shown ( verberkmoes et al . the greatest difficulty is the selection of customized subcollections from public resources since it has to be based on assumptions on the metaproteome composition . thus , resulting protein dbs are generally large ( > > 10 sequences ) . to overcome this , an iterative db search method that uses matches from a primary db search to assemble a customized database of reduced size this example shows a reduction in db size to < 0.1% of the original size . ( 2013 ) evaluated the impact of different protein database types , one based on matched metagenome data and another one based on sequences of expected genes from trembl . an interesting yet alarming result was that an overlap of only 36% of all identified peptides was found when using both protein dbs . there are various algorithms available to compare experimental and theoretic peptide fragmentation spectra , the computational basics of which are comprehensibly described elsewhere ( colinge and bennett 2007 ) . all have in common that they produce multiple testing effects increasing the number of wrongly accepted psms . the proportion of false positives can be controlled by the fdr ( benjamini and hochberg 1995 ) . meanwhile , various methods for fdr assessment have been entered in metaproteomics analyses ( nesvizhskii 2010 ) . for instance , target - decoy dbs composed of all protein sequences in forward ( target ) and reverse ( decoy ) direction have been applied as an easy and powerful method ( elias and gygi 2007 ) . however , this strategy leads to a doubling of the target db size that in turn increases the search space ( see above ) . with percolator , a semi - supervised machine learning algorithm trained by scrambled decoy peptides and best scoring target peptides is available ( kall et al . 2007 ; combined with accurate scoring functions for psm , the use of this approach can increase the number of peptide identifications in a variety of data sets as previously shown ( granholm et al . protein inference problem and provided a statistical model for ms - based protein identification ( nesvizhskii et al . distinct protein identifications need at least one identified peptide that uniquely maps to the respective protein . the proportion of unique peptides drops with an increasing number of closely related organisms considered by the target db , which complicates soil metaproteome data analyses . meanwhile , there are several approaches to calculate probabilities of identified proteins ( higdon and kolker 2007 ; serang and noble 2012 ; shi and wu 2012 ; yang , he and yu 2013 ) . however , it should be noted that protein probabilities are experiment specific since they correlate with factors such as spectra number , protein db size and protein abundances ( xue et al . this strategy is followed by the scaffold software ( searle 2010 ) , for instance . an alternative approach is provided by proteomediscoverer ( thermo scientific , waltham , massachusetts , usa ) assigning peptides to all possible proteins matching the quality criteria , and a combination of db searches and de novo sequencing is provided to maximize metaproteome coverage . however , at least peptides matching to proteins with equal probabilities can not be uniquely attributed . ( 2011 ) introduced a hierarchical protein clustering approach by means of those shared peptide matches . peptide - sharing proteins are grouped together and represented by a single anchor protein . however , at this time , this approach is beneficial rather for single - organism proteomics than metaproteomics where resulting clusters can be taxonomically and functionally diverse . the knowledge about the abundance of proteins is essential for a systems biological perspective on microbial consortia . various technologies have been established to assess whole protein inventories ( von bergen et al . 2013 ; otto , becher and schmidt 2014 ) . however , only a few are applicable in a scale needed for environmental proteomics . using 1d gel - based or gel - free approaches , protein amounts can be estimated based on spectral counts . normalized spectral abundance factors ( nsaf ) account for protein length and sample - to - sample variation ( zybailov et al . considers shared peptides by distributing shared spectral counts based on the number of unique spectral counts ( fig . 2b ) ( zhang et al . 2010 ; mcilwain et al . the application of metabolic labeling in environmental proteomics is hindered by the fact that the metabolic label has to be provided in sufficient amounts . in contrast to metagenomics , metaproteomics provides insights into the metabolically active species and their metabolic performance within the analyzed microbial consortium or ecosystem . however , the vast mass of data provided by metaproteome analyses complicates data interpretation ( fig . for both functional and taxonomic analyses ( which should ideally be combined ) , quality of protein annotation is crucial and should be considered already during protein db assembly . several online resources provide expertly curated data sets for a high number of proteins ( e.g. swissprot , refseq ) ( table 2 ) . however , two major problems persist(i ) limited ( functional ) annotation standards and ( ii ) missing global ( db - independent ) sequence identifiers which both considerably complicate meta - physiological research . thus , approaches to globalize sequence identifiers ( e.g. seguid ; babnigg and giometti 2006 ) or to classify functions ( e.g. tigr role categories ; haft et al . different repositories provide functional categories and corresponding profiles . with the cluster of orthologous groups , a widely distributed classification system is available for prokaryotic ( cog ) and eukaryotic ( kog ) proteins ( tatusov et al . however , this system has not been updated since 2003 ; therefore , eggnog as actively curated derivate can be recommended ( powell et al . expertly curated hidden markov models based on multiple sequence alignments of proteins fulfilling the same function are available ( haft et al . combined with tigr roles , an excellent classification system organized in ( i ) main roles ( e.g. energy metabolism ) , ( ii ) subroles ( e.g. glycolysis ) and ( iii ) functions ( e.g. enolase ) is provided ( fig . considering data from resources specialized to distinct protein functions can support detailed analyses on specific activities . a prominent example for such specialized resources is cazy ( http://www.cazy.org/ ) ( cantarel et al . 2014 ) listing more than 330 families of carbohydrate - active enzymes that have been already successfully employed in several environmental studies to estimate the amount of polymer - degrading enzymes ( aylward et al . 2012 different repositories such as kegg , bigg or biocyc are available ( schellenberger et al . voronoi treemaps can visualize highly complex hierarchically organized data in a space optimized manner . here , functional classification of tigrfams ( release 15.0 ) is depicted based on tigr roles main ( left ) and ( right ) subclasses . number of annotated protein sequences provided by uniprot and ncbi ( as of 28 january 2015 ) . including unclassified and other sequences . the national center of biotechnology information ( http://www.ncbi.nlm.nih.gov ) , as of 19 february 2013 . biologically non - redundant , annotation partially curated by experts . based on standardized taxonomic annotation , proteins in peptide sharing clusters can be reduced to the lowest common anchor ( lca ) ( fig . the unipept web application provides a robust lca approach considering all occurrences of identified tryptic peptides in uniprotkb . alternatively , pipasic estimates the peptide level similarity between reference proteomes allowing differentiation on strain level . the prophane web service provides a combined fully automated workflow for both ( i ) functional analyses using various resources ( cog / kog , tigrfams and pfams ) and ( ii ) lca - based taxonomic assessment ( www.prophane.de ) ( schneider et al . in addition , metaproteomeanalyzer software is a tool that features four freely available db search algorithms ( x!tandem , omssa , crux , inspect ) , and is also highly suitable for comprehensive analysis and visualization of metaproteomic datasets ( https://code.google.com/archive/p/meta-proteome-analyzer/ ) ( muth et al . the generated data take valuable space and are barely standardized that both makes data handling and integration difficult ( jimenez and vizcaino 2013 ) . meanwhile , several commercial ( stephan et al . 2010 ) and open - source ( perez - riverol et al . however , at least after publication spectral data should be made publicly available making online repositories such as pride , peptideatlas and tranche ( for review , see jimenez and vizcaino 2013 ) . furthermore , the enormous progress of analytical tools and the tremendous increase of available protein sequences require non - traditional data storage for keeping the data in an active state . thus , data storage should be never the end of the analysis pipeline but much more the beginning of a new improved analysis circle ( see also muth et al . voronoi treemaps have proven to be an excellent tool to visualize hierarchical data structures in a space optimized manner ( fig . stream graphs allow even one more dimension and are , thus , perfectly suited for time courses . however , there are two major drawbacks . first , biological data can not be always reduced to a non - redundant hierarchical organization . for instance , proteins can have more than one function and , thus , have multiple places in a treemap . second , with increasing data complexity , the human eye is overtaxed , particularly when viewing print media where space and resolution is limited . there are several important steps that must be carefully planned when employing soil metaproteome analysis . first , the sampling strategy must be well considered to cover the spatial and temporal heterogeneity of ( i ) the soil matrix and ( ii ) the microbial community that varies in diversity , size , generation time , functions and favored soil physical and chemical conditions . second , an optimal sample handling procedure has to be established and should be discussed within the scientific community to generate comparable data for meta - metanalysis . studies that compare storage conditions for soil and leaf litter from a wide variety of climates are still missing , but would be highly useful . we have reviewed the application of different extraction protocols for proteins present in soil and litter , and how soil characteristics may influence the protein extraction . however , it is important to mention that protein extraction methods need to be further explored and improved . in particular , more emphasis in the identification of extracellular proteins is required , as those are directly linked to biogeochemistry processes . so far dynamic succession of soil and leaf litter microbial populations , including their community structure and respective functions , are poorly investigated . in this regard , metaproteomics allows the untargeted assignment of proteins involved in a broad variety of biochemical processes . we thus expect that environmental metaproteomics , so far a mainly descriptive approach , will significantly contribute to hypothesis - driven research aiming at a deeper understanding of the highly complex metabolic network and multispecies interactions in terrestrial habitats . subsequent research aiming to develop sophisticated bioinformatic tools constantly facilitates the application of metaproteomics even in such complex habitats such as soil and leaf litter and will be a central prerequisite for the hypothesis - driven evaluation of metaproteome data . the power of metaproteomics can even be further enhanced , when combined or complemented with other omics technologies , i.e. metagenomics and metatranscriptomics and also classical soil analytics such as microbial biomass , potential enzyme activities and physico - chemical indicators . given the environmental challenges facing society today , the need for in - depth understanding of soil functioning is critical . this review therefore concludes that the continued and increased application of soil metaproteomes within holistic ecosystem experimental frameworks constitutes a research priority . this work was supported by the austrian science fund fwf [ p 25438 ] and the austrian climate research program [ kr13ac6k11008 ] .

the increasing application of soil metaproteomics is providing unprecedented , in - depth characterization of the composition and functionality of in situ microbial communities . despite recent advances in high - resolution mass spectrometry , soil metaproteomics still suffers from a lack of effective and reproducible protein extraction protocols and standardized data analyses . this review discusses the opportunities and limitations of selected techniques in soil- , and leaf litter metaproteomics , and presents a step - by - step guideline on their application , covering sampling , sample preparation , extraction and data evaluation strategies . in addition , we present recent applications of soil metaproteomics and discuss how such approaches , linking phylogenetics and functionality , can help gain deeper insights into terrestrial microbial ecology . finally , we strongly recommend that to maximize the insights environmental metaproteomics may provide , such methods should be employed within a holistic experimental approach considering relevant aboveground and belowground ecosystem parameters .

INTRODUCTION CONCEPTUALIZATION OF SOIL PROTEOMICS BY BASIC SOIL DATA FROM DATA TO UNDERSTANDING CONCLUDING REMARKS FUNDING

moreover , soil is an important habitat for soil microbes and animals , and serves as physical and cultural environment for humankind ( blum , busing and montanarella 2004 ) . every gram of soil harbors thousands of bacterial , archaeal and eukaryotic taxa , and this taxonomic diversity is mirrored by the diversity of their physiologies , life styles ( i.e. microbial diversity is highly variable in terrestrial ecosystems , depending on many factors , such as plant cover , animal activity , soil moisture , temperature , aeration , porosity , nutrient availability , ph and salinity ( kennedy et al . when comparing a broad range of soil ecosystem types acidobacteria and verrucomicrobia turned out to be the most abundant taxonomic groups followed by actinobacteria , bacteroidetes , planctomycetes and archaea ( barbern et al . 2012 ) , while desert microbial communities are characterized by a high abundance of genes associated with osmoregulation and dormancy , and genes associated with nutrient cycling and catabolism of plant - derived organic compounds are less abundant ( fierer et al . metagenome information thus represents only the functional potential and giving no indication of the relative activity of the phyla present . provided the number of studies that have successfully applied metaproteomics on soil and leaf litter environments continues to grow , including e.g. 2015a ) , soil restoration and ecosystem processes ( bastida et al . a bacterial dominated short - term assimilation of plant - derived n was shown , and oligotrophic and copitrophic life styles of soil organisms in terms of temporal leaf litter n utilization patterns illustrate a new cutting edge approach to determine ecological attributes of soil microbes ( starke et al . due to its large potential for providing a link between functional and phylogenetic information of soil microbial communities , as exemplified by the aforementioned studies , there has been growing interest in the application of metaproteomics in soil ecology to study microbially driven ecosystem functions ( e.g. however , soil metaproteomics still faces several challenges , including the heterogeneity of soil matrices , high microbial diversity , the ecosystem - specific dominance of few microbial species and limited metagenomic information and data handling ( keller and hettich 2009 ; schneider and riedel 2010 ; siggins , gunnigle and abram 2012 ; becher et al . protein extraction of soils is often difficult due to the presence of other organic compounds , such as complex carbohydrates , lipids and phenolic compounds ( e.g. lignin ) , and humic substances ( hs ) as well as inorganic compounds from the soil matrix , such as silt and clay minerals . clay ) not only complicate protein extraction but also interfere with the separation of peptides ( bastida et al . these limitations for extraction are due to the fact that proteins can be adsorbed , linked anchored or embedded on / to / in solid particles such as clay , clay minerals , and soil om organo - mineral complexes ( nielsen , calamai and pietramellara 2006 ; tomaszewski , schwarzenbach and sander 2011 ) , which thereby reduce extraction efficiency ( sander , tomaszewski and schwarzenbach 2011 ) . adsorption of proteins to clays is a rapid process , which is only partly reversible ( nielsen , calamai and pietramellara 2006 ) , and is based on the large specific surface area of clay minerals ( giagnoni et al . clays ) , which depend on the ph , the charge of the clay mineral and the mg concentration ( jiang et al . hs and proteins are bound reversibly by a cation exchange process , which depends on the cation exchange capacity ( cec ) of the soil , the amino acid composition and the isoelectric point of the target proteins . to this end , the choice of purification methods or the extraction buffer and additives to it depends not only on the soil type but also on the goal of the investigation . 1b ) , ( iii ) processing of soil protein extracts ( including removal of interfering substances , pre - fractionation of proteins or peptides and mass spectrometry ( ms ) analysis ( fig . all steps are crucial for obtaining , holding and sharing high - quality soil metaproteome data , and some of these steps have recently been reviewed in detail ( keller and hettich 2009 ; schneider and riedel 2010 ; siggins , gunnigle and abram 2012 ; becher et al . we focus on differences in sample preparation and published protocols ( table 1 ) and try to synthesize knowledge to provide a step - by - step guideline of how to best proceed in soil and leaf litter protein extraction ( fig . , the current work presents recent advances in data analysis and data interpretation using novel bioinformatic tools ( fig . the wider objective of the present work is to ( i ) highlight the need for standardized methodology , which would ensure better comparability of future soil metaproteomic analyses , and to ( ii ) provide the basis for future meta - analysis by including additional environmental parameters and different ecosystem properties into metaproteome datasets . researchers are confronted with various sampling methods and procedures that have to be carefully selected and combined for ( a ) sample preparation , including soil sampling homogenization and storage , ( b ) protein isolation and ( c ) shotgun proteomics ( from top to down ) . researchers can select or combine various methods for ( a ) data analysis , and ( b ) data interpretation ( from top to down ) . consecutive steps are connected by lines ( dashed lines represent workflows not suitable for high - throughput analyses ) . as soils of the globe are multifaceted , they are classified into groups based on their soil morphology , behavior or genesis in soil science . due to their varying characteristics in multiple scales , a case - by - case evaluation of sample handling as well as protein extraction strategies ( see also fig . to minimize artificially introduced variability , sample handling and preparation should involve as few steps as possible . in the following paragraphs , we will guide the reader step by step from the soil sampling to the analysis of metaproteomic data . we believe that meta - omics studies of soil ecosystems should also provide contextual data such as soil ph , organic carbon ( corg ) , n - content , sampling depth , soil texture and cec ( for soils ) ( table 1 ) . in addition , information on the study site including latitude and longitude , altitude , climate including mean annual temperature and precipitation , nutrient concentrations and bedrock material should be provided . however , for choosing an appropriate protein extraction , protocol knowledge on the before mentioned parameters ( partly displayed in table 1 ) is needed . as with any technique , metaproteomics per se hence , we need to implement additional data i.e. chemical background , soil history , microbial biomass and enzyme activities , to provide the basis to unravel the major biotic and abiotic drivers of the active abundant communities , not only for individual experiments but also for future , cross - biome meta - analysis . the spatial and temporal heterogeneity of the soil matrix need to be considered by obtaining a representative sample of the natural situation for metaproteome analysis . however , without giving any further details , such strategies might include sampling time , sample amount , sampling device , stratified sampling ( horizontal and vertical distribution ) , composite samples ( pooling ) when appropriate ( pettitt and mcbratney 1993 ) and/or apply a replicated sampling design ( for details , see boeddinghaus et al . given the spatio - temporal variability of climatic and pedologic characteristics in the field scale that shape the active soil microbial community , we highlight the importance of measuring these covariables in metaproteomic studies as already mentioned before . high clay and/or moisture content , however , can inhibit effective sieving in which case removal of visible organic debris and sample homogenization has to be done manually . unfortunately , om content and texture of the soils processed are not always given ( table 1 ) hampering systematic investigations of storage conditions on soil metaproteomes . however , a detailed comparison of the influence of storage conditions in terms of temperature and time on the stability and activity of soil proteins is urgently needed . an optimal protein extraction protocol contains at least three important steps : ( i ) quantitative extraction of proteins from the environmental matrix ( including steps for cell lysis , choice of buffer for solubilization and chemical reduction ) , ( ii ) protein purification ( i.e. several studies aimed at extracting the entire protein complement of an environmental sample by employing different strategies such as ( i ) indirect extraction , where microbes become enriched prior to extraction ( see table 1 , i.e # 9 , 10 ) , ( ii ) separation by means of density gradient centrifugation ( dgc ) prior to protein extraction ( to separate microorganisms from the environmental matrix , table 1 , i.e. to this end , grinding seems to be most applicable for plant material , leaf litter and soils with high humic and low sand content or compost . among physical procedures , sonication is a commonly used method for protein extraction from soils , as it favors the solubilization of stabilized proteins , and also breaks soil aggregates ( nannipieri 2006 ; ogunseitan 2006 ) . sds ) with other additives , such as enzymes and/or protease inhibitors cocktails , is a good strategy for direct cell lysis in soil samples , depending on the target cells and soil type and further downstream processing . basic knowledge of soil and environmental characteristics might aid the choice of an extraction procedure appropriate for the research question . thus , it will be at least possible to evaluate which challenges during protein extraction can be expected ( such as high humus content or clay - rich soils with high cec ) and to adopt existing protocols that provided promising results on similar soils , in comparable habitats . this illustrates the importance of the ph of the soil and the extraction buffer , as it governs sorption of proteins to minerals and removal of interfering substances , and it also influences protein structure ( bastida et al . the ph of the extraction buffer has a strong influence on cell extraction , and considerably increases with ph in the range from 5 to 8 ( bakken and frostegrd 2006 ) . the application to samples that contain interfering compounds resulted in more protein bands or spots on the gels and less proteolysis , and also downstream processing including bioinformatic analysis resulted better results for phenol - extracted proteins for plant tissue ( pavokovi , krinik and krsnik - rasol 2012 ) . the major drawbacks of phenol - based extractions are the corrosivity and toxicity of the chemical , and the time intensive extraction with the phase separation . these strategies can be easily applied with various protein extraction buffers either before ( using pvpp during grinding in liquid nitrogen ; keiblinger et al . elimination of coextractants consequently may also reduce target proteins ; to this end , recoveries of extraction should be monitored during all extractions by adding a standard protein spike to evaluate the extraction efficiency . cell lysis , ph and detergents of the extraction buffer , denaturation agents and application of phenol and precipitation method , table 1 ) . table 1 lists different extraction protocols for forest soils , agricultural soils and rhizosphere soils together with soil physicochemical parameters such as ph , cec , organic c , n content , soil texture , extraction strategy applied , extracted protein concentration or number of proteins ( spots ) or ( if applicable ) assigned proteins . owing to the complexity of the soil matrix , the reader would not be surprised that a unified extraction protocol for soils can not be recommended at the moment . although some suggestions are given above , based on the strong variation of conditions for sample handling and extraction , and further downstream processing as well as samples from strongly differing biomes given in table 1 , it is not even possible for agricultural and forest soils . many extracellular proteins have successfully been recovered from cultures grown on leaf litter ( schneider et al . while studies on leaf litter ( keiblinger et al . in contrast to leaf litter , the complexity of the soil matrix ( vos et al . in general , it should be mentioned that alkaline conditions are unfavorable for extraction of extracellular enzymes as cell lysis can occur , thereby including untargeted intracellular proteins . extracellular proteins have been isolated from a greenhouse soil and forest soils , using extraction buffers containing phosphate ( murase et al . to this end , proteins can either be concentrated by reducing the sample volume ( through freeze drying , heating , ultrafiltration or by vacuum centrifugation ; criquet , farnet and ferre 2002 ) by dialysis or desalting methods ( ogunseitan 2006 ) ; however , most commonly in soil metaproteomics is precipitation ( chourey et al . for soil protein extracts , most often trichloroacetic acid ( tca ) or methanol ammonium acetate precipitation ( table 1 ) tca precipitation is achieved by changing the ph , and reducing the solubility of proteins in solution . ; pavokovi , krinik and krsnik - rasol 2012 ) , and might be more suitable for soils with large amounts of hs . prior to further processing , the evaluation of the protein concentration is helpful . as most colorimetric assays such as bradford ( whiffen , midgley and mcgee 2007 ) interfere with hs , roberts and jones ( 2008 ) suggested that total protein concentrations should be determined by acid hydrolysis followed by amino acid measurements . this strategy has been successfully applied recently in soil metaproteomics ( bastida , hernandez and garcia 2014 ) . gel - free approaches include different protein extraction procedures , followed by in - solution digestion to peptides . proteins separated by gels can be enzymatically digested in - gel while gel - free approaches take advantage of in - solution or on - filter protein digestion ( fig . weston , bauer and hummon ( 2013 ) showed that filter - based digestion resulted in an 18% higher protein identification rate compared to in - solution digestion , which might be due to an additional denaturating protein solubilization step . the advantage of a gel - based fractionation is the combination of protein denaturation and separation , while it is more time consuming than gel - free fractionation that benefits from reduced processing time , and therefore has a greater high - throughput potential . one of the most frequently used strategies in such proteomic experiments is tandem ms of peptides after enzymatic protein digestion . however , this approach does not allow direct protein identifications but is based on two matching steps : ( i ) matching the experimental spectra to theoretical spectra obtained from a given protein db after in silico digestion and ( ii ) inferring the original proteins based on the resulting peptide - to - spectrum matches ( psms ) . these reference spectra have to meet high - quality criteria and , thus , their generation is costly and not practicable in dimensions demanded by metaproteomics . however , high - quality spectra can be used as a reference even if they are identified not yet . tools such as scanranker support selection of unidentified high - quality spectra by automatic routines ( ma et al . as mentioned before , correlating experimental spectra with theoretic spectra of peptides from a given protein db is the most frequently used proteomics approach . quality and performance of spectra correlation crucially depend on the size of the search space that is defined by both ( i ) the number of recorded spectra to compare and ( ii ) the number of theoretic or reference spectra compared to . using a six - frame translation of the metagenome sequence produces more complex protein dbs , but can be helpful to increase the metaproteome coverage . finally , unmatched metagenome data can be also successfully used for protein db assembly as previously shown ( verberkmoes et al . to overcome this , an iterative db search method that uses matches from a primary db search to assemble a customized database of reduced size this example shows a reduction in db size to < 0.1% of the original size . however , this strategy leads to a doubling of the target db size that in turn increases the search space ( see above ) . the proportion of unique peptides drops with an increasing number of closely related organisms considered by the target db , which complicates soil metaproteome data analyses . however , it should be noted that protein probabilities are experiment specific since they correlate with factors such as spectra number , protein db size and protein abundances ( xue et al . an alternative approach is provided by proteomediscoverer ( thermo scientific , waltham , massachusetts , usa ) assigning peptides to all possible proteins matching the quality criteria , and a combination of db searches and de novo sequencing is provided to maximize metaproteome coverage . using 1d gel - based or gel - free approaches , protein amounts can be estimated based on spectral counts . the application of metabolic labeling in environmental proteomics is hindered by the fact that the metabolic label has to be provided in sufficient amounts . in contrast to metagenomics , metaproteomics provides insights into the metabolically active species and their metabolic performance within the analyzed microbial consortium or ecosystem . for both functional and taxonomic analyses ( which should ideally be combined ) , quality of protein annotation is crucial and should be considered already during protein db assembly . the unipept web application provides a robust lca approach considering all occurrences of identified tryptic peptides in uniprotkb . in addition , metaproteomeanalyzer software is a tool that features four freely available db search algorithms ( x!tandem , omssa , crux , inspect ) , and is also highly suitable for comprehensive analysis and visualization of metaproteomic datasets ( https://code.google.com/archive/p/meta-proteome-analyzer/ ) ( muth et al . however , at least after publication spectral data should be made publicly available making online repositories such as pride , peptideatlas and tranche ( for review , see jimenez and vizcaino 2013 ) . thus , data storage should be never the end of the analysis pipeline but much more the beginning of a new improved analysis circle ( see also muth et al . second , an optimal sample handling procedure has to be established and should be discussed within the scientific community to generate comparable data for meta - metanalysis . studies that compare storage conditions for soil and leaf litter from a wide variety of climates are still missing , but would be highly useful . we have reviewed the application of different extraction protocols for proteins present in soil and litter , and how soil characteristics may influence the protein extraction . however , it is important to mention that protein extraction methods need to be further explored and improved . so far dynamic succession of soil and leaf litter microbial populations , including their community structure and respective functions , are poorly investigated . we thus expect that environmental metaproteomics , so far a mainly descriptive approach , will significantly contribute to hypothesis - driven research aiming at a deeper understanding of the highly complex metabolic network and multispecies interactions in terrestrial habitats . subsequent research aiming to develop sophisticated bioinformatic tools constantly facilitates the application of metaproteomics even in such complex habitats such as soil and leaf litter and will be a central prerequisite for the hypothesis - driven evaluation of metaproteome data . given the environmental challenges facing society today , the need for in - depth understanding of soil functioning is critical . this review therefore concludes that the continued and increased application of soil metaproteomes within holistic ecosystem experimental frameworks constitutes a research priority .

they consume native drink ( e.g. , sajpani , kiad etc . ) virtually in every occasion of life such as various festivals , marriage ceremony and even in funeral . almost all tribes prepare and consume traditional drink but their names , ingredients , and mode of preparation differ greatly from community to community and region to region . however , the tribals use local medicinal plant parts along with rice to prepare their native liquor and believe that the beverage prepared in this process prevents headache , insomnia , body ache , urinary troubles and cholera [ 2 - 4 ] . the north - eastern provinces of india , colonized by native bodo , garo , rabha , karbi , ahom , deori , dimasa , kachari , etc . communities are best known for the production of household liquors . however , there is virtually no data available regarding the traditional alcoholic beverage preparation in the sub - himalayan non - hilly region of bengal . malda , district of west bengal in india , is characterized by its diversified historical dynasty , mango production , rich wetland , natural vegetation and its diversified ethnic groups including santala , oraon , rajbanshi , namasudre , polia , mundas , malpaharias etc . amongst these tribes , oraon community in fact chullu production is a part of their culture , identity , myths , and spiritual practices . their own traditional way of preparing the brew with medicinally important plants distinguished them from rest of the tribal communities of the region . the recipe of brew preparation is however a secret and passed on generation after generation orally . besides so far there is no authentic documentation of chullu preparation technology used by oraons of malda district of west bengal , india . the plants those were used in preparing chullu , either known medicinally or have ethnic use among oraons. therefore , a semi - structured questionnaire process was also intended to focus the local use of the plants , their parts involved in medicinal practices and preparation of drugs from those plant parts . another aspect which was considered in this study was the economic significance of chullu production and sale . hence , it may be a pioneer study to explore the chullu procedure technology and its economic impact among oraon community of the district along with ethnomedicinal value of used plant species . malda ( latitude and longitude of 244020n to 253208n and 882810e to 874550e respectively ) , a district of west bengal , india with a total land area of 3455.66 km2 . is known as mango district for its wide array of mango variety and production . it consists of two municipalities , 15 blocks or sub - divisions and 3701 villages with a population of more than forty lakh . the adh soi wetland ( beel ) , located at harischandrapur - ii block of the region , is one of the largest among the wetlands of the state comprising rich vegetation due to its macrophytic diversity . a few small forest areas are also scattered in old malda , habibpur , harishchandrapur and in gajol blocks . most of ethnic communities live mainly in the four blocks of this province namely , gazole , bamongola , habibpur and old malda comprising more than 85% of total tribal population . hence we considered these four blocks as our study area [ figure 1 ] due to its noticeable oraon population . the district has a hot summer ( 35 - 42c ) from march to september and a very cold winter ( 6 - 12c ) from november to february . the monsoon starts from june and continues to the mid - september and the average rainfall is approximately 1453.1 mm . the map of study area ( malda district ) showing four main oraon populated zones ( 1 : gazole block ; 2 : bamongola block ; 3 : old maldah block ; 4 : habibpur block ) oraon community is one of the largest tribal groups in india , possessing a unique tradition and culture . in malda district , a sizeable portion of the tribal communities are the oraons who mostly inhabits in the remote villages . they have distinctive lifestyle and are fond of festivals of various kinds like jatrapala ( one kind of play portraying colorful stories ) , gambhira ( a kind of play portraying the social satire , political circumstances or the life story of god and goddesses ) , folk songs and dances , traditional musical instruments etc . consumption of chullu prepared mainly from rice is common for oraons during these occasions . besides , they regularly consume this drink during marriage ceremony , birth of a child or even in funeral . in fact , a section of the community makes a good living by preparing chullu . prior to survey , several meetings were held with the community members to explain the purposes of the study being conducted and to obtain their prior informed consent ( pic ) . the survey was carried out among the 19 villages ( chaknagar , haspukur , rishipur , bhabuk , parameshpur , dhumpur , nityanandapur , aktail , dangapara , lakhitur , habibpur , jhinjhinipukur , pakuahut , jagdala , kanturka , kenpukur , salaidanga , majhra , baidyapur ) of above mentioned four blocks ( gazole , bamongola , habibpur and old malda ) during last year ( april 2012 to june 2013 ) . hundreds of informants were interviewed to get the information regarding the chullu preparation and plants and plant - parts involved therein . the whole survey procedure involved several levels of interviewing such as semi - structured individual interviews , informal interviews , open - ended questionnaires , and group discussion with the local informants . hundreds of villagers of the study area were interviewed , but the information given by professional chullu producers and persons with proven knowledge on plants involved in chullu production were only recorded . after cross verification , the information obtained only from 201 chullu producers based on their experiences ( 174 female , 27 male ) of 19 villages were recorded . among the ethnomedicinal practitioners , we found 27 healers who were involved in traditional healing practices , gave information regarding the ethnic use of the same plants involved in chullu production for treating ailments . to survey the economic aspect of the chullu production , authors also interviewed 56 vendors of 19 villages . in order to evaluate the importance of the medicinal plants as per the local informants of the villages , ivs index measures the importance of a plant species based on how many informants cite one species as the most important one among the total number of informants ( value varies from 0 to 1 ) . ivs= nis / n ; where , nis= number of informants who consider the species to be the most important , and n= total number of informants . malda ( latitude and longitude of 244020n to 253208n and 882810e to 874550e respectively ) , a district of west bengal , india with a total land area of 3455.66 km2 . is known as mango district for its wide array of mango variety and production . it consists of two municipalities , 15 blocks or sub - divisions and 3701 villages with a population of more than forty lakh . the adh soi wetland ( beel ) , located at harischandrapur - ii block of the region , is one of the largest among the wetlands of the state comprising rich vegetation due to its macrophytic diversity . a few small forest areas are also scattered in old malda , habibpur , harishchandrapur and in gajol blocks . most of ethnic communities live mainly in the four blocks of this province namely , gazole , bamongola , habibpur and old malda comprising more than 85% of total tribal population . hence we considered these four blocks as our study area [ figure 1 ] due to its noticeable oraon population . the district has a hot summer ( 35 - 42c ) from march to september and a very cold winter ( 6 - 12c ) from november to february . the monsoon starts from june and continues to the mid - september and the average rainfall is approximately 1453.1 mm . the map of study area ( malda district ) showing four main oraon populated zones ( 1 : gazole block ; 2 : bamongola block ; 3 : old maldah block ; 4 : habibpur block ) oraon community is one of the largest tribal groups in india , possessing a unique tradition and culture . in malda district , a sizeable portion of the tribal communities are the oraons who mostly inhabits in the remote villages . they have distinctive lifestyle and are fond of festivals of various kinds like jatrapala ( one kind of play portraying colorful stories ) , gambhira ( a kind of play portraying the social satire , political circumstances or the life story of god and goddesses ) , folk songs and dances , traditional musical instruments etc . consumption of chullu prepared mainly from rice is common for oraons during these occasions . besides , they regularly consume this drink during marriage ceremony , birth of a child or even in funeral . in fact , a section of the community makes a good living by preparing chullu . prior to survey , several meetings were held with the community members to explain the purposes of the study being conducted and to obtain their prior informed consent ( pic ) . the survey was carried out among the 19 villages ( chaknagar , haspukur , rishipur , bhabuk , parameshpur , dhumpur , nityanandapur , aktail , dangapara , lakhitur , habibpur , jhinjhinipukur , pakuahut , jagdala , kanturka , kenpukur , salaidanga , majhra , baidyapur ) of above mentioned four blocks ( gazole , bamongola , habibpur and old malda ) during last year ( april 2012 to june 2013 ) . hundreds of informants were interviewed to get the information regarding the chullu preparation and plants and plant - parts involved therein . the sociocultural and marketing value of this drink has also been considered . the whole survey procedure involved several levels of interviewing such as semi - structured individual interviews , informal interviews , open - ended questionnaires , and group discussion with the local informants . hundreds of villagers of the study area were interviewed , but the information given by professional chullu producers and persons with proven knowledge on plants involved in chullu production were only recorded . after cross verification , the information obtained only from 201 chullu producers based on their experiences ( 174 female , 27 male ) of 19 villages were recorded . among the ethnomedicinal practitioners , we found 27 healers who were involved in traditional healing practices , gave information regarding the ethnic use of the same plants involved in chullu production for treating ailments . to survey the economic aspect of the chullu production , authors also interviewed 56 vendors of 19 villages . in order to evaluate the importance of the medicinal plants as per the local informants of the villages , the value of importance ( ivs ) index was determined . ivs index measures the importance of a plant species based on how many informants cite one species as the most important one among the total number of informants ( value varies from 0 to 1 ) . ivs= nis / n ; where , nis= number of informants who consider the species to be the most important , and n= total number of informants . after interviewing with 201 chullu producers , the local beverage procedure technology by oraon people was summarized under two main sub - legends : preparation of chullu -starter and preparation of chullu . it was observed that chullu production which has great impact on oraon people , indirectly help to uplift the village economy . a total of four medicinal plants including holarrhena pubescens , wattakaka volubilis , ichnocarpus frutescens and clerodendrum viscosum along with rice ( oryza sativa ) which were used in the preparation of this drink had great ethnomedicinal value in the locality . to prepare chullu - starter ( locally known as modguli ) , rice grains and 4 different plant parts are mixed together in a 2:1 ratios and dusted . briefly , rice grains are taken in earthen pot and cleaned in water , followed by drying under sunlight for 1 - 2 days . different plant parts like bark and leaves of h. pubescens , fruit and bark of w. volubilis , leaves of i. frutescens and c. viscosum are also cleaned well to remove dust particles and dried . then , the plant parts along with rice grains are powdered by dheki ( a wooden mortar with a large wooden handle ) . water ( 1/3 of the total powder ) is added to modgura to make dough and thick tablet like structures of 5 - 8 cm . in diameter the modgulis are kept in between two layers of straw for 4 - 5 days or until the pungent smell comes ( the process is known as jag - dewa ) . these modgulis are kept on clean cloth under sunlight for another 7 - 10 days . finally , the sundried chullu - starters are packaged depending upon their sizes for marketing [ figure 2 ] . preparation of chullu - starter to prepare chullu , cooked rice is the main ingredient . briefly , starters ( four to five pieces for 1 kg of cooked rice ) are dusted and mixed properly with cooked rice and taken in an earthen pot ( handi ) . water ( 400 - 500 ml ) is added into the pot covering the mouth with a banana leaf , followed by an earthen lid and left for fermentation . a yellowish watery juice with a strong alcoholic pungent smell comes out after 3 - 4 days , which is filtered with a clean cloth into another pot . fresh water ( 2 - 2.5 lit ) is added in the same earthen pot containing fermented rice and hanria and left for 12 - 18 h [ figure 3 ] . preparation of chullu now to prepare typical chullu , oraon follow their unique distillation process . in this preparation , the lowest pot containing the fermented rice with hanria , the middle one being an empty earthen pot with several pores ( known as jhanjhi ) at the bottom and the topmost aluminum pot filled with cold water . a pipe is inserted and sealed with mud at the side bottom of the middle earthen pot . the entire preparatory set is then placed over earthen oven starting the heating process [ figure 3 ] . after heating , the vapor goes up from the lower - most pot passes through the pores of the middle pot and comes in contact with upper - most pot containing cold water . due to cooling , the vapor condenses into water which comes down and is collected in the bottle through the pipe of the middle pot . thus , the prepared beverage is watery in color possessing alcoholic odor and is known as chullu or mod . finally , this alcoholic beverage is packaged in glass - bottle for selling in the market . during survey , we found that the plants used in chullu preparation had great local medicinal values . therefore , a semi - structured questionnaire and individual interview [ figure 4 ] among the healers had been carried out among 19 villages to obtain the information . after interviewing with some experienced traditional healers authors found a massive ethnomedicinal value in the region . however , as per our title concerned , we focused only on the above mentioned five plant species [ table 1 ] as those are used in chullu preparation . a total of 27 traditional experienced healers were chosen after cross verification for obtaining the information regarding ethnic use of those species only . questionnaire datasheet of ethnomedicinal use of plants in studied region list of medicinal plants investigated for chullu preparation and their respective ethnic uses it was observed that the herbal formulation from the bark of h. pubescens was prescribed by 22 healers out of 27 to treat chronic diarrhoea , chronic dysentery , urinary troubles , bleeding of piles etc . whereas c. viscosum was prescribed by 21 healers to treat several disorders . similarly , w. volubilis , i. frutescens , o. sativa were also used in various purposes as shown in table 1 . the ivs result exhibited high iv for all the species [ table 1 ] establishing greater ethnic knowledge regarding plant resources in the studied area . however , the ivs value of o. sativa is low ( 0.49 ) in comparison to others and prescribed only by only 13 healers . the study attempts to highlight that the above mentioned five plants are most valued species in the studied region and if sustainably harvested , they could be used as an alternative livelihood strategy for poor people . to prepare chullu - starter ( locally known as modguli ) , rice grains and 4 different plant parts are mixed together in a 2:1 ratios and dusted . briefly , rice grains are taken in earthen pot and cleaned in water , followed by drying under sunlight for 1 - 2 days . different plant parts like bark and leaves of h. pubescens , fruit and bark of w. volubilis , leaves of i. frutescens and c. viscosum are also cleaned well to remove dust particles and dried . then , the plant parts along with rice grains are powdered by dheki ( a wooden mortar with a large wooden handle ) . the powdered material is then sieved . the sieved material is locally known as modgura . water ( 1/3 of the total powder ) is added to modgura to make dough and thick tablet like structures of 5 - 8 cm . in diameter the modgulis are kept in between two layers of straw for 4 - 5 days or until the pungent smell comes ( the process is known as jag - dewa ) . these modgulis are kept on clean cloth under sunlight for another 7 - 10 days . finally , the sundried chullu - starters are packaged depending upon their sizes for marketing [ figure 2 ] . to prepare chullu - starter ( locally known as modguli ) , rice grains and 4 different plant parts are mixed together in a 2:1 ratios and dusted . briefly , rice grains are taken in earthen pot and cleaned in water , followed by drying under sunlight for 1 - 2 days . different plant parts like bark and leaves of h. pubescens , fruit and bark of w. volubilis , leaves of i. frutescens and c. viscosum are also cleaned well to remove dust particles and dried . then , the plant parts along with rice grains are powdered by dheki ( a wooden mortar with a large wooden handle ) . the powdered material is then sieved . the sieved material is locally known as modgura . water ( 1/3 of the total powder ) is added to modgura to make dough and thick tablet like structures of 5 - 8 cm . in diameter the modgulis are kept in between two layers of straw for 4 - 5 days or until the pungent smell comes ( the process is known as jag - dewa ) . these modgulis are kept on clean cloth under sunlight for another 7 - 10 days . finally , the sundried chullu - starters are packaged depending upon their sizes for marketing [ figure 2 ] . briefly , starters ( four to five pieces for 1 kg of cooked rice ) are dusted and mixed properly with cooked rice and taken in an earthen pot ( handi ) . water ( 400 - 500 ml ) is added into the pot covering the mouth with a banana leaf , followed by an earthen lid and left for fermentation . a yellowish watery juice with a strong alcoholic pungent smell comes out after 3 - 4 days , which is filtered with a clean cloth into another pot . fresh water ( 2 - 2.5 lit ) is added in the same earthen pot containing fermented rice and hanria and left for 12 - 18 h [ figure 3 ] . preparation of chullu now to prepare typical chullu , oraon follow their unique distillation process . in this preparation , the lowest pot containing the fermented rice with hanria , the middle one being an empty earthen pot with several pores ( known as jhanjhi ) at the bottom and the topmost aluminum pot filled with cold water . a pipe is inserted and sealed with mud at the side bottom of the middle earthen pot . the entire preparatory set is then placed over earthen oven starting the heating process [ figure 3 ] . after heating , the vapor goes up from the lower - most pot passes through the pores of the middle pot and comes in contact with upper - most pot containing cold water . due to cooling , the vapor condenses into water which comes down and is collected in the bottle through the pipe of the middle pot . thus , the prepared beverage is watery in color possessing alcoholic odor and is known as chullu or mod . finally , this alcoholic beverage is packaged in glass - bottle for selling in the market . during survey , we found that the plants used in chullu preparation had great local medicinal values . therefore , a semi - structured questionnaire and individual interview [ figure 4 ] among the healers had been carried out among 19 villages to obtain the information . after interviewing with some experienced traditional healers authors found a massive ethnomedicinal value in the region . however , as per our title concerned , we focused only on the above mentioned five plant species [ table 1 ] as those are used in chullu preparation . a total of 27 traditional experienced healers were chosen after cross verification for obtaining the information regarding ethnic use of those species only . questionnaire datasheet of ethnomedicinal use of plants in studied region list of medicinal plants investigated for chullu preparation and their respective ethnic uses it was observed that the herbal formulation from the bark of h. pubescens was prescribed by 22 healers out of 27 to treat chronic diarrhoea , chronic dysentery , urinary troubles , bleeding of piles etc . whereas c. viscosum was prescribed by 21 healers to treat several disorders . similarly , w. volubilis , i. frutescens , o. sativa were also used in various purposes as shown in table 1 . the ivs result exhibited high iv for all the species [ table 1 ] establishing greater ethnic knowledge regarding plant resources in the studied area . however , the ivs value of o. sativa is low ( 0.49 ) in comparison to others and prescribed only by only 13 healers . the study attempts to highlight that the above mentioned five plants are most valued species in the studied region and if sustainably harvested , they could be used as an alternative livelihood strategy for poor people . the local traditional liquor , chullu occupies a sizable portion of village economy , especially the economy of poor tribal people . the oraon community is actively involved in production and marketing of chullu . though the oraons are involved in chullu production , sometimes non - tribal agents provide funds to the tribals to produce liquor in a large scale and collect from them to be sold in the urban areas . the starters are processed into two different packets depending upon their sizes and sold at market . it was found that the large packets ( 5 - 7 cm diameter each ) of starter are sold @ 15 or usd 0.24 of per packet containing 8 pieces whereas small packets ( 2 - 3 cm diameter each ) are sold @ 8 or usd 0.13 of per packet with 8 pieces . hanria , the first alcoholic product during preparation of chullu is also sold @ 5 - 7 or usd 0.08 to 0.11 per glass of 100 ml whereas the typical chullu is sold @ 30 or usd 0.48 per bottle of 550 ml . during survey , we found that though the tribals consume chullu throughout the year , production of this drink usually at its peak in dry season like summer mainly because drying of starter is relatively easy in dry season . it was also observed that commercialization of chullu occurred when a groups of villagers from different parts gathered in local fairs , ritual ceremonies , jatrapalas , folk songs or dance programs or in other social activities . the other means of selling occurred when someone or a group of villagers go to the urban areas due to their personal purposes carry the native drink and sell those during their stay in urban areas . sales also occur through the agents who directly purchase the indigenous brew . as evident from table 2 , inhabitants of haspukur , baidyapur , kanturka , dhumpur , lakhitur , pakuahat , bhabuk and habibpur villages amongst 19 are more interested than others in preparing the local brew which indirectly helps to boost up their economic condition . however , the frequency of alcohol preparation in the villages was more than 65 percent suggesting high concern to prepare the traditional liquor . excess amount of local brew are also produced in those areas as per the demand of agents . hence , commercialization of local drink would be a good alternative way to the ethnic people if properly manufactured . frequency of chullu preparation in studied villages in selling local traditional brew , the most critical factors are the lack of proper infrastructure , proper management , communication , transportation , local market or beer - shops etc . it has been observed that some of the villages are in such remote areas that the transportation facilities are inaccessible . therefore it becomes difficult to reach to the desired places or sometimes become detached due to some natural calamities . as a result seasonality , especially rainy season is also a great factor . due to the presence of heavy moisture in the environment and/or inadequate sunlight , the starters the local traditional liquor , chullu occupies a sizable portion of village economy , especially the economy of poor tribal people . the oraon community is actively involved in production and marketing of chullu . though the oraons are involved in chullu production , sometimes non - tribal agents provide funds to the tribals to produce liquor in a large scale and collect from them to be sold in the urban areas . the starters are processed into two different packets depending upon their sizes and sold at market . it was found that the large packets ( 5 - 7 cm diameter each ) of starter are sold @ 15 or usd 0.24 of per packet containing 8 pieces whereas small packets ( 2 - 3 cm diameter each ) are sold @ 8 or usd 0.13 of per packet with 8 pieces . hanria , the first alcoholic product during preparation of chullu is also sold @ 5 - 7 or usd 0.08 to 0.11 per glass of 100 ml whereas the typical chullu is sold @ 30 or usd 0.48 per bottle of 550 ml . during survey , we found that though the tribals consume chullu throughout the year , production of this drink usually at its peak in dry season like summer mainly because drying of starter is relatively easy in dry season . it was also observed that commercialization of chullu occurred when a groups of villagers from different parts gathered in local fairs , ritual ceremonies , jatrapalas , folk songs or dance programs or in other social activities . the other means of selling occurred when someone or a group of villagers go to the urban areas due to their personal purposes carry the native drink and sell those during their stay in urban areas . sales also occur through the agents who directly purchase the indigenous brew . as evident from table 2 , inhabitants of haspukur , baidyapur , kanturka , dhumpur , lakhitur , pakuahat , bhabuk and habibpur villages amongst 19 are more interested than others in preparing the local brew which indirectly helps to boost up their economic condition . however , the frequency of alcohol preparation in the villages was more than 65 percent suggesting high concern to prepare the traditional liquor . excess amount of local brew are also produced in those areas as per the demand of agents . hence , commercialization of local drink would be a good alternative way to the ethnic people if properly manufactured . frequency of chullu preparation in studied villages in selling local traditional brew , the most critical factors are the lack of proper infrastructure , proper management , communication , transportation , local market or beer - shops etc . it has been observed that some of the villages are in such remote areas that the transportation facilities are inaccessible . therefore it becomes difficult to reach to the desired places or sometimes become detached due to some natural calamities . as a result seasonality , especially rainy season is also a great factor . due to the presence of heavy moisture in the environment and/or inadequate sunlight , the starters the knowledge of the wild medicinal plants used in various purposes are based on regular practices , oral transmission and are also influenced by several factors such as age , gender , relationship and other sociocultural factors generating variability in a particular zone . the use of selected plant species in chullu preparation distinguishes the oraon people from other communities in the studied region and it confirms that the knowledge is confined within this community . the open transfer of indigenous knowledge could only take place verbally along the family line , usually from older knowledgeable person to younger ones . the transfer of knowledge takes place hardly to the people outside the family and passed only on substantial cash payment . through the present survey we intended to have a detailed account of local drink production in selected regions of rural bengal . rural malda district to be precise , we have also given special emphasis to the tribal community and their way of preparing local drink , chullu . we found that chullu production , trade and marketing are a popular occupation among oraon communities . any kind of traditional alcoholic drink is popular among the tribal communities and the non - tribals because of its cheap cost and high alcohol content . the tribal communities use native brew virtually in every occasion , from birth to funeral . oraons have a popular belief that the plant parts used in chullu actually help them in combating against various ailments such as headache , insomnia etc . overall , it is apparent that the present survey is some of the most comprehensive one on chullu production and marketing in bengal and certainly the most exhaustive for rural malda district of west bengal .

aim : preparation of daily traditional drink by the indigenous tribes is a common phenomenon in india . oraon tribes in malda district of west bengal , india are very much practiced in making of their own native brew , known as chullu . therefore , the aim of this study was to explore the whole chullu procedure technology of the region and its socioeconomic effect on oraon . ethnomedicinal investigation of local plants involved in chullu preparation was another aspect of this study.materials and methods : the present study was conducted from april 2012 to june 2013 . consecutive field surveys were performed to collect information from chullu producers to focus the procedure technology of local brew by means of semi - structured individual interviews , informal interviews and group discussion . a semi - structured questionnaire process was also performed to obtain the information regarding the ethnic use of plant species involved in chullu preparation.results:the present study revealed that four medicinal plant species along with rice having strong local ethnomedicinal value were used to prepare this indigenous drink . oraon prepare the brew using their unique home - made distillation process . commercialization of this local brew represents an alternative income to develop their economic condition , especially for poor households . the index of importance value was considered to evaluate the importance , usage , and knowledge of the five studied species.conclusion:it could be concluded that practices of chullu preparation represent a bonding between ethnic knowledge and oraon people of the province . commercialization of chullu may be considered as a source of alternative way of income for poor households in the region .

INTRODUCTION MATERIALS AND METHODS Study Area Oraon Community Data Collection Total Key Informants Data Analysis RESULTS Chullu Procedure Technology Preparation of Chullu-starter Preparation of Chullu Ethnomedicinal Uses of Plants Involved in Chullu DISCUSSION Chullu and Village Economy Transmission of Knowledge CONCLUSION

almost all tribes prepare and consume traditional drink but their names , ingredients , and mode of preparation differ greatly from community to community and region to region . however , the tribals use local medicinal plant parts along with rice to prepare their native liquor and believe that the beverage prepared in this process prevents headache , insomnia , body ache , urinary troubles and cholera [ 2 - 4 ] . however , there is virtually no data available regarding the traditional alcoholic beverage preparation in the sub - himalayan non - hilly region of bengal . malda , district of west bengal in india , is characterized by its diversified historical dynasty , mango production , rich wetland , natural vegetation and its diversified ethnic groups including santala , oraon , rajbanshi , namasudre , polia , mundas , malpaharias etc . amongst these tribes , oraon community in fact chullu production is a part of their culture , identity , myths , and spiritual practices . their own traditional way of preparing the brew with medicinally important plants distinguished them from rest of the tribal communities of the region . besides so far there is no authentic documentation of chullu preparation technology used by oraons of malda district of west bengal , india . the plants those were used in preparing chullu , either known medicinally or have ethnic use among oraons. therefore , a semi - structured questionnaire process was also intended to focus the local use of the plants , their parts involved in medicinal practices and preparation of drugs from those plant parts . another aspect which was considered in this study was the economic significance of chullu production and sale . hence , it may be a pioneer study to explore the chullu procedure technology and its economic impact among oraon community of the district along with ethnomedicinal value of used plant species . malda ( latitude and longitude of 244020n to 253208n and 882810e to 874550e respectively ) , a district of west bengal , india with a total land area of 3455.66 km2 . the adh soi wetland ( beel ) , located at harischandrapur - ii block of the region , is one of the largest among the wetlands of the state comprising rich vegetation due to its macrophytic diversity . most of ethnic communities live mainly in the four blocks of this province namely , gazole , bamongola , habibpur and old malda comprising more than 85% of total tribal population . the map of study area ( malda district ) showing four main oraon populated zones ( 1 : gazole block ; 2 : bamongola block ; 3 : old maldah block ; 4 : habibpur block ) oraon community is one of the largest tribal groups in india , possessing a unique tradition and culture . in malda district , a sizeable portion of the tribal communities are the oraons who mostly inhabits in the remote villages . prior to survey , several meetings were held with the community members to explain the purposes of the study being conducted and to obtain their prior informed consent ( pic ) . the survey was carried out among the 19 villages ( chaknagar , haspukur , rishipur , bhabuk , parameshpur , dhumpur , nityanandapur , aktail , dangapara , lakhitur , habibpur , jhinjhinipukur , pakuahut , jagdala , kanturka , kenpukur , salaidanga , majhra , baidyapur ) of above mentioned four blocks ( gazole , bamongola , habibpur and old malda ) during last year ( april 2012 to june 2013 ) . hundreds of informants were interviewed to get the information regarding the chullu preparation and plants and plant - parts involved therein . the whole survey procedure involved several levels of interviewing such as semi - structured individual interviews , informal interviews , open - ended questionnaires , and group discussion with the local informants . hundreds of villagers of the study area were interviewed , but the information given by professional chullu producers and persons with proven knowledge on plants involved in chullu production were only recorded . after cross verification , the information obtained only from 201 chullu producers based on their experiences ( 174 female , 27 male ) of 19 villages were recorded . among the ethnomedicinal practitioners , we found 27 healers who were involved in traditional healing practices , gave information regarding the ethnic use of the same plants involved in chullu production for treating ailments . to survey the economic aspect of the chullu production , authors also interviewed 56 vendors of 19 villages . in order to evaluate the importance of the medicinal plants as per the local informants of the villages , ivs index measures the importance of a plant species based on how many informants cite one species as the most important one among the total number of informants ( value varies from 0 to 1 ) . malda ( latitude and longitude of 244020n to 253208n and 882810e to 874550e respectively ) , a district of west bengal , india with a total land area of 3455.66 km2 . the adh soi wetland ( beel ) , located at harischandrapur - ii block of the region , is one of the largest among the wetlands of the state comprising rich vegetation due to its macrophytic diversity . most of ethnic communities live mainly in the four blocks of this province namely , gazole , bamongola , habibpur and old malda comprising more than 85% of total tribal population . the map of study area ( malda district ) showing four main oraon populated zones ( 1 : gazole block ; 2 : bamongola block ; 3 : old maldah block ; 4 : habibpur block ) oraon community is one of the largest tribal groups in india , possessing a unique tradition and culture . in malda district , a sizeable portion of the tribal communities are the oraons who mostly inhabits in the remote villages . prior to survey , several meetings were held with the community members to explain the purposes of the study being conducted and to obtain their prior informed consent ( pic ) . the survey was carried out among the 19 villages ( chaknagar , haspukur , rishipur , bhabuk , parameshpur , dhumpur , nityanandapur , aktail , dangapara , lakhitur , habibpur , jhinjhinipukur , pakuahut , jagdala , kanturka , kenpukur , salaidanga , majhra , baidyapur ) of above mentioned four blocks ( gazole , bamongola , habibpur and old malda ) during last year ( april 2012 to june 2013 ) . hundreds of informants were interviewed to get the information regarding the chullu preparation and plants and plant - parts involved therein . the whole survey procedure involved several levels of interviewing such as semi - structured individual interviews , informal interviews , open - ended questionnaires , and group discussion with the local informants . hundreds of villagers of the study area were interviewed , but the information given by professional chullu producers and persons with proven knowledge on plants involved in chullu production were only recorded . after cross verification , the information obtained only from 201 chullu producers based on their experiences ( 174 female , 27 male ) of 19 villages were recorded . among the ethnomedicinal practitioners , we found 27 healers who were involved in traditional healing practices , gave information regarding the ethnic use of the same plants involved in chullu production for treating ailments . to survey the economic aspect of the chullu production , authors also interviewed 56 vendors of 19 villages . in order to evaluate the importance of the medicinal plants as per the local informants of the villages , the value of importance ( ivs ) index was determined . ivs index measures the importance of a plant species based on how many informants cite one species as the most important one among the total number of informants ( value varies from 0 to 1 ) . ivs= nis / n ; where , nis= number of informants who consider the species to be the most important , and n= total number of informants . after interviewing with 201 chullu producers , the local beverage procedure technology by oraon people was summarized under two main sub - legends : preparation of chullu -starter and preparation of chullu . it was observed that chullu production which has great impact on oraon people , indirectly help to uplift the village economy . a total of four medicinal plants including holarrhena pubescens , wattakaka volubilis , ichnocarpus frutescens and clerodendrum viscosum along with rice ( oryza sativa ) which were used in the preparation of this drink had great ethnomedicinal value in the locality . to prepare chullu - starter ( locally known as modguli ) , rice grains and 4 different plant parts are mixed together in a 2:1 ratios and dusted . then , the plant parts along with rice grains are powdered by dheki ( a wooden mortar with a large wooden handle ) . finally , the sundried chullu - starters are packaged depending upon their sizes for marketing [ figure 2 ] . preparation of chullu - starter to prepare chullu , cooked rice is the main ingredient . preparation of chullu now to prepare typical chullu , oraon follow their unique distillation process . in this preparation , the lowest pot containing the fermented rice with hanria , the middle one being an empty earthen pot with several pores ( known as jhanjhi ) at the bottom and the topmost aluminum pot filled with cold water . after heating , the vapor goes up from the lower - most pot passes through the pores of the middle pot and comes in contact with upper - most pot containing cold water . due to cooling , the vapor condenses into water which comes down and is collected in the bottle through the pipe of the middle pot . thus , the prepared beverage is watery in color possessing alcoholic odor and is known as chullu or mod . during survey , we found that the plants used in chullu preparation had great local medicinal values . therefore , a semi - structured questionnaire and individual interview [ figure 4 ] among the healers had been carried out among 19 villages to obtain the information . after interviewing with some experienced traditional healers authors found a massive ethnomedicinal value in the region . however , as per our title concerned , we focused only on the above mentioned five plant species [ table 1 ] as those are used in chullu preparation . a total of 27 traditional experienced healers were chosen after cross verification for obtaining the information regarding ethnic use of those species only . questionnaire datasheet of ethnomedicinal use of plants in studied region list of medicinal plants investigated for chullu preparation and their respective ethnic uses it was observed that the herbal formulation from the bark of h. pubescens was prescribed by 22 healers out of 27 to treat chronic diarrhoea , chronic dysentery , urinary troubles , bleeding of piles etc . the ivs result exhibited high iv for all the species [ table 1 ] establishing greater ethnic knowledge regarding plant resources in the studied area . the study attempts to highlight that the above mentioned five plants are most valued species in the studied region and if sustainably harvested , they could be used as an alternative livelihood strategy for poor people . to prepare chullu - starter ( locally known as modguli ) , rice grains and 4 different plant parts are mixed together in a 2:1 ratios and dusted . then , the plant parts along with rice grains are powdered by dheki ( a wooden mortar with a large wooden handle ) . the sieved material is locally known as modgura . water ( 1/3 of the total powder ) is added to modgura to make dough and thick tablet like structures of 5 - 8 cm . to prepare chullu - starter ( locally known as modguli ) , rice grains and 4 different plant parts are mixed together in a 2:1 ratios and dusted . then , the plant parts along with rice grains are powdered by dheki ( a wooden mortar with a large wooden handle ) . the sieved material is locally known as modgura . preparation of chullu now to prepare typical chullu , oraon follow their unique distillation process . in this preparation , the lowest pot containing the fermented rice with hanria , the middle one being an empty earthen pot with several pores ( known as jhanjhi ) at the bottom and the topmost aluminum pot filled with cold water . after heating , the vapor goes up from the lower - most pot passes through the pores of the middle pot and comes in contact with upper - most pot containing cold water . due to cooling , the vapor condenses into water which comes down and is collected in the bottle through the pipe of the middle pot . thus , the prepared beverage is watery in color possessing alcoholic odor and is known as chullu or mod . during survey , we found that the plants used in chullu preparation had great local medicinal values . therefore , a semi - structured questionnaire and individual interview [ figure 4 ] among the healers had been carried out among 19 villages to obtain the information . after interviewing with some experienced traditional healers authors found a massive ethnomedicinal value in the region . however , as per our title concerned , we focused only on the above mentioned five plant species [ table 1 ] as those are used in chullu preparation . a total of 27 traditional experienced healers were chosen after cross verification for obtaining the information regarding ethnic use of those species only . questionnaire datasheet of ethnomedicinal use of plants in studied region list of medicinal plants investigated for chullu preparation and their respective ethnic uses it was observed that the herbal formulation from the bark of h. pubescens was prescribed by 22 healers out of 27 to treat chronic diarrhoea , chronic dysentery , urinary troubles , bleeding of piles etc . the ivs result exhibited high iv for all the species [ table 1 ] establishing greater ethnic knowledge regarding plant resources in the studied area . the study attempts to highlight that the above mentioned five plants are most valued species in the studied region and if sustainably harvested , they could be used as an alternative livelihood strategy for poor people . the oraon community is actively involved in production and marketing of chullu . though the oraons are involved in chullu production , sometimes non - tribal agents provide funds to the tribals to produce liquor in a large scale and collect from them to be sold in the urban areas . hanria , the first alcoholic product during preparation of chullu is also sold @ 5 - 7 or usd 0.08 to 0.11 per glass of 100 ml whereas the typical chullu is sold @ 30 or usd 0.48 per bottle of 550 ml . it was also observed that commercialization of chullu occurred when a groups of villagers from different parts gathered in local fairs , ritual ceremonies , jatrapalas , folk songs or dance programs or in other social activities . the other means of selling occurred when someone or a group of villagers go to the urban areas due to their personal purposes carry the native drink and sell those during their stay in urban areas . as evident from table 2 , inhabitants of haspukur , baidyapur , kanturka , dhumpur , lakhitur , pakuahat , bhabuk and habibpur villages amongst 19 are more interested than others in preparing the local brew which indirectly helps to boost up their economic condition . however , the frequency of alcohol preparation in the villages was more than 65 percent suggesting high concern to prepare the traditional liquor . excess amount of local brew are also produced in those areas as per the demand of agents . hence , commercialization of local drink would be a good alternative way to the ethnic people if properly manufactured . frequency of chullu preparation in studied villages in selling local traditional brew , the most critical factors are the lack of proper infrastructure , proper management , communication , transportation , local market or beer - shops etc . it has been observed that some of the villages are in such remote areas that the transportation facilities are inaccessible . as a result seasonality , especially rainy season is also a great factor . due to the presence of heavy moisture in the environment and/or inadequate sunlight , the starters the local traditional liquor , chullu occupies a sizable portion of village economy , especially the economy of poor tribal people . the oraon community is actively involved in production and marketing of chullu . though the oraons are involved in chullu production , sometimes non - tribal agents provide funds to the tribals to produce liquor in a large scale and collect from them to be sold in the urban areas . hanria , the first alcoholic product during preparation of chullu is also sold @ 5 - 7 or usd 0.08 to 0.11 per glass of 100 ml whereas the typical chullu is sold @ 30 or usd 0.48 per bottle of 550 ml . it was also observed that commercialization of chullu occurred when a groups of villagers from different parts gathered in local fairs , ritual ceremonies , jatrapalas , folk songs or dance programs or in other social activities . the other means of selling occurred when someone or a group of villagers go to the urban areas due to their personal purposes carry the native drink and sell those during their stay in urban areas . as evident from table 2 , inhabitants of haspukur , baidyapur , kanturka , dhumpur , lakhitur , pakuahat , bhabuk and habibpur villages amongst 19 are more interested than others in preparing the local brew which indirectly helps to boost up their economic condition . however , the frequency of alcohol preparation in the villages was more than 65 percent suggesting high concern to prepare the traditional liquor . excess amount of local brew are also produced in those areas as per the demand of agents . hence , commercialization of local drink would be a good alternative way to the ethnic people if properly manufactured . frequency of chullu preparation in studied villages in selling local traditional brew , the most critical factors are the lack of proper infrastructure , proper management , communication , transportation , local market or beer - shops etc . as a result seasonality , especially rainy season is also a great factor . due to the presence of heavy moisture in the environment and/or inadequate sunlight , the starters the knowledge of the wild medicinal plants used in various purposes are based on regular practices , oral transmission and are also influenced by several factors such as age , gender , relationship and other sociocultural factors generating variability in a particular zone . the use of selected plant species in chullu preparation distinguishes the oraon people from other communities in the studied region and it confirms that the knowledge is confined within this community . through the present survey we intended to have a detailed account of local drink production in selected regions of rural bengal . rural malda district to be precise , we have also given special emphasis to the tribal community and their way of preparing local drink , chullu . overall , it is apparent that the present survey is some of the most comprehensive one on chullu production and marketing in bengal and certainly the most exhaustive for rural malda district of west bengal .

mobile health ( mhealth ) is a promising means of improving adherence to medication and clinic appointments thereby potentially improving clinical outcomes . mobile technology allows health care providers to communicate remotely with patients and more easily reach rural populations with poor or challenging transportation infrastructure . the number of mobile phone subscriptions in sub - saharan africa has been rising more rapidly than anywhere else in the world , allowing for innovative use of inexpensive mhealth technologies such as short - text messaging service ( sms ) . however , the results of recent studies using sms reminders for antiretroviral therapy ( art ) adherence from resource - limited settings have been mixed , with some studies showing an improvement and others showing no benefit . recent meta - analyses using evidence from randomized controlled trials have demonstrated that weekly sms is efficacious at improving self - reported art adherence . the start tb patients on art and retain on treatment ( start ) study is an innovative mixed methods cluster - randomized trial that evaluated the effectiveness , cost - effectiveness , and acceptability of a combination intervention package ( cip ) versus standard of care ( soc ) to improve early art initiation and retention during tuberculosis ( tb ) treatment , as well as tb treatment success , among patients with hiv / tb in lesotho . study methods have been described elsewhere , but in brief , 12 health facilities in the berea district were randomized to receive cip vs. soc from april 2013 to august 2015 . the cip included a range of programmatic , structural , and psychosocial components , including real - time patient adherence support using mhealth technology and trained village health workers ( vhws ) . vhws are community - based lay health workers who , subsequent to being trained under the ministry of health , provide essential health services at the community and household level , including health education , social welfare , and preventive health care services and the timely referral of individuals who are in need of facility - based medical care . the aim of this article is to describe the use and acceptability of the mhealth component of the start study intervention at the 6 cip health facilities , where the intervention was implemented . the mhealth intervention comprised the design and delivery of a standardized , automated sms system to provide real - time adherence support to patients on hiv and tb treatment . the study team developed a simple sms reminder application using commcare , an open source mobile platform developed by dimagi ( http://www.dimagi.com/ ) , a boston - based health informatics service provider that has supported many public health projects in developing countries . tools available online were used to build an application to send appointment and medication sms reminders to mobile phones based on a predefined algorithm . , patients with hiv and tb routinely identify a treatment supporter , usually a family member , to assist with day - to - day treatment support . for this study , patients and treatment supporters were given a choice to have the sms reminders sent to either one of them , or simultaneously to both . two types of sms reminders were sent at the recipients ' preferred time of day : appointment reminders were sent 2 days and 1 day before patients ' scheduled monthly clinic appointments and medication reminders were sent daily for the first 6 weeks of art , and daily or weekly thereafter according to individual preferences . patients and treatment supporters selected the time of day they wanted to receive appointment and medication reminders . medication reminders used code words ( did you eat your meal today ? ) to protect patients ' confidentiality . patients were not issued study phones because mobile phone usage at study initiation was found to be relatively high ( > 85% ) ; additionally , it was assumed that there was mobile phone reception throughout the study district . patients with access to a working phone were provided with approximately $ 3.70 in monthly airtime to facilitate communication with their vhw or nurse in case of difficulties ; incoming sms messages did not incur a charge . at each start cip facility , 12 vhws were selected to be facility - based vhws and to coordinate the activities of vhws providing community - based patient support . facility - based vhws were provided with password - protected mobile phones with the customized commcare application and sms text message capability , as well as monthly airtime to facilitate patient follow - up and support and to contact community - based vhws . facility - based vhws were trained to log patients ' information and preferred message time and frequency in the mobile phone application . data were transmitted to a secure cloud server in south africa that received the parameters entered by the vhws and sent outgoing messages in bulk to patients and treatment supporters ( fig . commcare provided access to patient data as well as data on outgoing messages on a secure web site , which helped the study team troubleshoot emergent problems , such as network and registration issues . the study team used the commcare reports module to monitor outgoing messages and to evaluate the implementation process . facility - based vhws provided detailed sms instructions to patients and treatment supporters , including practice exercises , to ensure their comfort and familiarity with mobile technology . mhealth use was quantitatively analyzed from study process data , drawn from a monthly program characteristics survey to track cip implementation , and an intervention receipt log for sms messages and mobile airtime to document the dosage of mhealth that was received by study patients and treatment supporters at the cip sites . mhealth acceptability was assessed using questions about intervention acceptability that were integrated into monthly follow - up interviews and qualitatively evaluated via in - depth interviews with a purposive , heterogeneous subset of patients and health care providers , 612 months post study initiation , at the cip sites . the use and acceptability of mhealth tools were contextualized by adherence data , drawn from monthly follow - up interviews with 371 patients ( measurement cohort ) at cip and soc sites during the course of tb treatment ( 69 months ) . all monthly follow - up interviews were based on standardized questionnaires and administered face - to - face by trained research assistants . all qualitative interviews were based on a semi - structured guide , conducted face - to - face by separate trained staff not directly involved in patient care , and thematically analyzed using a grounded theory approach . the study was reviewed and approved by the columbia university medical center institutional review board and the lesotho national health research and ethics committee . written informed consent was obtained from all participants completing study questionnaires and interviews . the mhealth intervention comprised the design and delivery of a standardized , automated sms system to provide real - time adherence support to patients on hiv and tb treatment . the study team developed a simple sms reminder application using commcare , an open source mobile platform developed by dimagi ( http://www.dimagi.com/ ) , a boston - based health informatics service provider that has supported many public health projects in developing countries . tools available online were used to build an application to send appointment and medication sms reminders to mobile phones based on a predefined algorithm . , patients with hiv and tb routinely identify a treatment supporter , usually a family member , to assist with day - to - day treatment support . for this study , patients and treatment supporters were given a choice to have the sms reminders sent to either one of them , or simultaneously to both . two types of sms reminders were sent at the recipients ' preferred time of day : appointment reminders were sent 2 days and 1 day before patients ' scheduled monthly clinic appointments and medication reminders were sent daily for the first 6 weeks of art , and daily or weekly thereafter according to individual preferences . patients and treatment supporters selected the time of day they wanted to receive appointment and medication reminders . medication reminders used code words ( did you eat your meal today ? ) to protect patients ' confidentiality . patients were not issued study phones because mobile phone usage at study initiation was found to be relatively high ( > 85% ) ; additionally , it was assumed that there was mobile phone reception throughout the study district . patients with access to a working phone were provided with approximately $ 3.70 in monthly airtime to facilitate communication with their vhw or nurse in case of difficulties ; incoming sms messages did not incur a charge . at each start cip facility , 12 vhws were selected to be facility - based vhws and to coordinate the activities of vhws providing community - based patient support . facility - based vhws were provided with password - protected mobile phones with the customized commcare application and sms text message capability , as well as monthly airtime to facilitate patient follow - up and support and to contact community - based vhws . facility - based vhws were trained to log patients ' information and preferred message time and frequency in the mobile phone application . data were transmitted to a secure cloud server in south africa that received the parameters entered by the vhws and sent outgoing messages in bulk to patients and treatment supporters ( fig . commcare provided access to patient data as well as data on outgoing messages on a secure web site , which helped the study team troubleshoot emergent problems , such as network and registration issues . the study team used the commcare reports module to monitor outgoing messages and to evaluate the implementation process . facility - based vhws provided detailed sms instructions to patients and treatment supporters , including practice exercises , to ensure their comfort and familiarity with mobile technology . mhealth use and acceptability were assessed using mixed methods . mhealth use was quantitatively analyzed from study process data , drawn from a monthly program characteristics survey to track cip implementation , and an intervention receipt log for sms messages and mobile airtime to document the dosage of mhealth that was received by study patients and treatment supporters at the cip sites . mhealth acceptability was assessed using questions about intervention acceptability that were integrated into monthly follow - up interviews and qualitatively evaluated via in - depth interviews with a purposive , heterogeneous subset of patients and health care providers , 612 months post study initiation , at the cip sites . the use and acceptability of mhealth tools were contextualized by adherence data , drawn from monthly follow - up interviews with 371 patients ( measurement cohort ) at cip and soc sites during the course of tb treatment ( 69 months ) . all monthly follow - up interviews were based on standardized questionnaires and administered face - to - face by trained research assistants . all qualitative interviews were based on a semi - structured guide , conducted face - to - face by separate trained staff not directly involved in patient care , and thematically analyzed using a grounded theory approach . the study was reviewed and approved by the columbia university medical center institutional review board and the lesotho national health research and ethics committee . over the study course of 29 months , the automated sms system successfully delivered 39,528 messages to 835 individuals who consented to receive study sms messages . of 713 patients who were eligible for sms reminders at cip sites , 657 ( 92.1% ) consented to receive the mhealth intervention , with no difference by gender . most patients received sms messages on their own ( n = 455 ) ; 24 treatment supporters received sms messages on behalf of their patients ; and in 178 cases , patients and their treatment supporters received the same sms message ( fig . ninety - six patients ( 15.2% ) switched from daily to weekly medication reminders and did so on average 45.4 39.1 days after enrollment ; a small number of patients [ n = 15 ( 15.6% ) ] subsequently switched back to daily medication reminders . patients ( pt ) and treatment supporters ( ts ) who received sms messages . during monthly follow - up interviews , 171 measurement cohort participants in cip sites were asked what had made it easier or helped them take their tb medications or art ; 41.9% stated the sms messages were a facilitator to adherence in at least 1 monthly follow - up interview . no measurement cohort participant reported that the sms messages posed a challenge to adherence in follow - up interviews . between february 2014 and january 2015 , in - depth qualitative interviews were completed with 30 health care providers and 30 patients , approximately equally distributed across the cip sites . health care provider participants included 10 nurses and 20 vhws with median 12 and 18 years of experience , respectively , providing services for patients and communities affected by hiv and/or tb . patient participants were on average 38.1 9.7 years old , 43.3% were women , and 73.3% had a primary education or no formal education ; these sociodemographic characteristics are representative of the populations served by participating health facilities ( table 1 ) . literacy , defined by the ability to read a full sentence , was found in 83.3% of patient participants . most patient participants ( 83.3% ) reported owning a mobile phone ; 30.0% reported electricity in the house . they perceived the appointment and medication reminders were complementary cues to adherence , alongside visits from vhws , and appreciated being able to self - select the time and frequency of messages . most patient participants also understood the coded text messages were a form of private health communication.it [ ie , sms ] helps me a lot because you may find that it is time when i get back home from work and by then i am tired , and i will just hear ringing phone , then when i take the phone to read i find out that it is the message that reminds me . ( male participant , age 35 years)i think the one that remind me to take my medication being the first class [ ie , excellent ] , i do n't make any mistake with the medication i fear and i ca n't miss them at all . ( male participant age 29 years)they do n't cause any problems because each and every one has his [ own ] phone and is private . ( female participant , age 26 years ) it [ ie , sms ] helps me a lot because you may find that it is time when i get back home from work and by then i am tired , and i will just hear ringing phone , then when i take the phone to read i find out that it is the message that reminds me . ( male participant , age 35 years ) i think the one that remind me to take my medication being the first class [ ie , excellent ] , i do n't make any mistake with the medication i fear and i ca n't miss them at all . ( male participant age 29 years ) they do n't cause any problems because each and every one has his [ own ] phone and is private . ( female participant , age 26 years ) patient participants were very appreciative of the airtime provided by the study . phone calls , above and beyond sms messages , empowered them to communicate with their health care providers and treatment supporters in a timely manner , without incurring a personal cost . patients said they more frequently called their provider to report a side effect , seek advice , or inform their clinic about potential delays to a clinic appointment . they also felt more inclined to call and request assistance from their treatment supporters.we are given airtime that we use it to communicate with our supporters if we have a problem . ( female participant , age 31 years ) we are given airtime that we use it to communicate with our supporters if we have a problem . ( female participant , age 31 years ) however , several participants reported routinely taking medications on their own with the aid of a phone alarm or were sufficiently reminded by their relatives , treatment supporter , or vhw . although they appreciated receiving reminders for times when they were preoccupied or fatigued , they perceived the sms service to be less necessary.my remembrance is that because i have a paper which i constantly mark in the morning and evening . it is the one which gives me understanding that in the morning when i have taken some , i mark there was no way i would say i have forgotten to take them ( male participant , age 28 years)you know these ones of tb i am still reminded by this person who is close to me , who is my wife , the one who comes and wakes me and also do things to help me . these ones of the afternoon which are the arvs i am still able to , even [ despite ] the sms enters to remind me . ( male participant , age 38 years ) my remembrance is that because i have a paper which i constantly mark in the morning and evening . it is the one which gives me understanding that in the morning when i have taken some , i mark there was no way i would say i have forgotten to take them ( male participant , age 28 years ) you know these ones of tb i am still reminded by this person who is close to me , who is my wife , the one who comes and wakes me and also do things to help me . these ones of the afternoon which are the arvs i am still able to , even [ despite ] the sms enters to remind me . ( male participant , age 38 years ) a few patient participants did not use or own a phone . they had registered their treatment supporter to receive sms reminders , who in turn , reminded them to take medications . as a result , they did not fully understand or appreciate the utility of the sms service.this little lady [ ie , my daughter ] is the one who supports me . ( female participant , age 56 years ) this little lady [ ie , my daughter ] is the one who supports me . ( female participant , age 56 years ) patients who were generally less familiar with mobile phones were at times confused by the coded messages . nonetheless , they managed to link the sms messages to medication intake.it [ ie , sms ] says have you eaten supper ( male participant , age 35 years ) it [ ie , sms ] says have you eaten supper ( male participant , age 35 years ) unstable access to electricity and temporary technical difficulties appeared to bar a few patient participants from receiving regular sms reminders.this phone has issue because at home we do n't have electricity it keeps giving us trouble as to where we charge and what to do like that . ( female participant , age 52 years)there was a time when they [ ie , sms ] were not coming through , but when i arrived here , i came to report it here and they said , there was a bit of a challenge but it is all well . ( male participant , age 53 years ) this phone has issue because at home we do n't have electricity it keeps giving us trouble as to where we charge and what to do like that . ( female participant , age 52 years ) there was a time when they [ ie , sms ] were not coming through , but when i arrived here , i came to report it here and they said , there was a bit of a challenge but it is all well . ( male participant , age 53 years ) health care provider participants reported receiving sufficient training and technical support to partake in phone - related study activities . they expressed support for the mhealth component of the intervention as it facilitated communication between patients , treatment supporters , and the various cadres of providers engaged in the cip . facility - based vhws appreciated the flexibility of being able to use study - issued phones for text messages as well as phone calls . nurses and facility - based vhws reported patients expressed feeling more cared for when they were followed up more frequently . provider bond.actually , the importance and goodness of airtime is communication , because , at times i left the patient not feeling well , so maybe when i 'm at home i will call to ask how she is feeling and she answers . even when it comes to the patients , i find it very good because it encourages them , showing them that they are cared for , they are assisted with health . even if they have nothing , they should have airtime . ( vhw ) actually , the importance and goodness of airtime is communication , because , at times i left the patient not feeling well , so maybe when i 'm at home i will call to ask how she is feeling and she answers . even when it comes to the patients , i find it very good because it encourages them , showing them that they are cared for , they are assisted with health . even if they have nothing , they should have airtime . ( vhw ) community - based vhws appreciated being able to monitor patients over phone , as they faced substantial geographic barriers and unpredictable weather when following patients living in disparate , remote rural areas . some vhws would call patients nearly every day to remind them to take daily doses and to ensure that they made it to their next clinic visit with the appropriate tools in hand such as sputum samples . this helped to avoid delays at the facility , capture and resolve potential reasons for missing a visit , and prevent nonadherence . routine communication helped them triage patients for emerging medical complications or adverse drug effects more efficiently and support patients through difficult periods in their treatment in a more meaningful way . vhws were also more easily able to track and follow - up with patients at risk for nonadherence or treatment interruption.the use of sms is very important suppose it rains heavily and i am unable to attend him , i text him and say , it is your time now . i will have taught him that when i say that , i mean it 's time to take his pills . so it is very helpful this sms thing , it helps us meet our patients . ( vhw ) the use of sms is very important suppose it rains heavily and i am unable to attend him , i text him and say , it is your time now . have you remembered your food? he already knows . i will have taught him that when i say that , i mean it 's time to take his pills . so it is very helpful this sms thing , it helps us meet our patients . ( vhw ) vhws were sensitive to patients ' experiences with hiv - related stigma and supported the use of simple , coded text messages that protected patients ' privacy and confidentiality . however , they worried that study calls could disrupt dynamics within households where patients had not disclosed their hiv status . in consultation with such patients , vhws developed innovative , patient - acceptable strategies to protect against inadvertent breaches in confidentiality.it is very important , the way it [ ie , sms ] is written . if ] you find that somone 's phone is in the wrong hands , then they get to know the patient 's issues too soon . [ name ] , when she was with the in - laws , her case was her secret . she was familiar with my number and if she did not respond to it i would know that the clouds have covered [ ie , there are people around ] but if i make her a callback and she does the same , i knew that it s clear and so we can talk . ( vhw ) it is very important , the way it [ ie , sms ] is written . if ] you find that somone 's phone is in the wrong hands , then they get to know the patient 's issues too soon . ( vhw ) the case of mrs . [ name ] , when she was with the in - laws , her case was her secret . she was familiar with my number and if she did not respond to it i would know that the clouds have covered [ ie , there are people around ] but if i make her a callback and she does the same , i knew that it s clear and so we can talk . ( vhw ) akin to reports from patient participants , health care providers also experienced technical challenges with the sms service ; most were quickly resolved after discussions with study staff or affected patients.when i was initially taught it was a bit of a struggle to understand as quick . but i ultimately got it even though a phone at times can give you problems here and there , and you find that here it gives a delivery report and at the head office where it is supposed to be sent to they say it does not arrive but when we fix that problem again you find that it goes through smoothly . for others you find that the patient has given you a certain number , in a blink of an eye he has changed it without telling you that he does n't use that number anymore . ( vhw ) when i was initially taught it was a bit of a struggle to understand as quick . but i ultimately got it even though a phone at times can give you problems here and there , and you find that here it gives a delivery report and at the head office where it is supposed to be sent to they say it does not arrive but when we fix that problem again you find that it goes through smoothly . for others you find that the patient has given you a certain number , in a blink of an eye he has changed it without telling you that he does n't use that number anymore . ( vhw ) data from monthly adherence assessment interviews among measurement cohort participants found that self - reported 30-day adherence to 100% of art were 86.3% in the cip versus 80.7% in soc , and to tb medications was 89.1% in cip versus 79.5% in soc ( table 2 ) ; adherence did not vary by gender . among patient participants in the qualitative evaluation ( n = 30 ) , the proportion reporting 100% adherence to art and tb medications was higher , at 90.0% and 93.3% , respectively . over the study course of 29 months , the automated sms system successfully delivered 39,528 messages to 835 individuals who consented to receive study sms messages . of 713 patients who were eligible for sms reminders at cip sites , 657 ( 92.1% ) consented to receive the mhealth intervention , with no difference by gender . most patients received sms messages on their own ( n = 455 ) ; 24 treatment supporters received sms messages on behalf of their patients ; and in 178 cases , patients and their treatment supporters received the same sms message ( fig . ninety - six patients ( 15.2% ) switched from daily to weekly medication reminders and did so on average 45.4 39.1 days after enrollment ; a small number of patients [ n = 15 ( 15.6% ) ] subsequently switched back to daily medication reminders . during monthly follow - up interviews , 171 measurement cohort participants in cip sites were asked what had made it easier or helped them take their tb medications or art ; 41.9% stated the sms messages were a facilitator to adherence in at least 1 monthly follow - up interview . no measurement cohort participant reported that the sms messages posed a challenge to adherence in follow - up interviews . between february 2014 and january 2015 , in - depth qualitative interviews were completed with 30 health care providers and 30 patients , approximately equally distributed across the cip sites . health care provider participants included 10 nurses and 20 vhws with median 12 and 18 years of experience , respectively , providing services for patients and communities affected by hiv and/or tb . patient participants were on average 38.1 9.7 years old , 43.3% were women , and 73.3% had a primary education or no formal education ; these sociodemographic characteristics are representative of the populations served by participating health facilities ( table 1 ) . literacy , defined by the ability to read a full sentence , was found in 83.3% of patient participants . most patient participants ( 83.3% ) reported owning a mobile phone ; 30.0% reported electricity in the house . they perceived the appointment and medication reminders were complementary cues to adherence , alongside visits from vhws , and appreciated being able to self - select the time and frequency of messages . most patient participants also understood the coded text messages were a form of private health communication.it [ ie , sms ] helps me a lot because you may find that it is time when i get back home from work and by then i am tired , and i will just hear ringing phone , then when i take the phone to read i find out that it is the message that reminds me . ( male participant , age 35 years)i think the one that remind me to take my medication being the first class [ ie , excellent ] , i do n't make any mistake with the medication i fear and i ca n't miss them at all . ( male participant age 29 years)they do n't cause any problems because each and every one has his [ own ] phone and is private . ( female participant , age 26 years ) it [ ie , sms ] helps me a lot because you may find that it is time when i get back home from work and by then i am tired , and i will just hear ringing phone , then when i take the phone to read i find out that it is the message that reminds me . ( male participant , age 35 years ) i think the one that remind me to take my medication being the first class [ ie , excellent ] , i do n't make any mistake with the medication i fear and i ca n't miss them at all . ( male participant age 29 years ) they do n't cause any problems because each and every one has his [ own ] phone and is private . ( female participant , age 26 years ) patient participants were very appreciative of the airtime provided by the study . phone calls , above and beyond sms messages , empowered them to communicate with their health care providers and treatment supporters in a timely manner , without incurring a personal cost . patients said they more frequently called their provider to report a side effect , seek advice , or inform their clinic about potential delays to a clinic appointment . they also felt more inclined to call and request assistance from their treatment supporters.we are given airtime that we use it to communicate with our supporters if we have a problem . ( female participant , age 31 years ) we are given airtime that we use it to communicate with our supporters if we have a problem . ( female participant , age 31 years ) however , several participants reported routinely taking medications on their own with the aid of a phone alarm or were sufficiently reminded by their relatives , treatment supporter , or vhw . although they appreciated receiving reminders for times when they were preoccupied or fatigued , they perceived the sms service to be less necessary.my remembrance is that because i have a paper which i constantly mark in the morning and evening . it is the one which gives me understanding that in the morning when i have taken some , i mark there was no way i would say i have forgotten to take them ( male participant , age 28 years)you know these ones of tb i am still reminded by this person who is close to me , who is my wife , the one who comes and wakes me and also do things to help me . these ones of the afternoon which are the arvs i am still able to , even [ despite ] the sms enters to remind me . ( male participant , age 38 years ) my remembrance is that because i have a paper which i constantly mark in the morning and evening . it is the one which gives me understanding that in the morning when i have taken some , i mark there was no way i would say i have forgotten to take them ( male participant , age 28 years ) you know these ones of tb i am still reminded by this person who is close to me , who is my wife , the one who comes and wakes me and also do things to help me . these ones of the afternoon which are the arvs i am still able to , even [ despite ] the sms enters to remind me . ( male participant , age 38 years ) a few patient participants did not use or own a phone . they had registered their treatment supporter to receive sms reminders , who in turn , reminded them to take medications . as a result , they did not fully understand or appreciate the utility of the sms service.this little lady [ ie , my daughter ] is the one who supports me . ( female participant , age 56 years ) this little lady [ ie , my daughter ] is the one who supports me . ( female participant , age 56 years ) patients who were generally less familiar with mobile phones were at times confused by the coded messages . nonetheless , they managed to link the sms messages to medication intake.it [ ie , sms ] says have you eaten supper ( male participant , age 35 years ) it [ ie , sms ] says have you eaten supper ( male participant , age 35 years ) unstable access to electricity and temporary technical difficulties appeared to bar a few patient participants from receiving regular sms reminders.this phone has issue because at home we do n't have electricity it keeps giving us trouble as to where we charge and what to do like that . ( female participant , age 52 years)there was a time when they [ ie , sms ] were not coming through , but when i arrived here , i came to report it here and they said , there was a bit of a challenge but it is all well . ( male participant , age 53 years ) this phone has issue because at home we do n't have electricity it keeps giving us trouble as to where we charge and what to do like that . ( female participant , age 52 years ) there was a time when they [ ie , sms ] were not coming through , but when i arrived here , i came to report it here and they said , there was a bit of a challenge but it is all well . ( male participant , age 53 years ) health care provider participants reported receiving sufficient training and technical support to partake in phone - related study activities . they expressed support for the mhealth component of the intervention as it facilitated communication between patients , treatment supporters , and the various cadres of providers engaged in the cip . facility - based vhws appreciated the flexibility of being able to use study - issued phones for text messages as well as phone calls . nurses and facility - based vhws reported patients expressed feeling more cared for when they were followed up more frequently . provider bond.actually , the importance and goodness of airtime is communication , because , at times i left the patient not feeling well , so maybe when i 'm at home i will call to ask how she is feeling and she answers . even when it comes to the patients , i find it very good because it encourages them , showing them that they are cared for , they are assisted with health . ( vhw ) actually , the importance and goodness of airtime is communication , because , at times i left the patient not feeling well , so maybe when i 'm at home i will call to ask how she is feeling and she answers . even when it comes to the patients , i find it very good because it encourages them , showing them that they are cared for , they are assisted with health . even if they have nothing , they should have airtime . ( vhw ) community - based vhws appreciated being able to monitor patients over phone , as they faced substantial geographic barriers and unpredictable weather when following patients living in disparate , remote rural areas . some vhws would call patients nearly every day to remind them to take daily doses and to ensure that they made it to their next clinic visit with the appropriate tools in hand such as sputum samples . this helped to avoid delays at the facility , capture and resolve potential reasons for missing a visit , and prevent nonadherence . routine communication helped them triage patients for emerging medical complications or adverse drug effects more efficiently and support patients through difficult periods in their treatment in a more meaningful way . vhws were also more easily able to track and follow - up with patients at risk for nonadherence or treatment interruption.the use of sms is very important suppose it rains heavily and i am unable to attend him , i text him and say , it is your time now . i will have taught him that when i say that , i mean it 's time to take his pills . so it is very helpful this sms thing , it helps us meet our patients . ( vhw ) the use of sms is very important suppose it rains heavily and i am unable to attend him , i text him and say , it is your time now . have you remembered your food? he already knows . i will have taught him that when i say that , i mean it 's time to take his pills . so it is very helpful this sms thing , it helps us meet our patients . ( vhw ) vhws were sensitive to patients ' experiences with hiv - related stigma and supported the use of simple , coded text messages that protected patients ' privacy and confidentiality . however , they worried that study calls could disrupt dynamics within households where patients had not disclosed their hiv status . in consultation with such patients , vhws developed innovative , patient - acceptable strategies to protect against inadvertent breaches in confidentiality.it is very important , the way it [ ie , sms ] is written . [ if ] you find that somone 's phone is in the wrong hands , then they get to know the patient 's issues too soon . [ name ] , when she was with the in - laws , her case was her secret . she was familiar with my number and if she did not respond to it i would know that the clouds have covered [ ie , there are people around ] but if i make her a callback and she does the same , i knew that it s clear and so we can talk . ( vhw ) it is very important , the way it [ ie , sms ] is written . [ if ] you find that somone 's phone is in the wrong hands , then they get to know the patient 's issues too soon . ( vhw ) the case of mrs . [ name ] , when she was with the in - laws , her case was her secret . she was familiar with my number and if she did not respond to it i would know that the clouds have covered [ ie , there are people around ] but if i make her a callback and she does the same , i knew that it s clear and so we can talk . ( vhw ) akin to reports from patient participants , health care providers also experienced technical challenges with the sms service ; most were quickly resolved after discussions with study staff or affected patients.when i was initially taught it was a bit of a struggle to understand as quick . but i ultimately got it even though a phone at times can give you problems here and there , and you find that here it gives a delivery report and at the head office where it is supposed to be sent to they say it does not arrive but when we fix that problem again you find that it goes through smoothly . for others you find that the patient has given you a certain number , in a blink of an eye he has changed it without telling you that he does n't use that number anymore . when i was initially taught it was a bit of a struggle to understand as quick . but i ultimately got it even though a phone at times can give you problems here and there , and you find that here it gives a delivery report and at the head office where it is supposed to be sent to they say it does not arrive but when we fix that problem again you find that it goes through smoothly . for others you find that the patient has given you a certain number , in a blink of an eye he has changed it without telling you that he does n't use that number anymore . ( vhw ) they perceived the appointment and medication reminders were complementary cues to adherence , alongside visits from vhws , and appreciated being able to self - select the time and frequency of messages . most patient participants also understood the coded text messages were a form of private health communication.it [ ie , sms ] helps me a lot because you may find that it is time when i get back home from work and by then i am tired , and i will just hear ringing phone , then when i take the phone to read i find out that it is the message that reminds me . ( male participant , age 35 years)i think the one that remind me to take my medication being the first class [ ie , excellent ] , i do n't make any mistake with the medication i fear and i ca n't miss them at all . ( male participant age 29 years)they do n't cause any problems because each and every one has his [ own ] phone and is private . ( female participant , age 26 years ) it [ ie , sms ] helps me a lot because you may find that it is time when i get back home from work and by then i am tired , and i will just hear ringing phone , then when i take the phone to read i find out that it is the message that reminds me . ( male participant , age 35 years ) i think the one that remind me to take my medication being the first class [ ie , excellent ] , i do n't make any mistake with the medication i fear and i ca n't miss them at all . ( male participant age 29 years ) they do n't cause any problems because each and every one has his [ own ] phone and is private . ( female participant , age 26 years ) patient participants were very appreciative of the airtime provided by the study . phone calls , above and beyond sms messages , empowered them to communicate with their health care providers and treatment supporters in a timely manner , without incurring a personal cost . patients said they more frequently called their provider to report a side effect , seek advice , or inform their clinic about potential delays to a clinic appointment . they also felt more inclined to call and request assistance from their treatment supporters.we are given airtime that we use it to communicate with our supporters if we have a problem . ( female participant , age 31 years ) we are given airtime that we use it to communicate with our supporters if we have a problem . ( female participant , age 31 years ) however , several participants reported routinely taking medications on their own with the aid of a phone alarm or were sufficiently reminded by their relatives , treatment supporter , or vhw . although they appreciated receiving reminders for times when they were preoccupied or fatigued , they perceived the sms service to be less necessary.my remembrance is that because i have a paper which i constantly mark in the morning and evening . it is the one which gives me understanding that in the morning when i have taken some , i mark there was no way i would say i have forgotten to take them ( male participant , age 28 years)you know these ones of tb i am still reminded by this person who is close to me , who is my wife , the one who comes and wakes me and also do things to help me . these ones of the afternoon which are the arvs i am still able to , even [ despite ] the sms enters to remind me . ( male participant , age 38 years ) my remembrance is that because i have a paper which i constantly mark in the morning and evening . it is the one which gives me understanding that in the morning when i have taken some , i mark there was no way i would say i have forgotten to take them ( male participant , age 28 years ) you know these ones of tb i am still reminded by this person who is close to me , who is my wife , the one who comes and wakes me and also do things to help me . these ones of the afternoon which are the arvs i am still able to , even [ despite ] the sms enters to remind me . ( male participant , age 38 years ) a few patient participants did not use or own a phone . they had registered their treatment supporter to receive sms reminders , who in turn , reminded them to take medications . as a result , they did not fully understand or appreciate the utility of the sms service.this little lady [ ie , my daughter ] is the one who supports me . she is the one who would be telling me that , hey , it 's time ( female participant , age 56 years ) this little lady [ ie , my daughter ] is the one who supports me . she is the one who would be telling me that , ( female participant , age 56 years ) patients who were generally less familiar with mobile phones were at times confused by the coded messages . nonetheless , they managed to link the sms messages to medication intake.it [ ie , sms ] says have you eaten supper ( male participant , age 35 years ) it [ ie , sms ] says have you eaten supper it reminds me that i have to eat before i take medication . ( male participant , age 35 years ) unstable access to electricity and temporary technical difficulties appeared to bar a few patient participants from receiving regular sms reminders.this phone has issue because at home we do n't have electricity it keeps giving us trouble as to where we charge and what to do like that . ( female participant , age 52 years)there was a time when they [ ie , sms ] were not coming through , but when i arrived here , i came to report it here and they said , there was a bit of a challenge but it is all well . ( male participant , age 53 years ) this phone has issue because at home we do n't have electricity it keeps giving us trouble as to where we charge and what to do like that . ( female participant , age 52 years ) there was a time when they [ ie , sms ] were not coming through , but when i arrived here , i came to report it here and they said , there was a bit of a challenge but it is all well . ( male participant , age 53 years ) health care provider participants reported receiving sufficient training and technical support to partake in phone - related study activities . they expressed support for the mhealth component of the intervention as it facilitated communication between patients , treatment supporters , and the various cadres of providers engaged in the cip . facility - based vhws appreciated the flexibility of being able to use study - issued phones for text messages as well as phone calls . nurses and facility - based vhws reported patients expressed feeling more cared for when they were followed up more frequently . provider bond.actually , the importance and goodness of airtime is communication , because , at times i left the patient not feeling well , so maybe when i 'm at home i will call to ask how she is feeling and she answers . even when it comes to the patients , i find it very good because it encourages them , showing them that they are cared for , they are assisted with health . even if they have nothing , they should have airtime . ( vhw ) actually , the importance and goodness of airtime is communication , because , at times i left the patient not feeling well , so maybe when i 'm at home i will call to ask how she is feeling and she answers . even when it comes to the patients , i find it very good because it encourages them , showing them that they are cared for , they are assisted with health . even if they have nothing , they should have airtime . ( vhw ) community - based vhws appreciated being able to monitor patients over phone , as they faced substantial geographic barriers and unpredictable weather when following patients living in disparate , remote rural areas . some vhws would call patients nearly every day to remind them to take daily doses and to ensure that they made it to their next clinic visit with the appropriate tools in hand such as sputum samples . this helped to avoid delays at the facility , capture and resolve potential reasons for missing a visit , and prevent nonadherence . routine communication helped them triage patients for emerging medical complications or adverse drug effects more efficiently and support patients through difficult periods in their treatment in a more meaningful way . vhws were also more easily able to track and follow - up with patients at risk for nonadherence or treatment interruption.the use of sms is very important suppose it rains heavily and i am unable to attend him , i text him and say , it is your time now . i will have taught him that when i say that , i mean it 's time to take his pills . so it is very helpful this sms thing , it helps us meet our patients . ( vhw ) the use of sms is very important suppose it rains heavily and i am unable to attend him , i text him and say , it is your time now . i will have taught him that when i say that , i mean it 's time to take his pills . so it is very helpful this sms thing , it helps us meet our patients . ( vhw ) vhws were sensitive to patients ' experiences with hiv - related stigma and supported the use of simple , coded text messages that protected patients ' privacy and confidentiality . however , they worried that study calls could disrupt dynamics within households where patients had not disclosed their hiv status . in consultation with such patients , vhws developed innovative , patient - acceptable strategies to protect against inadvertent breaches in confidentiality.it is very important , the way it [ ie , sms ] is written . if ] you find that somone 's phone is in the wrong hands , then they get to know the patient 's issues too soon . [ name ] , when she was with the in - laws , her case was her secret . she was familiar with my number and if she did not respond to it i would know that the clouds have covered [ ie , there are people around ] but if i make her a callback and she does the same , i knew that it s clear and so we can talk . ( vhw ) it is very important , the way it [ ie , sms ] is written . [ if ] you find that somone 's phone is in the wrong hands , then they get to know the patient 's issues too soon . ( vhw ) the case of mrs . [ name ] , when she was with the in - laws , her case was her secret . she was familiar with my number and if she did not respond to it i would know that the clouds have covered [ ie , there are people around ] but if i make her a callback and she does the same , i knew that it s clear and so we can talk . ( vhw ) akin to reports from patient participants , health care providers also experienced technical challenges with the sms service ; most were quickly resolved after discussions with study staff or affected patients.when i was initially taught it was a bit of a struggle to understand as quick . but i ultimately got it even though a phone at times can give you problems here and there , and you find that here it gives a delivery report and at the head office where it is supposed to be sent to they say it does not arrive but when we fix that problem again you find that it goes through smoothly . for others you find that the patient has given you a certain number , in a blink of an eye he has changed it without telling you that he does n't use that number anymore . ( vhw ) when i was initially taught it was a bit of a struggle to understand as quick . but i ultimately got it even though a phone at times can give you problems here and there , and you find that here it gives a delivery report and at the head office where it is supposed to be sent to they say it does not arrive but when we fix that problem again you find that it goes through smoothly . for others you find that the patient has given you a certain number , in a blink of an eye he has changed it without telling you that he does n't use that number anymore . ( vhw ) data from monthly adherence assessment interviews among measurement cohort participants found that self - reported 30-day adherence to 100% of art were 86.3% in the cip versus 80.7% in soc , and to tb medications was 89.1% in cip versus 79.5% in soc ( table 2 ) ; adherence did not vary by gender . among patient participants in the qualitative evaluation ( n = 30 ) , the proportion reporting 100% adherence to art and tb medications was higher , at 90.0% and 93.3% , respectively . in this mixed - methods implementation science study evaluating the use and acceptability of a cip to improve early art initiation and retention , and tb treatment success among hiv / tb patients in lesotho , the mhealth component of the intervention package was considered beneficial by both patients and health care providers . intervention delivery process data highlighted very high uptake of the sms intervention among both male and female patients . the perceived importance of sms messages in supporting adherence also arose during patients ' monthly follow - up interviews and in - depth qualitative interviews . qualitative interviews revealed many overlaps between the viewpoints of patient and health care provider participants . drivers of mhealth use included : cues to adherence , for medication intake and clinic appointments ; access to airtime , via the issuance of vouchers ; ability to place outgoing calls as well as to send sms messages ; personal choice in selecting the time and frequency of incoming sms messages ; and the use of private sms messages ( fig . 3 ) . these factors were perceived to enhance the quality and frequency of communication between patients and providers and between vhws and nurses , with positive impacts on patient provider interactions , provider workload , and quality of care . the interviews also allowed us to identify 2 groups of patients among whom the mhealth component of the cip was of less perceived value or less appreciated . this included patients with existing adherence supports who may have had less need for additional cues and patients who had not disclosed their hiv status to household members and were thus less comfortable reading text messages at home or in public places . technical challenges and confusion related to general phone use may have also impeded optimal utilization of the sms service . the optimal frequency of providing sms messages to patients has not been clear . in early 2012 when this study was designed , the only 2 sms trials published were from lester et al ( 2010 ) , who tested a weekly sms message and found it effective , and pop - eleches et al ( 2011 ) , who compared weekly and daily sms messages and found that weekly messages had a positive effect on adherence , whereas daily messages did not have an effect on adherence . in the start study , we chose to provide daily messages for the first 6 weeks of hiv / tb cotreatment and then to allow patients choose whether to continue daily messages or switch to weekly messages . it is interesting to note that only 15.2% of patients elected to switch from daily to weekly medication reminders and a percentage of those ( 15.6% ) subsequently switched back to daily reminders . overall , delivery of the mhealth intervention was found to be inexpensive , as one server can provide sms messages to thousands of patients in a large geographic area with very few human resources needed beyond the initial setup . the application was developed using an open source mobile platform . during 29 months of study implementation , the costs for troubleshooting the application before intervention launch and providing sms services to 835 individuals totaled $ 3124 ; study - issued airtime vouchers totaled $ 2233 per patient or treatment supporter , depending on the length of cotreatment . during dissemination of study results , the lesotho ministry of health ( moh ) indicated an interest in rolling out implementation of the sms intervention in a programmatic setting ; however , they did not commit to the provision of airtime . a 2011 systematic review of art adherence in 20 countries found the average rate of reporting 90% art adherence is 62% . this level of adherence in hiv monoinfected individuals is significantly lower than what was seen in our study , where 86.3% and 89.1% of hiv / tb co - infected patients in the cip arm reported adherence levels of 100% to hiv and tb medications , respectively ; a trend of higher adherence was noted in the cip arm versus the soc arm . the study design precludes evaluation of the effectiveness of individual components of the cip ; however , process data and qualitative analyses have highlighted patient and health care provider perspectives on the utility and acceptability of the mhealth intervention . first , mhealth interventions are more likely to be successful if they allow for flexibility in sms / call capability , frequency , and timing and are accompanied with training and technical support , routine patient instruction ( eg , on phone use and coded messages ) , and consideration of regional infrastructure ( eg , network , electricity ) . although airtime provision may increase intervention uptake and is likely to enhance acceptability , it is more costly and its adoption would depend on local feasibility and resources . second , the potential challenge of hiv stigma should be recognized and addressed in context - specific ways as hiv nondisclosure , an established proxy for stigma , may interfere with mhealth uptake . third , although sms technology is relatively inexpensive , in highly resource - constrained settings , it may be reserved for those patients who lack other sources of social and adherence support . the study has several limitations . unlike other studies that either provided a mobile phone or required access to one , phone ownership was not an inclusion criterion for the cip . patients without mobile phones may be different from those who have access to phones , thus limiting generalizability of our findings . however , given our implementation science framework and focus on sustainability , we did not find it feasible to issue study phones in this resource - limited setting . another limitation is in the lack of precision in measuring dosage of the mhealth intervention . similar to other mhealth studies , participants in the cip were not asked to respond to sms messages . one - way messages precluded us from being certain that messages were received and read by the individual patient . adherence may have been overreported in monthly follow - up interviews due to social desirability bias . to protect patient confidentiality , we did not link patients ' phone numbers and study ids , and thus we could not compare adherence levels of patients who received daily versus weekly adherence reminders . we were also unable to interview treatment supporters and learn from their unique experiences as recipients of the sms intervention . as such , we carefully measured intervention implementation processes as well as dosage of the intervention received . additionally , we integrated mixed methods , which allowed us to assess barriers and facilitators for medication adherence and to evaluate the acceptability and utilization of the sms intervention . second , the participating health facilities have adequate heterogeneity , covering both urban and rural locations , which strengthens the external validity of findings . third , more than half of the study sample were men , a group that is often hard to reach and support , and thus our findings can inform adherence and retention strategies targeting this priority population to achieve epidemic control . finally , having the support and engagement of the moh in the design and implementation phases of this study has fostered moh ownership at the national and district levels and will help to ensure sustainability of the mhealth intervention . the mhealth intervention for hiv / tb treatment support in lesotho was found to be a low - technology , user - friendly intervention , which was acceptable to patients and health care providers .

background : mhealth is a promising means of supporting adherence to treatment . the start tb patients on art and retain on treatment ( start ) study included real - time adherence support using short - text messaging service ( sms ) text messaging and trained village health workers ( vhws ) . we describe the use and acceptability of mhealth by patients with hiv / tuberculosis and health care providers.methods:patients and treatment supporters received automated , coded medication and appointment reminders at their preferred time and frequency , using their own phones , and $ 3.70 in monthly airtime . facility - based vhws were trained to log patient information and text message preferences into a mobile application and were given a password - protected mobile phone and airtime to communicate with community - based vhws . the use of mhealth tools was analyzed from process data over the study course . acceptability was evaluated during monthly follow - up interviews with all participants and during qualitative interviews with a subset of 30 patients and 30 health care providers at intervention sites . use and acceptability were contextualized by monthly adherence data.findings:from april 2013 to august 2015 , the automated sms system successfully delivered 39,528 messages to 835 individuals , including 633 patients and 202 treatment supporters . uptake of the sms intervention was high , with 92.1% of 713 eligible patients choosing to receive sms messages . patient and provider interviews yielded insight into barriers and facilitators to mhealth utilization . the intervention improved the quality of health communication between patients , treatment supporters , and providers . hiv - related stigma and technical challenges were identified as potential barriers.conclusions:the mhealth intervention for hiv / tuberculosis treatment support in lesotho was found to be a low - tech , user - friendly intervention , which was acceptable to patients and health care providers .

BACKGROUND METHODS mHealth Intervention Data Collection and Analysis Ethics and Consent Process FINDINGS Evaluation of mHealth Use Evaluation of Intervention Acceptability Patient Acceptability Health Care Provider Acceptability Evaluation of Adherence DISCUSSION CONCLUSION

mobile health ( mhealth ) is a promising means of improving adherence to medication and clinic appointments thereby potentially improving clinical outcomes . mobile technology allows health care providers to communicate remotely with patients and more easily reach rural populations with poor or challenging transportation infrastructure . the number of mobile phone subscriptions in sub - saharan africa has been rising more rapidly than anywhere else in the world , allowing for innovative use of inexpensive mhealth technologies such as short - text messaging service ( sms ) . the start tb patients on art and retain on treatment ( start ) study is an innovative mixed methods cluster - randomized trial that evaluated the effectiveness , cost - effectiveness , and acceptability of a combination intervention package ( cip ) versus standard of care ( soc ) to improve early art initiation and retention during tuberculosis ( tb ) treatment , as well as tb treatment success , among patients with hiv / tb in lesotho . study methods have been described elsewhere , but in brief , 12 health facilities in the berea district were randomized to receive cip vs. soc from april 2013 to august 2015 . the cip included a range of programmatic , structural , and psychosocial components , including real - time patient adherence support using mhealth technology and trained village health workers ( vhws ) . vhws are community - based lay health workers who , subsequent to being trained under the ministry of health , provide essential health services at the community and household level , including health education , social welfare , and preventive health care services and the timely referral of individuals who are in need of facility - based medical care . the aim of this article is to describe the use and acceptability of the mhealth component of the start study intervention at the 6 cip health facilities , where the intervention was implemented . the mhealth intervention comprised the design and delivery of a standardized , automated sms system to provide real - time adherence support to patients on hiv and tb treatment . for this study , patients and treatment supporters were given a choice to have the sms reminders sent to either one of them , or simultaneously to both . two types of sms reminders were sent at the recipients ' preferred time of day : appointment reminders were sent 2 days and 1 day before patients ' scheduled monthly clinic appointments and medication reminders were sent daily for the first 6 weeks of art , and daily or weekly thereafter according to individual preferences . patients and treatment supporters selected the time of day they wanted to receive appointment and medication reminders . patients were not issued study phones because mobile phone usage at study initiation was found to be relatively high ( > 85% ) ; additionally , it was assumed that there was mobile phone reception throughout the study district . patients with access to a working phone were provided with approximately $ 3.70 in monthly airtime to facilitate communication with their vhw or nurse in case of difficulties ; incoming sms messages did not incur a charge . at each start cip facility , 12 vhws were selected to be facility - based vhws and to coordinate the activities of vhws providing community - based patient support . facility - based vhws were provided with password - protected mobile phones with the customized commcare application and sms text message capability , as well as monthly airtime to facilitate patient follow - up and support and to contact community - based vhws . facility - based vhws were trained to log patients ' information and preferred message time and frequency in the mobile phone application . data were transmitted to a secure cloud server in south africa that received the parameters entered by the vhws and sent outgoing messages in bulk to patients and treatment supporters ( fig . facility - based vhws provided detailed sms instructions to patients and treatment supporters , including practice exercises , to ensure their comfort and familiarity with mobile technology . mhealth use was quantitatively analyzed from study process data , drawn from a monthly program characteristics survey to track cip implementation , and an intervention receipt log for sms messages and mobile airtime to document the dosage of mhealth that was received by study patients and treatment supporters at the cip sites . mhealth acceptability was assessed using questions about intervention acceptability that were integrated into monthly follow - up interviews and qualitatively evaluated via in - depth interviews with a purposive , heterogeneous subset of patients and health care providers , 612 months post study initiation , at the cip sites . the use and acceptability of mhealth tools were contextualized by adherence data , drawn from monthly follow - up interviews with 371 patients ( measurement cohort ) at cip and soc sites during the course of tb treatment ( 69 months ) . all monthly follow - up interviews were based on standardized questionnaires and administered face - to - face by trained research assistants . the mhealth intervention comprised the design and delivery of a standardized , automated sms system to provide real - time adherence support to patients on hiv and tb treatment . for this study , patients and treatment supporters were given a choice to have the sms reminders sent to either one of them , or simultaneously to both . two types of sms reminders were sent at the recipients ' preferred time of day : appointment reminders were sent 2 days and 1 day before patients ' scheduled monthly clinic appointments and medication reminders were sent daily for the first 6 weeks of art , and daily or weekly thereafter according to individual preferences . patients and treatment supporters selected the time of day they wanted to receive appointment and medication reminders . patients were not issued study phones because mobile phone usage at study initiation was found to be relatively high ( > 85% ) ; additionally , it was assumed that there was mobile phone reception throughout the study district . patients with access to a working phone were provided with approximately $ 3.70 in monthly airtime to facilitate communication with their vhw or nurse in case of difficulties ; incoming sms messages did not incur a charge . at each start cip facility , 12 vhws were selected to be facility - based vhws and to coordinate the activities of vhws providing community - based patient support . facility - based vhws were provided with password - protected mobile phones with the customized commcare application and sms text message capability , as well as monthly airtime to facilitate patient follow - up and support and to contact community - based vhws . facility - based vhws were trained to log patients ' information and preferred message time and frequency in the mobile phone application . data were transmitted to a secure cloud server in south africa that received the parameters entered by the vhws and sent outgoing messages in bulk to patients and treatment supporters ( fig . facility - based vhws provided detailed sms instructions to patients and treatment supporters , including practice exercises , to ensure their comfort and familiarity with mobile technology . mhealth use and acceptability were assessed using mixed methods . mhealth use was quantitatively analyzed from study process data , drawn from a monthly program characteristics survey to track cip implementation , and an intervention receipt log for sms messages and mobile airtime to document the dosage of mhealth that was received by study patients and treatment supporters at the cip sites . mhealth acceptability was assessed using questions about intervention acceptability that were integrated into monthly follow - up interviews and qualitatively evaluated via in - depth interviews with a purposive , heterogeneous subset of patients and health care providers , 612 months post study initiation , at the cip sites . the use and acceptability of mhealth tools were contextualized by adherence data , drawn from monthly follow - up interviews with 371 patients ( measurement cohort ) at cip and soc sites during the course of tb treatment ( 69 months ) . all monthly follow - up interviews were based on standardized questionnaires and administered face - to - face by trained research assistants . over the study course of 29 months , the automated sms system successfully delivered 39,528 messages to 835 individuals who consented to receive study sms messages . of 713 patients who were eligible for sms reminders at cip sites , 657 ( 92.1% ) consented to receive the mhealth intervention , with no difference by gender . most patients received sms messages on their own ( n = 455 ) ; 24 treatment supporters received sms messages on behalf of their patients ; and in 178 cases , patients and their treatment supporters received the same sms message ( fig . patients ( pt ) and treatment supporters ( ts ) who received sms messages . during monthly follow - up interviews , 171 measurement cohort participants in cip sites were asked what had made it easier or helped them take their tb medications or art ; 41.9% stated the sms messages were a facilitator to adherence in at least 1 monthly follow - up interview . no measurement cohort participant reported that the sms messages posed a challenge to adherence in follow - up interviews . between february 2014 and january 2015 , in - depth qualitative interviews were completed with 30 health care providers and 30 patients , approximately equally distributed across the cip sites . phone calls , above and beyond sms messages , empowered them to communicate with their health care providers and treatment supporters in a timely manner , without incurring a personal cost . they also felt more inclined to call and request assistance from their treatment supporters.we are given airtime that we use it to communicate with our supporters if we have a problem . ( female participant , age 31 years ) we are given airtime that we use it to communicate with our supporters if we have a problem . nonetheless , they managed to link the sms messages to medication intake.it [ ie , sms ] says have you eaten supper ( male participant , age 35 years ) it [ ie , sms ] says have you eaten supper ( male participant , age 35 years ) unstable access to electricity and temporary technical difficulties appeared to bar a few patient participants from receiving regular sms reminders.this phone has issue because at home we do n't have electricity it keeps giving us trouble as to where we charge and what to do like that . they expressed support for the mhealth component of the intervention as it facilitated communication between patients , treatment supporters , and the various cadres of providers engaged in the cip . ( vhw ) community - based vhws appreciated being able to monitor patients over phone , as they faced substantial geographic barriers and unpredictable weather when following patients living in disparate , remote rural areas . vhws were also more easily able to track and follow - up with patients at risk for nonadherence or treatment interruption.the use of sms is very important suppose it rains heavily and i am unable to attend him , i text him and say , it is your time now . ( vhw ) vhws were sensitive to patients ' experiences with hiv - related stigma and supported the use of simple , coded text messages that protected patients ' privacy and confidentiality . ( vhw ) akin to reports from patient participants , health care providers also experienced technical challenges with the sms service ; most were quickly resolved after discussions with study staff or affected patients.when i was initially taught it was a bit of a struggle to understand as quick . among patient participants in the qualitative evaluation ( n = 30 ) , the proportion reporting 100% adherence to art and tb medications was higher , at 90.0% and 93.3% , respectively . over the study course of 29 months , the automated sms system successfully delivered 39,528 messages to 835 individuals who consented to receive study sms messages . of 713 patients who were eligible for sms reminders at cip sites , 657 ( 92.1% ) consented to receive the mhealth intervention , with no difference by gender . most patients received sms messages on their own ( n = 455 ) ; 24 treatment supporters received sms messages on behalf of their patients ; and in 178 cases , patients and their treatment supporters received the same sms message ( fig . during monthly follow - up interviews , 171 measurement cohort participants in cip sites were asked what had made it easier or helped them take their tb medications or art ; 41.9% stated the sms messages were a facilitator to adherence in at least 1 monthly follow - up interview . no measurement cohort participant reported that the sms messages posed a challenge to adherence in follow - up interviews . between february 2014 and january 2015 , in - depth qualitative interviews were completed with 30 health care providers and 30 patients , approximately equally distributed across the cip sites . they perceived the appointment and medication reminders were complementary cues to adherence , alongside visits from vhws , and appreciated being able to self - select the time and frequency of messages . phone calls , above and beyond sms messages , empowered them to communicate with their health care providers and treatment supporters in a timely manner , without incurring a personal cost . they also felt more inclined to call and request assistance from their treatment supporters.we are given airtime that we use it to communicate with our supporters if we have a problem . ( female participant , age 31 years ) we are given airtime that we use it to communicate with our supporters if we have a problem . ( male participant , age 53 years ) health care provider participants reported receiving sufficient training and technical support to partake in phone - related study activities . they expressed support for the mhealth component of the intervention as it facilitated communication between patients , treatment supporters , and the various cadres of providers engaged in the cip . facility - based vhws appreciated the flexibility of being able to use study - issued phones for text messages as well as phone calls . nurses and facility - based vhws reported patients expressed feeling more cared for when they were followed up more frequently . ( vhw ) community - based vhws appreciated being able to monitor patients over phone , as they faced substantial geographic barriers and unpredictable weather when following patients living in disparate , remote rural areas . vhws were also more easily able to track and follow - up with patients at risk for nonadherence or treatment interruption.the use of sms is very important suppose it rains heavily and i am unable to attend him , i text him and say , it is your time now . ( vhw ) vhws were sensitive to patients ' experiences with hiv - related stigma and supported the use of simple , coded text messages that protected patients ' privacy and confidentiality . ( vhw ) akin to reports from patient participants , health care providers also experienced technical challenges with the sms service ; most were quickly resolved after discussions with study staff or affected patients.when i was initially taught it was a bit of a struggle to understand as quick . ( vhw ) they perceived the appointment and medication reminders were complementary cues to adherence , alongside visits from vhws , and appreciated being able to self - select the time and frequency of messages . phone calls , above and beyond sms messages , empowered them to communicate with their health care providers and treatment supporters in a timely manner , without incurring a personal cost . they also felt more inclined to call and request assistance from their treatment supporters.we are given airtime that we use it to communicate with our supporters if we have a problem . ( male participant , age 53 years ) health care provider participants reported receiving sufficient training and technical support to partake in phone - related study activities . they expressed support for the mhealth component of the intervention as it facilitated communication between patients , treatment supporters , and the various cadres of providers engaged in the cip . facility - based vhws appreciated the flexibility of being able to use study - issued phones for text messages as well as phone calls . nurses and facility - based vhws reported patients expressed feeling more cared for when they were followed up more frequently . ( vhw ) community - based vhws appreciated being able to monitor patients over phone , as they faced substantial geographic barriers and unpredictable weather when following patients living in disparate , remote rural areas . vhws were also more easily able to track and follow - up with patients at risk for nonadherence or treatment interruption.the use of sms is very important suppose it rains heavily and i am unable to attend him , i text him and say , it is your time now . ( vhw ) vhws were sensitive to patients ' experiences with hiv - related stigma and supported the use of simple , coded text messages that protected patients ' privacy and confidentiality . ( vhw ) akin to reports from patient participants , health care providers also experienced technical challenges with the sms service ; most were quickly resolved after discussions with study staff or affected patients.when i was initially taught it was a bit of a struggle to understand as quick . ( vhw ) data from monthly adherence assessment interviews among measurement cohort participants found that self - reported 30-day adherence to 100% of art were 86.3% in the cip versus 80.7% in soc , and to tb medications was 89.1% in cip versus 79.5% in soc ( table 2 ) ; adherence did not vary by gender . in this mixed - methods implementation science study evaluating the use and acceptability of a cip to improve early art initiation and retention , and tb treatment success among hiv / tb patients in lesotho , the mhealth component of the intervention package was considered beneficial by both patients and health care providers . intervention delivery process data highlighted very high uptake of the sms intervention among both male and female patients . the perceived importance of sms messages in supporting adherence also arose during patients ' monthly follow - up interviews and in - depth qualitative interviews . qualitative interviews revealed many overlaps between the viewpoints of patient and health care provider participants . drivers of mhealth use included : cues to adherence , for medication intake and clinic appointments ; access to airtime , via the issuance of vouchers ; ability to place outgoing calls as well as to send sms messages ; personal choice in selecting the time and frequency of incoming sms messages ; and the use of private sms messages ( fig . these factors were perceived to enhance the quality and frequency of communication between patients and providers and between vhws and nurses , with positive impacts on patient provider interactions , provider workload , and quality of care . technical challenges and confusion related to general phone use may have also impeded optimal utilization of the sms service . the optimal frequency of providing sms messages to patients has not been clear . overall , delivery of the mhealth intervention was found to be inexpensive , as one server can provide sms messages to thousands of patients in a large geographic area with very few human resources needed beyond the initial setup . during dissemination of study results , the lesotho ministry of health ( moh ) indicated an interest in rolling out implementation of the sms intervention in a programmatic setting ; however , they did not commit to the provision of airtime . the study design precludes evaluation of the effectiveness of individual components of the cip ; however , process data and qualitative analyses have highlighted patient and health care provider perspectives on the utility and acceptability of the mhealth intervention . first , mhealth interventions are more likely to be successful if they allow for flexibility in sms / call capability , frequency , and timing and are accompanied with training and technical support , routine patient instruction ( eg , on phone use and coded messages ) , and consideration of regional infrastructure ( eg , network , electricity ) . adherence may have been overreported in monthly follow - up interviews due to social desirability bias . we were also unable to interview treatment supporters and learn from their unique experiences as recipients of the sms intervention . additionally , we integrated mixed methods , which allowed us to assess barriers and facilitators for medication adherence and to evaluate the acceptability and utilization of the sms intervention . third , more than half of the study sample were men , a group that is often hard to reach and support , and thus our findings can inform adherence and retention strategies targeting this priority population to achieve epidemic control . the mhealth intervention for hiv / tb treatment support in lesotho was found to be a low - technology , user - friendly intervention , which was acceptable to patients and health care providers .

a study design was developed and implemented in canada , england , the netherlands , and the united states ( us ) between september 2010 and august 2011 . values for eq-5d-5l health states were elicited by means of tto ( not presented ) , visual analog scales ( not presented ) , and a choice model based on paired comparisons . this study was part of a larger pilot project that tested the performance of the software and it infrastructure for running multinational online surveys . the eq-5d-5l descriptive system comprises the same 5 dimensions as the eq-5d with 3 levels , that is , mobility ( mo ) , self - care ( sc ) , usual activities ( ua ) , pain / discomfort ( pd ) , anxiety / depression ( ad ) . each dimension has 5 levels : no problems , slight problems , moderate problems , severe problems , and extreme problems / unable to.13 on the basis of responses to the eq-5d health - state classifier , a preference - based scoring function can be applied that generates a single value for health . the current study investigates the feasibility of an alternative method for a scoring function that could be used to derive values . however , the values reported are not endorsed by the euroqol group or intended to replace existing value sets . in each of the 4 countries , at least 400 persons participated in the study . representative samples were recruited from the general population ( stratified by age , education , and sex ) , with a minimum age of 18 years . for the us cohort participants were recruited from the chicago area , a populous , ethnically diverse urban area . in the england study , the sample was recruited by approaching through email or telephone members of a panel of individuals ( belonging to the agency responsible for the fieldwork ) who had previously indicated a willingness to participate in research studies . in canada , participants were recruited by random cold phone call in 2 multiethnic cities : hamilton and montreal . english was used as the survey language in hamilton , whereas french was used in montreal . in the netherlands respondents the netherlands , and the us , participants self - completed the tasks in groups with limited interviewer assistance ( in particular intended for the tto task ) . for these computer - based assessments , about 3 trained interviewers oversaw groups of approximately 15 respondents in 68 sessions per day . in england , identical software was used ; however , a team of 8 home - based interviewers conducted the assessments in face - to - face interviews.14 respondents were paid a small sum for completing the survey ; the exact amount differed by country , ranging from $ 20 to $ 60 . a bayesian algorithm was used to generate an efficient design consisting of 200 paired comparisons ( ie , 400 health states ) for which priors were adapted based on an earlier study.15 constraints were applied to get a roughly level - balanced design . the number of very mild states for the eq-5d-5l generated by the algorithm was low , and some frequently observed health states were not included . because this could lead to less precise estimates of the lower levels of the domains , 10 pairs were constructed manually and included in the design ( ie , 1 or 2 domains at level 2 , whereas the other levels are at level 1 ) . the 200 paired comparisons were subdivided into 20 blocks so that each respondent would make 10 paired comparisons ( appendix ) . the order of the pairs and order within each pair were fully randomized within a digital setting by using a computer - assisted personal interview mode of administration : the euroqol valuation technology.12 we were also able to examine the efficiency of the design by comparing the predicted health - state values of all pairs . first , the respondent was asked to complete the eq-5d-5l measure for their own health as a warm - up exercise . next , they were given the most simple response task in the framework of choice modeling : a paired comparison between 2 different eq-5d health states ( fig . these paired comparison tasks did not include dead or duration statements ( see the discussion section ) . example of the paired comparison task for the eq-5d-5l ( top ) and the localization ( based on logistic regression resulting in predictions for all 3125 eq-5d-5l health states ) of this pair ( eq-5d-5l states 43534 vs. 32125 ) in relation to the other 199 pairs ( dashed 45-degree line indicates equal values for state a and b ; x - axis and y - axis sorted on predicted values for all 3125 states ) ; dark area roughly represents the combinations of the most informative pairs of health states ( approximately 70% vs. 30% preferred by respondents ) , around diagonal ( 50% vs. 50% ) and in the 2 corners ( approximately 90% preference for one of the 2 health states ) the noninformative pairs . the data were analyzed with a multinomial probit regression model ( asmprobit , stata ) yielding parameter estimates ( regression coefficients ) and estimated values for each health state ( applying these coefficients ) . the parameter estimates were relevant to evaluate the consistency of the discrete choice model and the similarity across countries , whereas the estimated values were only studied to examine similarity across countries . the main - effects model included 20 dummy variables representing level 2 , 3 , 4 , and 5 for each of the 5 domains : mo , sc , ua , pd , and ad . it also included an alternative - specific constant , capturing a tendency to always choose the first option , which can be considered as an indicator of feasibility . expressed in a formula , the model predicts latent values or utilities v of individuals choosing health state s ; represents a single vector of unknown regression coefficients ; and zrs indicates a vector of alternative - specific explanatory variables ( eg , dummies ) for individual r.models were run separately by country to assess the degree of variability across settings . a model was also run on the pooled dataset , including all 4 countries with the same 20 dummies . logical ordering of parameter estimates in all countries was used as criterion to assess the consistency of the models . differences in parameter estimates between countries were tested with independent t tests ( pooled variance ) , where p<0.01 was considered statistically significant , to correct for multiple testing . pearson correlations were estimated for the predicted 3125 eq-5d-5l health - state values to express the similarity between the countries , as well as intraclass correlations ( iccs ; 2-way mixed - effects , individual ratings , absolute agreement ) . in addition , graphs for the pooled data of the 4 countries combined with the data of the individual countries and their regression functions were made in sigmaplot ( version 11.0 ; systat software inc . respondents were asked to rate the ease and clarity of the exercise using a 5-point likert scale ( 1=agree , 5=disagree ) and drop - out rates were computed . a study design was developed and implemented in canada , england , the netherlands , and the united states ( us ) between september 2010 and august 2011 . values for eq-5d-5l health states were elicited by means of tto ( not presented ) , visual analog scales ( not presented ) , and a choice model based on paired comparisons . this study was part of a larger pilot project that tested the performance of the software and it infrastructure for running multinational online surveys . the eq-5d-5l descriptive system comprises the same 5 dimensions as the eq-5d with 3 levels , that is , mobility ( mo ) , self - care ( sc ) , usual activities ( ua ) , pain / discomfort ( pd ) , anxiety / depression ( ad ) . each dimension has 5 levels : no problems , slight problems , moderate problems , severe problems , and extreme problems / unable to.13 on the basis of responses to the eq-5d health - state classifier , a preference - based scoring function can be applied that generates a single value for health . the current study investigates the feasibility of an alternative method for a scoring function that could be used to derive values . however , the values reported are not endorsed by the euroqol group or intended to replace existing value sets . in each of the 4 countries , at least 400 persons participated in the study . representative samples were recruited from the general population ( stratified by age , education , and sex ) , with a minimum age of 18 years . for the us cohort participants were recruited from the chicago area , a populous , ethnically diverse urban area . in the england study , the sample was recruited by approaching through email or telephone members of a panel of individuals ( belonging to the agency responsible for the fieldwork ) who had previously indicated a willingness to participate in research studies . in canada , participants were recruited by random cold phone call in 2 multiethnic cities : hamilton and montreal . english was used as the survey language in hamilton , whereas french was used in montreal . in the netherlands respondents the netherlands , and the us , participants self - completed the tasks in groups with limited interviewer assistance ( in particular intended for the tto task ) . for these computer - based assessments , about 3 trained interviewers oversaw groups of approximately 15 respondents in 68 sessions per day . in england , identical software was used ; however , a team of 8 home - based interviewers conducted the assessments in face - to - face interviews.14 respondents were paid a small sum for completing the survey ; the exact amount differed by country , ranging from $ 20 to $ 60 . a bayesian algorithm was used to generate an efficient design consisting of 200 paired comparisons ( ie , 400 health states ) for which priors were adapted based on an earlier study.15 constraints were applied to get a roughly level - balanced design . the number of very mild states for the eq-5d-5l generated by the algorithm was low , and some frequently observed health states were not included . because this could lead to less precise estimates of the lower levels of the domains , 10 pairs were constructed manually and included in the design ( ie , 1 or 2 domains at level 2 , whereas the other levels are at level 1 ) . the 200 paired comparisons were subdivided into 20 blocks so that each respondent would make 10 paired comparisons ( appendix ) . the order of the pairs and order within each pair were fully randomized within a digital setting by using a computer - assisted personal interview mode of administration : the euroqol valuation technology.12 we were also able to examine the efficiency of the design by comparing the predicted health - state values of all pairs . first , the respondent was asked to complete the eq-5d-5l measure for their own health as a warm - up exercise . next , they were given the most simple response task in the framework of choice modeling : a paired comparison between 2 different eq-5d health states ( fig . these paired comparison tasks did not include dead or duration statements ( see the discussion section ) . example of the paired comparison task for the eq-5d-5l ( top ) and the localization ( based on logistic regression resulting in predictions for all 3125 eq-5d-5l health states ) of this pair ( eq-5d-5l states 43534 vs. 32125 ) in relation to the other 199 pairs ( dashed 45-degree line indicates equal values for state a and b ; x - axis and y - axis sorted on predicted values for all 3125 states ) ; dark area roughly represents the combinations of the most informative pairs of health states ( approximately 70% vs. 30% preferred by respondents ) , around diagonal ( 50% vs. 50% ) and in the 2 corners ( approximately 90% preference for one of the 2 health states ) the noninformative pairs . the data were analyzed with a multinomial probit regression model ( asmprobit , stata ) yielding parameter estimates ( regression coefficients ) and estimated values for each health state ( applying these coefficients ) . the parameter estimates were relevant to evaluate the consistency of the discrete choice model and the similarity across countries , whereas the estimated values were only studied to examine similarity across countries . the main - effects model included 20 dummy variables representing level 2 , 3 , 4 , and 5 for each of the 5 domains : mo , sc , ua , pd , and ad . it also included an alternative - specific constant , capturing a tendency to always choose the first option , which can be considered as an indicator of feasibility . expressed in a formula , the model predicts latent values or utilities v of individuals choosing health state s ; represents a single vector of unknown regression coefficients ; and zrs indicates a vector of alternative - specific explanatory variables ( eg , dummies ) for individual r.models were run separately by country to assess the degree of variability across settings . a model was also run on the pooled dataset , including all 4 countries with the same 20 dummies . logical ordering of parameter estimates in all countries was used as criterion to assess the consistency of the models . differences in parameter estimates between countries were tested with independent t tests ( pooled variance ) , where p<0.01 was considered statistically significant , to correct for multiple testing . pearson correlations were estimated for the predicted 3125 eq-5d-5l health - state values to express the similarity between the countries , as well as intraclass correlations ( iccs ; 2-way mixed - effects , individual ratings , absolute agreement ) . in addition , graphs for the pooled data of the 4 countries combined with the data of the individual countries and their regression functions were made in sigmaplot ( version 11.0 ; systat software inc . , san jose , ca ) to investigate differences in constant and slope . respondents were asked to rate the ease and clarity of the exercise using a 5-point likert scale ( 1=agree , 5=disagree ) and drop - out rates were computed . the number of individuals who entered the survey was 547 for canada , 404 for england , 407 for the netherlands , and 417 for the us . the number of judgments for each separate health state in the 4 countries ranged from 15 to 42 ( median , 22.5 ; sd 2.68 ) . in the dutch study , 1 block of states ( block 11 ) was not assessed due to a programming error . age distribution was similar in the 4 countries , although the netherlands had a smaller proportion of younger participants and a larger proportion of middle - aged ones ( table 1 ) . the mean age in the entire dataset was 40 ( sd 16 ) years , with a range of 18100 . the england sample included a larger proportion of degree - educated and employed individuals compared with the general population in england , but the sample was broadly representative of the general population in terms of other background characteristics , such as ethnicity.16 among us respondents , 70.8% reported that they received education beyond high school ; 65.8% were non - hispanic white ( n=273 ) , 17.6% were african american ( n=73 ) , and 16.6% were all other ethnicities . characteristics of the 4 samples the drop - out ( not completing all of the valuation tasks ) was low in all countries ( england 4 , the netherlands 14 ) . for the netherlands and england , the average duration ( s ) per task was 32.5 and 45.2 , respectively . for canada it was 35.85 ( sd 39.50 ; minimum , 0.81 ; maximum , 494.1 ) , and for the us it was 29.16 ( sd 37.07 ; minimum , 0.91 ; maximum 332.88 ) . mean responses for canada , england , the netherlands , and us on the 4 feasibility questions were as follows : the instructions that were given on the computer made it clear what i needed to do 2.34 , 2.27 , 2.31 , 2.31 ; it was easy to understand the questions i was asked 3.19 , 3.60 , 3.32 , 3.09 ; i found it difficult to decide 3.87 , 3.44 , 3.60 , 3.86 ; i found it easy to tell the difference between the health states i was asked to think about 2.63 , 2.47 , 2.60 , 2.53 . the alternative - specific constant parameter of the regression model ( table 2 ) showed a significant tendency among a subgroup of respondents in each country to choose the first health state . parameter estimates ( probit regression ) for the 4 countries separately and the 4 countries together four regression coefficients with illogical ordering were observed in the national datasets ( the netherlands : level 3 mo and pd were considered less severe than level 2 ; us : level 3 ua and pd was considered less severe than level 2 ) and one in the pooled data ( levels 2 and 3 pd were reversed ) . the spread of parameter estimates within each domain of health consistently followed the same patterns across domains and across countries : levels 2 and 3 lowered the values slightly and levels 4 and 5 even more so . all 20 parameters were statistically significant in all countries , with the exception of level 2 for ua in the netherlands ( p=0.51 ) ( table 2 ) . in comparing the relative value weights assigned to each dimension , sc and ua were generally assigned less weight than the other 3 domains , although there were differences between countries . dutch respondents were more concerned about severe and extreme pd and ad than about problems in the other domains . significant differences in coefficients were noted for pd level 4 ( canada vs. england : p<0.001 ) and level 5 ( canada vs. england , canada vs. the netherlands , england vs. us , and the netherlands vs. us : p<0.001 ) ; ad level 4 ( canada vs. england , england vs. us : p<0.001 ) and level 5 ( canada vs. england , england vs. us , the netherlands vs. us : p<0.001 ) ; mo level 5 ( england vs. the netherlands , the netherlands vs. us ( p<0.001 ) , and canada vs. us ( p=0.001 ) ; and ua level 2 ( canada vs. the netherlands , england vs. the netherlands : p<0.001 ) . likelihood ratio tests suggested that a pooled model offered a significantly worse fit to the data than a model with all parameters estimated separately for each country . inclusion of interaction terms for all combinations of the 20 dummies with country ( reference : england ) revealed that 9 interactions were statistically significant ( results not presented in table 2 ) . seven of these involved the domains pd and ad on levels 4 and 5 for england and the netherlands . separate analyses were performed for the 3 countries ( canada , england , us ) in which the pairs of states from block 11 were excluded . comparison with the dutch sample showed that omitting those states led to somewhat higher p - values for the regression coefficients , particularly for levels 2 and 3 . after the analyses without block 11 , however , comparable differences remained between the 3 countries . the cross - country comparison of predictions for the complete set of 3125 eq-5d-5l states demonstrated strong agreement across the 4 countries ( icc=0.89 ) ( fig . point estimates for agreement between any 2 countries were strong ( icc>0.5 ) , ranging from 0.80 for england versus us to 0.98 for canada versus us . pearson correlation coefficients reflected strength of agreement among 4 countries compared with the icc results ( table 3 ) . correlations , intraclass correlations , and regression functions between the 4 countries based on the 3125 estimated eq-5d-5l health states relationships between the 3125 estimated ( choice model , asmprobit ) eq-5d-5l values ( 0=best health state ) for the 4 countries compared with the pooled results of the 4 countries ( x - axis ) . the predicted values of the health states suggested that the assumptions underlying the efficient design ( plus the manual changes that were made to it ) were reasonable . the pooled predictions for the 400 states that were part of the 200 paired comparison tasks showed that many separate health states of the paired comparison tasks fulfilled efficient design assumptions ( approximately 70% vs. 30% preference ) . however , many paired comparison tasks also consisted of health states that were relatively close to each other in attributed value ( fig . the number of individuals who entered the survey was 547 for canada , 404 for england , 407 for the netherlands , and 417 for the us . the number of judgments for each separate health state in the 4 countries ranged from 15 to 42 ( median , 22.5 ; sd 2.68 ) . in the dutch study , 1 block of states ( block 11 ) was not assessed due to a programming error . age distribution was similar in the 4 countries , although the netherlands had a smaller proportion of younger participants and a larger proportion of middle - aged ones ( table 1 ) . the mean age in the entire dataset was 40 ( sd 16 ) years , with a range of 18100 . the england sample included a larger proportion of degree - educated and employed individuals compared with the general population in england , but the sample was broadly representative of the general population in terms of other background characteristics , such as ethnicity.16 among us respondents , 70.8% reported that they received education beyond high school ; 65.8% were non - hispanic white ( n=273 ) , 17.6% were african american ( n=73 ) , and 16.6% were all other ethnicities . the drop - out ( not completing all of the valuation tasks ) was low in all countries ( england 4 , the netherlands 14 ) . for the netherlands and england , the average duration ( s ) per task was 32.5 and 45.2 , respectively . for canada it was 35.85 ( sd 39.50 ; minimum , 0.81 ; maximum , 494.1 ) , and for the us it was 29.16 ( sd 37.07 ; minimum , 0.91 ; maximum 332.88 ) . mean responses for canada , england , the netherlands , and us on the 4 feasibility questions were as follows : the instructions that were given on the computer made it clear what i needed to do 2.34 , 2.27 , 2.31 , 2.31 ; it was easy to understand the questions i was asked 3.19 , 3.60 , 3.32 , 3.09 ; i found it difficult to decide 3.87 , 3.44 , 3.60 , 3.86 ; i found it easy to tell the difference between the health states i was asked to think about 2.63 , 2.47 , 2.60 , 2.53 . the alternative - specific constant parameter of the regression model ( table 2 ) showed a significant tendency among a subgroup of respondents in each country to choose the first health state . parameter estimates ( probit regression ) for the 4 countries separately and the 4 countries together four regression coefficients with illogical ordering were observed in the national datasets ( the netherlands : level 3 mo and pd were considered less severe than level 2 ; us : level 3 ua and pd was considered less severe than level 2 ) and one in the pooled data ( levels 2 and 3 pd were reversed ) . the spread of parameter estimates within each domain of health consistently followed the same patterns across domains and across countries : levels 2 and 3 lowered the values slightly and levels 4 and 5 even more so . all 20 parameters were statistically significant in all countries , with the exception of level 2 for ua in the netherlands ( p=0.51 ) ( table 2 ) . in comparing the relative value weights assigned to each dimension , sc and ua were generally assigned less weight than the other 3 domains , although there were differences between countries . dutch respondents were more concerned about severe and extreme pd and ad than about problems in the other domains . in the us , mo was the most important domain . significant differences in coefficients were noted for pd level 4 ( canada vs. england : p<0.001 ) and level 5 ( canada vs. england , canada vs. the netherlands , england vs. us , and the netherlands vs. us : p<0.001 ) ; ad level 4 ( canada vs. england , england vs. us : p<0.001 ) and level 5 ( canada vs. england , england vs. us , the netherlands vs. us : p<0.001 ) ; mo level 5 ( england vs. the netherlands , the netherlands vs. us ( p<0.001 ) , and canada vs. us ( p=0.001 ) ; and ua level 2 ( canada vs. the netherlands , england vs. the netherlands : p<0.001 ) . likelihood ratio tests suggested that a pooled model offered a significantly worse fit to the data than a model with all parameters estimated separately for each country . inclusion of interaction terms for all combinations of the 20 dummies with country ( reference : england ) revealed that 9 interactions were statistically significant ( results not presented in table 2 ) . seven of these involved the domains pd and ad on levels 4 and 5 for england and the netherlands . separate analyses were performed for the 3 countries ( canada , england , us ) in which the pairs of states from block 11 were excluded . comparison with the dutch sample showed that omitting those states led to somewhat higher p - values for the regression coefficients , particularly for levels 2 and 3 . after the analyses without block 11 , however , comparable differences remained between the 3 countries . the cross - country comparison of predictions for the complete set of 3125 eq-5d-5l states demonstrated strong agreement across the 4 countries ( icc=0.89 ) ( fig . point estimates for agreement between any 2 countries were strong ( icc>0.5 ) , ranging from 0.80 for england versus us to 0.98 for canada versus us . pearson correlation coefficients reflected strength of agreement among 4 countries compared with the icc results ( table 3 ) . correlations , intraclass correlations , and regression functions between the 4 countries based on the 3125 estimated eq-5d-5l health states relationships between the 3125 estimated ( choice model , asmprobit ) eq-5d-5l values ( 0=best health state ) for the 4 countries compared with the pooled results of the 4 countries ( x - axis ) . the predicted values of the health states suggested that the assumptions underlying the efficient design ( plus the manual changes that were made to it ) were reasonable . the pooled predictions for the 400 states that were part of the 200 paired comparison tasks showed that many separate health states of the paired comparison tasks fulfilled efficient design assumptions ( approximately 70% vs. 30% preference ) . however , many paired comparison tasks also consisted of health states that were relatively close to each other in attributed value ( fig . we found that it was feasible to implement a standardized protocol for computer - based assessment of a choice - based selection of health states for the eq-5d-5l across countries . models generated parameter estimates that logically supported the structure of the descriptive system and values for eq-5d-5l health states strongly agreed across countries , with only minor differences across the 4 countries . feedback from respondents indicated that they understood the tasks and interviewers did not report any concerns about the acceptability of the choice - based tasks . most respondents completed the paired comparisons , and few complained about the difficulty of the information they had to retain and compare . nevertheless , the feasibility questions revealed that choosing between the 2 options was not considered an easy task . in addition , the constant in the model indicates that a proportion of the respondents were not performing the tasks carefully . we expected logical ordering of coefficients in all countries , but observed a few inconsistencies . these were all related to illogical ordering from level 2 to level 3 and may be a result of our modest sample size in each country or other unidentified reasons . examining the parameter estimates across countries revealed high levels of agreement in the value ( overall impact ) of the various dimensions . however , differences between countries were observed , and likelihood ratio tests supported separate models by country rather than a single pooled model . large confidence intervals in agreement based on iccs between england and the other 3 countries suggest a structural difference between data from england and the other countries . after raising the number of levels in the eq-5d from 3 to 5 five - level states are much harder to think about than 3-level states , and a paired comparison of eq-5d-5l states that differ only subtly is more difficult than the task using the previous version ( eq-5d-3l ) . in addition , the bayesian approach to determine the set of paired comparisons was programmed in such a way that choice between states for these pairs would produce a high level of information but also make it harder to choose . there is a revival of interest in these methods due to the relative simplicity of eliciting ordinal responses and a widening range of analytic tools to accommodate them.7,1721 one previous study suggested that choice models have a practical advantage : when conducting choice experiments , health states may be evaluated in a self - completion format using online panels.22 this study confirmed that result . in the english arm of this study , data were obtained through face - to - face interviews . in the other 3 countries , data were collected largely in a self - completion format . nonetheless , we observed no appreciable differences between england and the other countries in data quality ( ie , number of complete responses , duration ) . this means that valuation studies based on discrete choices appear to have an important advantage over tto techniques because the latter require face - to - face interviews to produce data of reasonable credibility.14 one of the most serious limitations of choice - based models is that they produce relative values . although differences between the values are meaningful , the positions of the top and bottom values are not interpretable . that is , they are on an undefined scale ( without meaningful anchors such as 0=dead and 1=full health ) . several attempts have been made to resolve this issue.15,17,23,24 a limitation of our study may be that interaction terms were not included in the modeling , yet they may be found in the available data . this effect seems moderate ; however , and to capture it would require a more elaborate study.17 another limitation is the disability to generalize to older individuals and to those with low education , as these were less represented in our study samples . the paired comparisons offered to respondents in this study did not specify the duration of the states . it is possible that the respondents imposed their own ideas about the duration of the states when making the paired comparisons . such concealed ideas about time are probably diverse among the population , theoretically increasing systematic errors and may be biasing the obtained health - state values . however , as the 2 states in each of pairs were similar in the present study , a duration statement may have minimal impact on the responses . from a measurement perspective , it may be better to describe the subject of interest ( eg , health states ) as uniformly and distinctively as possible.25 interest in cross - country variation in health - state valuations is growing . there is some evidence that the results from one country can not be transferred to other countries.2629 these studies suggest that differences exist in the values given to the same states . however , it is hard to say whether any differences in these values are due to cultural notions , methodological differences , or to translational issues ( eg , dutch wording may make levels 2 and 3 seem closer together than in other language versions ) . in this study , the 5 levels in the dutch and french ( canada ) language eq-5d-5l may not exactly match the 5 levels in the english language eq-5d-5l because of language differences.14 interestingly , a recent study to measure disability weights for a wide array of health outcomes across a diverse range of populations showed that , based on the same measurement framework that we used in our study , the differences between countries were modest.30 to conclude , parameter estimates modeled from a choice - based approach were generally consistent and logical , although some deviations were observed . the estimated values were similar between the countries , and the differences may be attributed to the administration of the valuation exercise in different countries , and also due to cultural differences . overall , results indicated that it is feasible to collect valid paired comparison data with limited interviewer assistance , supporting the possibility of data collection by means of online panels .

aims : to investigate the feasibility of discrete choice experiments for valuing eq-5d-5l states using computer - based data collection , the consistency of the estimated regression coefficients produced after modeling the preference data , and to examine the similarity of the values derived across countries.methods:data were collected in canada , england , the netherlands , and the united states ( us ) . interactive software was developed to standardize the format of the choice tasks across countries , except for face - to - face interviewing in england . the choice task required respondents to choose between 2 suboptimal health states . a bayesian design was used to generate 200 pairs of states that were randomly grouped into 20 blocks . each respondent completed 1 block of 10 pairs . a main - effects probit model was used to estimate regression coefficients and to derive values.results:approximately 400 respondents participated from each country . the mean time to perform 1 choice task was between 29.2 ( us ) and 45.2 ( england ) seconds . all regression coefficients were statistically significant , except level 2 for usual activities in the netherlands ( p=0.51 ) . predictions for the complete set of 3125 eq-5d-5l health states were similar for the 4 countries . intraclass correlation coefficients between the countries were high : from 0.80 ( england vs. us ) through 0.98 ( canada vs. us).conclusions : derivation of value sets from the general population using computer - based choice tasks for the eq-5d-5l is feasible . parameter estimates were generally consistent and logical , and health - state values were similar across the 4 countries .

METHODS Overview EuroQol-5D-5L Respondents Experimental Design Data Collection Analysis RESULTS Completion Demographics Feasibility Parameters of Choice Models Similarity of Health-State Values Design Efficiency DISCUSSION

a study design was developed and implemented in canada , england , the netherlands , and the united states ( us ) between september 2010 and august 2011 . values for eq-5d-5l health states were elicited by means of tto ( not presented ) , visual analog scales ( not presented ) , and a choice model based on paired comparisons . each dimension has 5 levels : no problems , slight problems , moderate problems , severe problems , and extreme problems / unable to.13 on the basis of responses to the eq-5d health - state classifier , a preference - based scoring function can be applied that generates a single value for health . the current study investigates the feasibility of an alternative method for a scoring function that could be used to derive values . in each of the 4 countries , at least 400 persons participated in the study . representative samples were recruited from the general population ( stratified by age , education , and sex ) , with a minimum age of 18 years . in the netherlands respondents the netherlands , and the us , participants self - completed the tasks in groups with limited interviewer assistance ( in particular intended for the tto task ) . in england , identical software was used ; however , a team of 8 home - based interviewers conducted the assessments in face - to - face interviews.14 respondents were paid a small sum for completing the survey ; the exact amount differed by country , ranging from $ 20 to $ 60 . a bayesian algorithm was used to generate an efficient design consisting of 200 paired comparisons ( ie , 400 health states ) for which priors were adapted based on an earlier study.15 constraints were applied to get a roughly level - balanced design . the number of very mild states for the eq-5d-5l generated by the algorithm was low , and some frequently observed health states were not included . because this could lead to less precise estimates of the lower levels of the domains , 10 pairs were constructed manually and included in the design ( ie , 1 or 2 domains at level 2 , whereas the other levels are at level 1 ) . the order of the pairs and order within each pair were fully randomized within a digital setting by using a computer - assisted personal interview mode of administration : the euroqol valuation technology.12 we were also able to examine the efficiency of the design by comparing the predicted health - state values of all pairs . next , they were given the most simple response task in the framework of choice modeling : a paired comparison between 2 different eq-5d health states ( fig . example of the paired comparison task for the eq-5d-5l ( top ) and the localization ( based on logistic regression resulting in predictions for all 3125 eq-5d-5l health states ) of this pair ( eq-5d-5l states 43534 vs. 32125 ) in relation to the other 199 pairs ( dashed 45-degree line indicates equal values for state a and b ; x - axis and y - axis sorted on predicted values for all 3125 states ) ; dark area roughly represents the combinations of the most informative pairs of health states ( approximately 70% vs. 30% preferred by respondents ) , around diagonal ( 50% vs. 50% ) and in the 2 corners ( approximately 90% preference for one of the 2 health states ) the noninformative pairs . the parameter estimates were relevant to evaluate the consistency of the discrete choice model and the similarity across countries , whereas the estimated values were only studied to examine similarity across countries . the main - effects model included 20 dummy variables representing level 2 , 3 , 4 , and 5 for each of the 5 domains : mo , sc , ua , pd , and ad . logical ordering of parameter estimates in all countries was used as criterion to assess the consistency of the models . differences in parameter estimates between countries were tested with independent t tests ( pooled variance ) , where p<0.01 was considered statistically significant , to correct for multiple testing . pearson correlations were estimated for the predicted 3125 eq-5d-5l health - state values to express the similarity between the countries , as well as intraclass correlations ( iccs ; 2-way mixed - effects , individual ratings , absolute agreement ) . in addition , graphs for the pooled data of the 4 countries combined with the data of the individual countries and their regression functions were made in sigmaplot ( version 11.0 ; systat software inc . a study design was developed and implemented in canada , england , the netherlands , and the united states ( us ) between september 2010 and august 2011 . values for eq-5d-5l health states were elicited by means of tto ( not presented ) , visual analog scales ( not presented ) , and a choice model based on paired comparisons . each dimension has 5 levels : no problems , slight problems , moderate problems , severe problems , and extreme problems / unable to.13 on the basis of responses to the eq-5d health - state classifier , a preference - based scoring function can be applied that generates a single value for health . the current study investigates the feasibility of an alternative method for a scoring function that could be used to derive values . in each of the 4 countries , at least 400 persons participated in the study . representative samples were recruited from the general population ( stratified by age , education , and sex ) , with a minimum age of 18 years . in the netherlands respondents the netherlands , and the us , participants self - completed the tasks in groups with limited interviewer assistance ( in particular intended for the tto task ) . in england , identical software was used ; however , a team of 8 home - based interviewers conducted the assessments in face - to - face interviews.14 respondents were paid a small sum for completing the survey ; the exact amount differed by country , ranging from $ 20 to $ 60 . a bayesian algorithm was used to generate an efficient design consisting of 200 paired comparisons ( ie , 400 health states ) for which priors were adapted based on an earlier study.15 constraints were applied to get a roughly level - balanced design . the number of very mild states for the eq-5d-5l generated by the algorithm was low , and some frequently observed health states were not included . because this could lead to less precise estimates of the lower levels of the domains , 10 pairs were constructed manually and included in the design ( ie , 1 or 2 domains at level 2 , whereas the other levels are at level 1 ) . the order of the pairs and order within each pair were fully randomized within a digital setting by using a computer - assisted personal interview mode of administration : the euroqol valuation technology.12 we were also able to examine the efficiency of the design by comparing the predicted health - state values of all pairs . next , they were given the most simple response task in the framework of choice modeling : a paired comparison between 2 different eq-5d health states ( fig . example of the paired comparison task for the eq-5d-5l ( top ) and the localization ( based on logistic regression resulting in predictions for all 3125 eq-5d-5l health states ) of this pair ( eq-5d-5l states 43534 vs. 32125 ) in relation to the other 199 pairs ( dashed 45-degree line indicates equal values for state a and b ; x - axis and y - axis sorted on predicted values for all 3125 states ) ; dark area roughly represents the combinations of the most informative pairs of health states ( approximately 70% vs. 30% preferred by respondents ) , around diagonal ( 50% vs. 50% ) and in the 2 corners ( approximately 90% preference for one of the 2 health states ) the noninformative pairs . the parameter estimates were relevant to evaluate the consistency of the discrete choice model and the similarity across countries , whereas the estimated values were only studied to examine similarity across countries . the main - effects model included 20 dummy variables representing level 2 , 3 , 4 , and 5 for each of the 5 domains : mo , sc , ua , pd , and ad . logical ordering of parameter estimates in all countries was used as criterion to assess the consistency of the models . differences in parameter estimates between countries were tested with independent t tests ( pooled variance ) , where p<0.01 was considered statistically significant , to correct for multiple testing . pearson correlations were estimated for the predicted 3125 eq-5d-5l health - state values to express the similarity between the countries , as well as intraclass correlations ( iccs ; 2-way mixed - effects , individual ratings , absolute agreement ) . in addition , graphs for the pooled data of the 4 countries combined with the data of the individual countries and their regression functions were made in sigmaplot ( version 11.0 ; systat software inc . the number of individuals who entered the survey was 547 for canada , 404 for england , 407 for the netherlands , and 417 for the us . in the dutch study , 1 block of states ( block 11 ) was not assessed due to a programming error . age distribution was similar in the 4 countries , although the netherlands had a smaller proportion of younger participants and a larger proportion of middle - aged ones ( table 1 ) . the england sample included a larger proportion of degree - educated and employed individuals compared with the general population in england , but the sample was broadly representative of the general population in terms of other background characteristics , such as ethnicity.16 among us respondents , 70.8% reported that they received education beyond high school ; 65.8% were non - hispanic white ( n=273 ) , 17.6% were african american ( n=73 ) , and 16.6% were all other ethnicities . characteristics of the 4 samples the drop - out ( not completing all of the valuation tasks ) was low in all countries ( england 4 , the netherlands 14 ) . for the netherlands and england , the average duration ( s ) per task was 32.5 and 45.2 , respectively . mean responses for canada , england , the netherlands , and us on the 4 feasibility questions were as follows : the instructions that were given on the computer made it clear what i needed to do 2.34 , 2.27 , 2.31 , 2.31 ; it was easy to understand the questions i was asked 3.19 , 3.60 , 3.32 , 3.09 ; i found it difficult to decide 3.87 , 3.44 , 3.60 , 3.86 ; i found it easy to tell the difference between the health states i was asked to think about 2.63 , 2.47 , 2.60 , 2.53 . parameter estimates ( probit regression ) for the 4 countries separately and the 4 countries together four regression coefficients with illogical ordering were observed in the national datasets ( the netherlands : level 3 mo and pd were considered less severe than level 2 ; us : level 3 ua and pd was considered less severe than level 2 ) and one in the pooled data ( levels 2 and 3 pd were reversed ) . all 20 parameters were statistically significant in all countries , with the exception of level 2 for ua in the netherlands ( p=0.51 ) ( table 2 ) . significant differences in coefficients were noted for pd level 4 ( canada vs. england : p<0.001 ) and level 5 ( canada vs. england , canada vs. the netherlands , england vs. us , and the netherlands vs. us : p<0.001 ) ; ad level 4 ( canada vs. england , england vs. us : p<0.001 ) and level 5 ( canada vs. england , england vs. us , the netherlands vs. us : p<0.001 ) ; mo level 5 ( england vs. the netherlands , the netherlands vs. us ( p<0.001 ) , and canada vs. us ( p=0.001 ) ; and ua level 2 ( canada vs. the netherlands , england vs. the netherlands : p<0.001 ) . inclusion of interaction terms for all combinations of the 20 dummies with country ( reference : england ) revealed that 9 interactions were statistically significant ( results not presented in table 2 ) . separate analyses were performed for the 3 countries ( canada , england , us ) in which the pairs of states from block 11 were excluded . the cross - country comparison of predictions for the complete set of 3125 eq-5d-5l states demonstrated strong agreement across the 4 countries ( icc=0.89 ) ( fig . point estimates for agreement between any 2 countries were strong ( icc>0.5 ) , ranging from 0.80 for england versus us to 0.98 for canada versus us . correlations , intraclass correlations , and regression functions between the 4 countries based on the 3125 estimated eq-5d-5l health states relationships between the 3125 estimated ( choice model , asmprobit ) eq-5d-5l values ( 0=best health state ) for the 4 countries compared with the pooled results of the 4 countries ( x - axis ) . the pooled predictions for the 400 states that were part of the 200 paired comparison tasks showed that many separate health states of the paired comparison tasks fulfilled efficient design assumptions ( approximately 70% vs. 30% preference ) . the number of individuals who entered the survey was 547 for canada , 404 for england , 407 for the netherlands , and 417 for the us . in the dutch study , 1 block of states ( block 11 ) was not assessed due to a programming error . age distribution was similar in the 4 countries , although the netherlands had a smaller proportion of younger participants and a larger proportion of middle - aged ones ( table 1 ) . the england sample included a larger proportion of degree - educated and employed individuals compared with the general population in england , but the sample was broadly representative of the general population in terms of other background characteristics , such as ethnicity.16 among us respondents , 70.8% reported that they received education beyond high school ; 65.8% were non - hispanic white ( n=273 ) , 17.6% were african american ( n=73 ) , and 16.6% were all other ethnicities . the drop - out ( not completing all of the valuation tasks ) was low in all countries ( england 4 , the netherlands 14 ) . for the netherlands and england , the average duration ( s ) per task was 32.5 and 45.2 , respectively . mean responses for canada , england , the netherlands , and us on the 4 feasibility questions were as follows : the instructions that were given on the computer made it clear what i needed to do 2.34 , 2.27 , 2.31 , 2.31 ; it was easy to understand the questions i was asked 3.19 , 3.60 , 3.32 , 3.09 ; i found it difficult to decide 3.87 , 3.44 , 3.60 , 3.86 ; i found it easy to tell the difference between the health states i was asked to think about 2.63 , 2.47 , 2.60 , 2.53 . parameter estimates ( probit regression ) for the 4 countries separately and the 4 countries together four regression coefficients with illogical ordering were observed in the national datasets ( the netherlands : level 3 mo and pd were considered less severe than level 2 ; us : level 3 ua and pd was considered less severe than level 2 ) and one in the pooled data ( levels 2 and 3 pd were reversed ) . all 20 parameters were statistically significant in all countries , with the exception of level 2 for ua in the netherlands ( p=0.51 ) ( table 2 ) . significant differences in coefficients were noted for pd level 4 ( canada vs. england : p<0.001 ) and level 5 ( canada vs. england , canada vs. the netherlands , england vs. us , and the netherlands vs. us : p<0.001 ) ; ad level 4 ( canada vs. england , england vs. us : p<0.001 ) and level 5 ( canada vs. england , england vs. us , the netherlands vs. us : p<0.001 ) ; mo level 5 ( england vs. the netherlands , the netherlands vs. us ( p<0.001 ) , and canada vs. us ( p=0.001 ) ; and ua level 2 ( canada vs. the netherlands , england vs. the netherlands : p<0.001 ) . inclusion of interaction terms for all combinations of the 20 dummies with country ( reference : england ) revealed that 9 interactions were statistically significant ( results not presented in table 2 ) . separate analyses were performed for the 3 countries ( canada , england , us ) in which the pairs of states from block 11 were excluded . the cross - country comparison of predictions for the complete set of 3125 eq-5d-5l states demonstrated strong agreement across the 4 countries ( icc=0.89 ) ( fig . point estimates for agreement between any 2 countries were strong ( icc>0.5 ) , ranging from 0.80 for england versus us to 0.98 for canada versus us . correlations , intraclass correlations , and regression functions between the 4 countries based on the 3125 estimated eq-5d-5l health states relationships between the 3125 estimated ( choice model , asmprobit ) eq-5d-5l values ( 0=best health state ) for the 4 countries compared with the pooled results of the 4 countries ( x - axis ) . the pooled predictions for the 400 states that were part of the 200 paired comparison tasks showed that many separate health states of the paired comparison tasks fulfilled efficient design assumptions ( approximately 70% vs. 30% preference ) . we found that it was feasible to implement a standardized protocol for computer - based assessment of a choice - based selection of health states for the eq-5d-5l across countries . models generated parameter estimates that logically supported the structure of the descriptive system and values for eq-5d-5l health states strongly agreed across countries , with only minor differences across the 4 countries . in the english arm of this study , data were obtained through face - to - face interviews . this means that valuation studies based on discrete choices appear to have an important advantage over tto techniques because the latter require face - to - face interviews to produce data of reasonable credibility.14 one of the most serious limitations of choice - based models is that they produce relative values . although differences between the values are meaningful , the positions of the top and bottom values are not interpretable . in this study , the 5 levels in the dutch and french ( canada ) language eq-5d-5l may not exactly match the 5 levels in the english language eq-5d-5l because of language differences.14 interestingly , a recent study to measure disability weights for a wide array of health outcomes across a diverse range of populations showed that , based on the same measurement framework that we used in our study , the differences between countries were modest.30 to conclude , parameter estimates modeled from a choice - based approach were generally consistent and logical , although some deviations were observed . the estimated values were similar between the countries , and the differences may be attributed to the administration of the valuation exercise in different countries , and also due to cultural differences .

. ninety percent of primary liver cancers are hcc , the majority of which develop on the background of cirrhosis . over the past decade , medical management of the patient with chronic liver disease has improved . in parallel , the prevalence of hepatitis b ( hbv ) , hepatitis c virus ( hcv ) , alcohol related liver disease , and nash has increased and combined with an ageing population has led to a surge in the number of cases worldwide [ 13 ] . as a consequence , hcc is an important complication of cirrhosis and a leading indication for liver transplantation ( lt ) , accounting for approximately a third of patients on transplant waiting lists . the introduction of surveillance using alphafetoprotein and ultrasound has led to the earlier recognition of hcc and increases the therapeutic options available . in the absence of treatment these include lt , resection , locoregional , and systemic therapies . for a solitary hcc with preserved liver function and low hepatic vein pressure gradient , historically , survival rates were 3562% at 3 years and 1750% at 5 years for patients with cirrhosis undergoing resection for hcc [ 6 , 7 ] . however , tumor recurrence rates were high , up to 70% , and progression to liver failure was common [ 6 , 810 ] . lt is an attractive treatment option as it treats both the cancer and underlying liver disease . in the 1980s , patients presenting with large hcc were considered good candidates for lt as they were in better condition than patients with chronic liver disease and were more likely to survive the perioperative period but they had large or multifocal tumors . this resulted in a high recurrence rate of up to 65% , a 5 year survival of 1035% , and a median survival of those with recurrence of 6 months , coupled with increasing demand for donor livers led to a more restrictive selection process [ 1113 ] . recognition that small tumors appeared to fare better after lt led mazzaferro to introduce the milan criteria to select patients leading to improved survival with low rates of tumor recurrence . by adhering to the milan criteria of undertaking lt for hcc with a solitary tumor 5 cm or 3 tumors 3 cm each , a 5 year survival greater than 70% and recurrence rates of < 10% were produced . a staging system for hcc poses problems because the presence of liver disease and tumor varies due to the different epidemiological backgrounds and risk factors . the ideal staging system needs to include prognostic information regarding both the cancer and liver functional status and take account of clinical factors that influence response to treatment . the tnm classification is an oncology standard useful in conjunction with the presence of microvascular invasion of examined resection or explanted tumors / liver and provides information regarding the risk of tumor recurrence but does not take account of the liver functional status . as tnm requires pathological data ( microvascular invasion ) and only 20% hcc are resected for which it is good at discriminating stages for , its usability is limited . it uses four criteria of ascites , albumin , bilirubin , and tumor size to assess liver functional status and tumor stage . it is a good system for stratifying advanced / symptomatic disease but less useful in early stage to guide treatment choices . other available systems are the french classification , the cancer of the liver italian program ( clip ) classification , and the barcelona - clnic liver cancer ( bclc ) staging system ; the chinese university prognostic index ( cupi score ) and the japan integrated staging ( jis ) or bm - jis if biomarkers are included . the cupi and clip scores mainly stratify patients at advanced stages ; only two include prognostic variables ( bclc , cupi ) and only one allocates treatment according to specific prognostic subclasses ( bclc ) . the bclc is emerging as the standard staging system for hcc in the west and has been externally validated and incorporates prognostic variables related to tumor ( size , number , vascular invasion , n1 , m1 ) , liver function ( child - pugh ) , and health status ( ecog - eastern cooperative oncology group performance status ) . as well as incorporating variables that influence therapy such as bilirubin , portal hypertension , and presence of symptoms to assist in treatment decision making ( see figure 1 ) . lt is the treatment of choice for small multifocal hcc 3 tumors and 3 cm or a single tumor 5 cm with significant liver functional impairment . better selection of patients has improved 5 year survival to > 70% and recurrence < 10% . the major limitation to lt as treatment for hcc is the scarcity of cadaveric donors and the associated waiting time that results in a 20% drop - out rate and potentially increases the risk of recurrence from extension of vascular invasion . the use of tumor size and number to try to reflect tumor biology has been successful . however , it is clear that some patients with favourable biology are excluded . a number of groups have tried to expand indications beyond the milan criteria and claim to achieve similar survival rates [ 16 , 17 ] . the university of california san francisco ( ucsf ) criteria are probably the best known and include one tumor 6.5 cm or multiple tumors of which the largest is 4.5 cm and the sum of all diameters is 8 cm . more recently the up - to-7 criteria , where the hcc scores 7 based on the sum of the largest tumor ( diameter cm ) and the total number of tumors , have been introduced . the majority of the studies supporting extension of the milan criteria are based on retrospective histological analysis of the tumor burden in the explant liver and have not been validated prospectively [ 1619 ] . another area where the principles of the milan criteria have been challenged is in salvage lt . salvage lt has been advocated by some to manage hcc within milan criteria after resection . in selected cases , similar overall 5 year survival for salvage lt as primary lt for hcc has been achieved ( provided the comparison is from time listed for lt rather than date of lt , that is , intention to treat bias ) . other groups have found salvage lt to have a high operative mortality , 23.5% versus 2.1% for primary lt , higher recurrence rates , and poorer overall 5 year survival of 41% . salvage lt remains controversial at a time of a limited resource with tumor characteristics , background liver ( cirrhotic or noncirrhotic ) , and centre experience appearing to be the main determinants of recurrence and survival . an alternative strategy to expanding the criteria for lt is to downstage to within milan criteria aiming to achieve patient survival and recurrence free survival rates similar to those treated at an earlier stage . bridging therapy is utilised to maintain the tumor within listing criteria while a suitable graft is awaited for on the waiting list . bridging therapy is a widely accepted practice whereas downstaging for lt is not [ 23 , 24 ] . to be eligible for downstaging locoregional therapy , there should be no radiological evidence of vascular invasion . predictors of downstaging failure are tumors with an infiltrative pattern and an afp > 1000 ng / ml . there is evidence to suggest that downstaging of hcc to within milan criteria can produce reasonable results [ 25 , 27 ] . but the data is difficult to interpret as the studies utilise different inclusion criteria ( tumor size and number ) , locoregional therapies either individually or in combination , and endpoints . current published data reveal that after downstaging , surgical resection rates vary widely between 7% and 18% producing 5 year survival rates of between 25% and 57% and lt rates range between 24% and 90% , with an intention to treat post hcc treatment survival of between 60% and 70% at 3 years [ 27 , 29 , 30 ] . living donor lt ( ldlt ) is a good graft option for hcc as it allows neoadjuvant treatment to be organized around a lt . it provides a high - quality graft and removes a competing hcc recipient from the waiting list . but higher recurrence rates and reduced survival have been reported when compared to cadaveric lt [ 3133 ] . explanations for this observation include growth factors released from the regenerating liver may stimulate cancer cell growth . the shorter waiting time for ldlt may remove the observation period that occurs on the waiting list to assess tumor biology and a 3 month cooling off period has been advocated before undertaking ldlt . surgical oncological clearance in addition , an element of institutional bias may lead to ldlt in hcc with a higher risk of recurrence . on multivariate analysis of published studies on ldlt versus cadaveric lt , graft type , and waiting time have not been found to be significant risk factors for recurrence post lt . if ldlt is undertaken for hcc outside regional criteria and the graft fails retransplantation with cadaveric lt is ethically contentious . the donor risk and the degree of benefit to the recipient needed to justify ldlt for advanced hcc are still undetermined and for now many centres have adopted the same criteria / therapeutic goals for ldlt as cadaveric lt . noncirrhotic hcc accounts for 5% of cases in the west and 40% of cases in asia . patients with cirrhosis suitable for resection need preserved liver function and a hepatic venous pressure gradient 10 mmhg . anatomic resection is advocated by some as being more preferable to nonanatomic , as it is thought to produce better outcome by eliminating intrahepatic metastases in the related portal vein tributary . in patients with cirrhosis selected on liver functional status , five - year survival for tumors 2 cm , 25 cm , and > 5 cm are 66% , 52% , and 37% , respectively . for single tumors the 5 year survival is 57% and for multiple 26% but some centres are achieving > 50% in multiple hcc within the milan criteria but otherwise are not suitable for lt . recurrence remains problematic occurring in 70% at 5 years ; true recurrence / intrahepatic metastases generally occur within 2 years of resection ; if greater than 2 years it is generally regarded as a de novo tumor or late recurrence . at present there is no evidence that neoadjuvant / adjuvant therapy has any efficacy in reducing recurrence after resection . downstaging locoregional therapy can be employed to facilitate resection in disease which is initially regarded as unresectable and can achieve reasonable outcome , with 5 year survivals of 2567% , with the possibility of cure . preoperative portal vein embolisation can be employed to increase future remnant liver volume to allow more extensive resections to be undertaken but the complication rate in cirrhotics is 1020% and its effectiveness in this patient group is not fully established . a laparoscopic approach to resection in cirrhotics has been proposed by some to reduce the operative insult and the risk of decompensation . there are a number of different locoregional strategies available or being developed but the largest experience is with transarterial chemoembolization ( tace ) and radiofrequency ablation ( rfa ) . percutaneous ethanol injection ( pei ) was the first chemical ablative technique utilised . when applied to small tumors < 2 cm pei produces 90% necroses and a 5-year survival of 4753% but chemical ablation has now been superseded by thermal techniques such as radiofrequency ablation ( rfa ) . rfa is the most well - studied alternative to pei producing better local tumor control with a 2 year recurrence of 218% and a 5 year survival of 4070% or better when the treatment groups have been selected . meta - analyses of randomised control trials have confirmed that rfa is a more effective way to obtain local tumor control and survival benefit compared to pei , establishing it as a standard locoregional treatment [ 44 , 45 ] . rfa can be performed percutaneously , laparoscopically , or at open surgery depending on tumor location [ 46 , 47 ] . rfa is effective for early small hcc <3 cm when resection or lt are not feasible [ 4851 ] , whereas larger tumors may be inadequately treated . overall 1025% of tumors will not be suitable for rfa because of location such as subcapsular , adjacent to the gall bladder or major vessels which increases the risk of complication and inadequate ablation because of heat sink . the recurrence rate after rfa for selected early small hcc can be comparable to that of surgery [ 50 , 51 ] . in highly selected hcc < 2 cm rfa has the potential to be curative with a rate of complete response approaching 97% and a 5 year survival of 68% . however , randomised control trials of rfa against resection for small hcc < 3 cm have failed to show that rfa is as effective as resection but the majority of studies were underpowered or had incomplete follow up [ 49 , 52 ] . increasingly , rfa is being considered as an alternative initial curative treatment option for small centrally placed hcc as it offers the advantages of preserving parenchyma , potentially removes competition from the transplant waiting list and based on location , effective tumor necrosis can be obtained [ 43 , 48 ] . solitary hcc > 3 cm but < 5 cm rfa becomes less effective . but when tace followed by rfa for this size tumor is applied , the therapeutic effect of rfa is significantly increased and reduces tumor progression rate to 6% compared to 39% for rfa alone . in larger hcc > 5 cm outside lt criteria or not suitable for resection , ablative strategies may not work in a predictable manner . tace also has inconsistent results and no advantage has been demonstrated by combining therapies . when rfa is not suitable either because of tumor location or size , novel thermal or nonthermal ablative techniques may overcome the limits of rfa . promising thermal ablative strategies include microwave producing large areas of ablation with less heat sink and high intensity focused us ( hifu ) that can be used in patients with ascites . in assessing the effectiveness of ablation radiologically , the widely used recist ( the response evaluation criteria in solid tumours ) has limitations as it includes both necrotic and viable tumor areas and the modified recist that includes the assessment of viable tumor showing uptake in the arterial phase is more reliable . based on a meta - analysis tace is emerging as the standard of care for asymptomatic hcc outside milan criteria , demonstrating improved survival compared to best supportive care . a partial response of 1555% can be observed producing a survival benefit , increasing median survival time from 16 months to 20 months with 49% survival at 2 years . but individually the studies did not clearly demonstrate a benefit , mainly because of heterogenous patient study groups and varying tace techniques . this implies that good results with tace are achieved when it is used on a selective basis . generally tace is not suitable in decompensated liver disease where there is ascites or jaundice to the avoid major complications and minimize treatment related deaths to less than 2% . for optimal results tace needs to be as selective as possible , producing sustained and high localised concentrations within the tumor minimizing systemic exposure . alternative ways to be delivering chemotherapy instead of the standard ethiodized oil ( lipiodol ) suspensions are drug - eluting beads . in the precision v trial , a randomized control trial , comparing drug - eluting beads with doxorubicin to conventional tace with doxorubicin found it was better tolerated , with reduced liver toxicity and improved treatment response . owing to the improved safety and tolerance drug - eluting beads could be applicable in higher risk patient groups . further ways of optimising the therapeutic benefit of tace is by combining with systemic drugs . using agents that target the angiogenic pathways that are switched on by the local hypoxia produced by tace if there is no response after two tace sessions , alternative treatment strategies should be considered , which in the majority will be systemic therapy . in highly selected patients consideration advanced hcc that is symptomatic , exhibiting vascular invasion and/or has extrahepatic disease have a short median survival of 6 months with 25% surviving a year . systemics are often the only treatment option for palliation but there is a subset that benefit from locoregional therapy such as where vascular invasion is limited to a venous branch receiving intra - arterial therapies such as tace or radioembolization . radioembolization using yttrium-90 ( 90y ) labelled microspheres a beta emitter , appears promising , and may also be effective as a precursor to radical therapy with outcomes similar to tace [ 30 , 63 ] and sorafenib . there is a need to be aware of intestinal and lung shunting which may provoke serious complications . there is a minimal embolic effect so when there is main portal vein involvement and tace is contraindicated radioembolization with yttrium may be a good option . in the absence of portal vein involvement radioembolization in child a survival is 15.5 months , child b is 13 months , with a portal vein involvement survival of both child a + b being 5.6 months with 2550% response rates . cyberknife is a new stereotactic body radiation therapy ( sbrt ) or stereotactic ablative radiotherapy ( sabr ) in combination with a robotic system that tracks the tumor during respiration and is able to deliver high dose radiation accurately sparing adjacent normal tissue in a small number of fractions . a number of studies in hcc not suitable for standard locoregional treatment or resection have reported promising results . in hcc < 100 ml progression free survival rates at 6 months , 1 year , and 3 years of 83% , 72% , and 68% respectively , with overall survival at 1 year , and 3 years of 92.9% and 58.6% have been reported . it also has utility as local salvage treatment after tace achieving local control in 95% . to this date no serious sbrt related toxicities being reported [ 6769 ] but it is not clear whether it can be applied to patients with more severe liver diseases as its threshold for tolerance is not defined . worldwide 78% of hcc are viral related with 53% attributed to hbv and 25% to hcv . risk of hcc recurrence after treatment is increased with progression of active hepatitis and fibrosis . antiviral therapy is used as an adjuvant treatment with the aim to reduce viral load and fibrosis with the aim of halting progression of viral induced liver disease and reducing the risk of further hcc developing . hepatitis b ( hbv ) infection increases the risk of hcc recurrence particularly for the patient who is hbeag + and/or has a high serum hbv dna level [ 71 , 72 ] . treatment with nucleoside analogues entecavir or tenofovir suppresses hbv dna levels improving liver function in decompensated liver disease and may reduce the risk of hcc development over time . hbv antiviral therapy reduces risk of recurrence by 41% and overall mortality by 73% , mainly because death from liver failure is reduced by 77% . use of longterm lamivudine treatment resistance can occur in 70% over 5 years has given way to alternative nucleoside analogues such as entecavir , telbivudine , and nucleotide analogues such as tenofovir but longterm data is lacking for their effect on reducing hcc recurrence . chronic infection with hcv appears to increase the rate of hcc development in a similar way to hbv . the risk does not change with genotype ( g ) but a recent meta - analysis suggests that hcv g1b maybe more at risk of hcc transformation . meta - analysis of adjuvant alpha ifn shows reduction in hcc recurrence and mortality in curatively ablated viral hepatitis related hcc . antiviral potency and the ability to produce a sustained viral response ( svr ) in hcv appear to be associated with reducing the risk of hcc recurrence . more longterm data is needed from the newer protease inhibitors ( boceprevir , telaprevir ) to determine whether the higher svr they are able to produce translates into lower risk of hcc longterm . mammalian target of rapamycin ( mtor ) inhibitors , for example , sirolimus is an exception to this rule . mtor is overexpressed in approximately 2/3 of hcc making it an attractive therapeutic target . to establish whether the immunosuppressive regime affects recurrence rates , data from the silver study the silver study is a randomised multicenter clinical trial comparing sirolimus containing to a mtor inhibitor free immunosuppressive regimes . , there is little evidence on whether the immunosuppression regime should be completely changed to a mtor inhibitor or whether these agents should be added to the preexisting immunosuppressive regime when recurrent hcc presents post lt [ 35 , 78 , 79 ] . cni exposure should be minimised as there is evidence that this reduces the risk of tumor recurrence long term . hcc is an unique chemoresistant tumor and until 2007 no systemic drug was recommended for its management . in the early 1990s , a number of randomised controlled studies assessed the role of adjuvant chemotherapy but no benefit or efficacy was demonstrated . multiple agents have been assessed but doxorubicin , an anthracycline , has been most rigorously studied [ 81 , 82 ] . the main patient groups that should be considered for adjuvant chemotherapy are those being transplanted for extended criteria or have a high risk of recurrence based on the explant pathology . as patients selected for lt should be at low risk of recurrence the majority will not gain any benefit from routine adjuvant chemotherapy . since 2007 sorafenib , an oral tyrosine kinase inhibitor , has become the standard of care . based upon the sharp study that demonstrated sorafenib improved median survival from 7.9 months to 10.7 months and slowed time to progression from 2.8 to 5.5 months . it is well tolerated with diarrhoea in 8 - 9% and hand - foot skin reaction in 816% , with side effects leading to its discontinuation in 15% . sorafenib is regarded as the standard therapy for metastatic disease and for hcc progressing despite optimal locoregional therapy . a number of ongoing studies are establishing sorafenib 's adjuvant role in resection , local ablation ( sorafenib as adjuvant treatment in the prevention of recurrence of hepatocellular carcinoma ( storm ) ) , and tace . additionally , a phase 1 study is being undertaken in high - risk patients post - lt that on explant are outside milan criteria with microvascular or macrovascular invasion or histologically poorly differentiated hcc . at present there is no evidence of increased toxicity in lt recipients and sorafenib can produce a response based on published case reports [ 85 , 86 ] . other biological agents entering phase 2 or 3 trials for hcc include egfr ( erlotinib ) and vegfr / fgfr ( brivanib ) tyrosine kinase inhibitors . management of hcc continues to evolve and interventional radiology in the form of tace rfa increasingly dominates management either as a bridge to lt or to downstage facilitating lt or resection . as locoregional therapy technology advances patients that can be considered either for palliation or potential cure will increase . criteria for lt listing need to become more sophisticated by incorporating tumor biology in decision making , presently inferred from clinical behaviour but in the future by the use of molecular markers . ultimately , the aim of lt , irrespective of disease etiology is to give the maximum benefit from a limited organ pool .

liver transplantation is the treatment of choice for selected patients with hepatocellular carcinoma ( hcc ) on a background of chronic liver disease . liver resection or locoregional ablative therapies may be indicated for patients with preserved synthetic function without significant portal hypertension . milan criteria were introduced to select suitable patients for liver transplant with low risk of tumor recurrence and 5-year survival in excess of 70% . currently the incidence of hcc is climbing rapidly and in a current climate of organ shortage has led to the re - evaluation of locoregional therapies and resectional surgery to manage the case load . the introduction of biological therapies has had a new dimension to care , adding to the complexities of multidisciplinary team working in the management of hcc . the aim of this paper is to give a brief overview of present day management strategies and decision making .

1. Introduction 2. Staging 3. Liver Transplant 4. Liver Resection 5. Locoregional Therapy: Ablation, TACE, and Radiation 6. Systemic Therapies: Antiviral Therapy, 7. Conclusions

ninety percent of primary liver cancers are hcc , the majority of which develop on the background of cirrhosis . over the past decade , medical management of the patient with chronic liver disease has improved . in parallel , the prevalence of hepatitis b ( hbv ) , hepatitis c virus ( hcv ) , alcohol related liver disease , and nash has increased and combined with an ageing population has led to a surge in the number of cases worldwide [ 13 ] . as a consequence , hcc is an important complication of cirrhosis and a leading indication for liver transplantation ( lt ) , accounting for approximately a third of patients on transplant waiting lists . the introduction of surveillance using alphafetoprotein and ultrasound has led to the earlier recognition of hcc and increases the therapeutic options available . in the absence of treatment these include lt , resection , locoregional , and systemic therapies . for a solitary hcc with preserved liver function and low hepatic vein pressure gradient , historically , survival rates were 3562% at 3 years and 1750% at 5 years for patients with cirrhosis undergoing resection for hcc [ 6 , 7 ] . however , tumor recurrence rates were high , up to 70% , and progression to liver failure was common [ 6 , 810 ] . lt is an attractive treatment option as it treats both the cancer and underlying liver disease . in the 1980s , patients presenting with large hcc were considered good candidates for lt as they were in better condition than patients with chronic liver disease and were more likely to survive the perioperative period but they had large or multifocal tumors . this resulted in a high recurrence rate of up to 65% , a 5 year survival of 1035% , and a median survival of those with recurrence of 6 months , coupled with increasing demand for donor livers led to a more restrictive selection process [ 1113 ] . recognition that small tumors appeared to fare better after lt led mazzaferro to introduce the milan criteria to select patients leading to improved survival with low rates of tumor recurrence . by adhering to the milan criteria of undertaking lt for hcc with a solitary tumor 5 cm or 3 tumors 3 cm each , a 5 year survival greater than 70% and recurrence rates of < 10% were produced . a staging system for hcc poses problems because the presence of liver disease and tumor varies due to the different epidemiological backgrounds and risk factors . the tnm classification is an oncology standard useful in conjunction with the presence of microvascular invasion of examined resection or explanted tumors / liver and provides information regarding the risk of tumor recurrence but does not take account of the liver functional status . as tnm requires pathological data ( microvascular invasion ) and only 20% hcc are resected for which it is good at discriminating stages for , its usability is limited . it uses four criteria of ascites , albumin , bilirubin , and tumor size to assess liver functional status and tumor stage . other available systems are the french classification , the cancer of the liver italian program ( clip ) classification , and the barcelona - clnic liver cancer ( bclc ) staging system ; the chinese university prognostic index ( cupi score ) and the japan integrated staging ( jis ) or bm - jis if biomarkers are included . the bclc is emerging as the standard staging system for hcc in the west and has been externally validated and incorporates prognostic variables related to tumor ( size , number , vascular invasion , n1 , m1 ) , liver function ( child - pugh ) , and health status ( ecog - eastern cooperative oncology group performance status ) . as well as incorporating variables that influence therapy such as bilirubin , portal hypertension , and presence of symptoms to assist in treatment decision making ( see figure 1 ) . lt is the treatment of choice for small multifocal hcc 3 tumors and 3 cm or a single tumor 5 cm with significant liver functional impairment . better selection of patients has improved 5 year survival to > 70% and recurrence < 10% . the major limitation to lt as treatment for hcc is the scarcity of cadaveric donors and the associated waiting time that results in a 20% drop - out rate and potentially increases the risk of recurrence from extension of vascular invasion . the use of tumor size and number to try to reflect tumor biology has been successful . however , it is clear that some patients with favourable biology are excluded . a number of groups have tried to expand indications beyond the milan criteria and claim to achieve similar survival rates [ 16 , 17 ] . the university of california san francisco ( ucsf ) criteria are probably the best known and include one tumor 6.5 cm or multiple tumors of which the largest is 4.5 cm and the sum of all diameters is 8 cm . the majority of the studies supporting extension of the milan criteria are based on retrospective histological analysis of the tumor burden in the explant liver and have not been validated prospectively [ 1619 ] . another area where the principles of the milan criteria have been challenged is in salvage lt . salvage lt has been advocated by some to manage hcc within milan criteria after resection . in selected cases , similar overall 5 year survival for salvage lt as primary lt for hcc has been achieved ( provided the comparison is from time listed for lt rather than date of lt , that is , intention to treat bias ) . salvage lt remains controversial at a time of a limited resource with tumor characteristics , background liver ( cirrhotic or noncirrhotic ) , and centre experience appearing to be the main determinants of recurrence and survival . an alternative strategy to expanding the criteria for lt is to downstage to within milan criteria aiming to achieve patient survival and recurrence free survival rates similar to those treated at an earlier stage . to be eligible for downstaging locoregional therapy , there should be no radiological evidence of vascular invasion . predictors of downstaging failure are tumors with an infiltrative pattern and an afp > 1000 ng / ml . there is evidence to suggest that downstaging of hcc to within milan criteria can produce reasonable results [ 25 , 27 ] . but the data is difficult to interpret as the studies utilise different inclusion criteria ( tumor size and number ) , locoregional therapies either individually or in combination , and endpoints . living donor lt ( ldlt ) is a good graft option for hcc as it allows neoadjuvant treatment to be organized around a lt . it provides a high - quality graft and removes a competing hcc recipient from the waiting list . explanations for this observation include growth factors released from the regenerating liver may stimulate cancer cell growth . surgical oncological clearance in addition , an element of institutional bias may lead to ldlt in hcc with a higher risk of recurrence . on multivariate analysis of published studies on ldlt versus cadaveric lt , graft type , and waiting time have not been found to be significant risk factors for recurrence post lt . if ldlt is undertaken for hcc outside regional criteria and the graft fails retransplantation with cadaveric lt is ethically contentious . the donor risk and the degree of benefit to the recipient needed to justify ldlt for advanced hcc are still undetermined and for now many centres have adopted the same criteria / therapeutic goals for ldlt as cadaveric lt . noncirrhotic hcc accounts for 5% of cases in the west and 40% of cases in asia . patients with cirrhosis suitable for resection need preserved liver function and a hepatic venous pressure gradient 10 mmhg . anatomic resection is advocated by some as being more preferable to nonanatomic , as it is thought to produce better outcome by eliminating intrahepatic metastases in the related portal vein tributary . in patients with cirrhosis selected on liver functional status , five - year survival for tumors 2 cm , 25 cm , and > 5 cm are 66% , 52% , and 37% , respectively . for single tumors the 5 year survival is 57% and for multiple 26% but some centres are achieving > 50% in multiple hcc within the milan criteria but otherwise are not suitable for lt . recurrence remains problematic occurring in 70% at 5 years ; true recurrence / intrahepatic metastases generally occur within 2 years of resection ; if greater than 2 years it is generally regarded as a de novo tumor or late recurrence . preoperative portal vein embolisation can be employed to increase future remnant liver volume to allow more extensive resections to be undertaken but the complication rate in cirrhotics is 1020% and its effectiveness in this patient group is not fully established . a laparoscopic approach to resection in cirrhotics has been proposed by some to reduce the operative insult and the risk of decompensation . there are a number of different locoregional strategies available or being developed but the largest experience is with transarterial chemoembolization ( tace ) and radiofrequency ablation ( rfa ) . when applied to small tumors < 2 cm pei produces 90% necroses and a 5-year survival of 4753% but chemical ablation has now been superseded by thermal techniques such as radiofrequency ablation ( rfa ) . rfa is the most well - studied alternative to pei producing better local tumor control with a 2 year recurrence of 218% and a 5 year survival of 4070% or better when the treatment groups have been selected . rfa can be performed percutaneously , laparoscopically , or at open surgery depending on tumor location [ 46 , 47 ] . rfa is effective for early small hcc <3 cm when resection or lt are not feasible [ 4851 ] , whereas larger tumors may be inadequately treated . overall 1025% of tumors will not be suitable for rfa because of location such as subcapsular , adjacent to the gall bladder or major vessels which increases the risk of complication and inadequate ablation because of heat sink . the recurrence rate after rfa for selected early small hcc can be comparable to that of surgery [ 50 , 51 ] . in highly selected hcc < 2 cm rfa has the potential to be curative with a rate of complete response approaching 97% and a 5 year survival of 68% . however , randomised control trials of rfa against resection for small hcc < 3 cm have failed to show that rfa is as effective as resection but the majority of studies were underpowered or had incomplete follow up [ 49 , 52 ] . increasingly , rfa is being considered as an alternative initial curative treatment option for small centrally placed hcc as it offers the advantages of preserving parenchyma , potentially removes competition from the transplant waiting list and based on location , effective tumor necrosis can be obtained [ 43 , 48 ] . solitary hcc > 3 cm but < 5 cm rfa becomes less effective . but when tace followed by rfa for this size tumor is applied , the therapeutic effect of rfa is significantly increased and reduces tumor progression rate to 6% compared to 39% for rfa alone . in larger hcc > 5 cm outside lt criteria or not suitable for resection , ablative strategies may not work in a predictable manner . tace also has inconsistent results and no advantage has been demonstrated by combining therapies . when rfa is not suitable either because of tumor location or size , novel thermal or nonthermal ablative techniques may overcome the limits of rfa . promising thermal ablative strategies include microwave producing large areas of ablation with less heat sink and high intensity focused us ( hifu ) that can be used in patients with ascites . in assessing the effectiveness of ablation radiologically , the widely used recist ( the response evaluation criteria in solid tumours ) has limitations as it includes both necrotic and viable tumor areas and the modified recist that includes the assessment of viable tumor showing uptake in the arterial phase is more reliable . based on a meta - analysis tace is emerging as the standard of care for asymptomatic hcc outside milan criteria , demonstrating improved survival compared to best supportive care . a partial response of 1555% can be observed producing a survival benefit , increasing median survival time from 16 months to 20 months with 49% survival at 2 years . this implies that good results with tace are achieved when it is used on a selective basis . generally tace is not suitable in decompensated liver disease where there is ascites or jaundice to the avoid major complications and minimize treatment related deaths to less than 2% . alternative ways to be delivering chemotherapy instead of the standard ethiodized oil ( lipiodol ) suspensions are drug - eluting beads . in the precision v trial , a randomized control trial , comparing drug - eluting beads with doxorubicin to conventional tace with doxorubicin found it was better tolerated , with reduced liver toxicity and improved treatment response . owing to the improved safety and tolerance drug - eluting beads could be applicable in higher risk patient groups . using agents that target the angiogenic pathways that are switched on by the local hypoxia produced by tace if there is no response after two tace sessions , alternative treatment strategies should be considered , which in the majority will be systemic therapy . in highly selected patients consideration advanced hcc that is symptomatic , exhibiting vascular invasion and/or has extrahepatic disease have a short median survival of 6 months with 25% surviving a year . radioembolization using yttrium-90 ( 90y ) labelled microspheres a beta emitter , appears promising , and may also be effective as a precursor to radical therapy with outcomes similar to tace [ 30 , 63 ] and sorafenib . there is a minimal embolic effect so when there is main portal vein involvement and tace is contraindicated radioembolization with yttrium may be a good option . in the absence of portal vein involvement radioembolization in child a survival is 15.5 months , child b is 13 months , with a portal vein involvement survival of both child a + b being 5.6 months with 2550% response rates . cyberknife is a new stereotactic body radiation therapy ( sbrt ) or stereotactic ablative radiotherapy ( sabr ) in combination with a robotic system that tracks the tumor during respiration and is able to deliver high dose radiation accurately sparing adjacent normal tissue in a small number of fractions . it also has utility as local salvage treatment after tace achieving local control in 95% . to this date no serious sbrt related toxicities being reported [ 6769 ] but it is not clear whether it can be applied to patients with more severe liver diseases as its threshold for tolerance is not defined . worldwide 78% of hcc are viral related with 53% attributed to hbv and 25% to hcv . risk of hcc recurrence after treatment is increased with progression of active hepatitis and fibrosis . antiviral therapy is used as an adjuvant treatment with the aim to reduce viral load and fibrosis with the aim of halting progression of viral induced liver disease and reducing the risk of further hcc developing . hepatitis b ( hbv ) infection increases the risk of hcc recurrence particularly for the patient who is hbeag + and/or has a high serum hbv dna level [ 71 , 72 ] . treatment with nucleoside analogues entecavir or tenofovir suppresses hbv dna levels improving liver function in decompensated liver disease and may reduce the risk of hcc development over time . hbv antiviral therapy reduces risk of recurrence by 41% and overall mortality by 73% , mainly because death from liver failure is reduced by 77% . chronic infection with hcv appears to increase the rate of hcc development in a similar way to hbv . the risk does not change with genotype ( g ) but a recent meta - analysis suggests that hcv g1b maybe more at risk of hcc transformation . meta - analysis of adjuvant alpha ifn shows reduction in hcc recurrence and mortality in curatively ablated viral hepatitis related hcc . antiviral potency and the ability to produce a sustained viral response ( svr ) in hcv appear to be associated with reducing the risk of hcc recurrence . more longterm data is needed from the newer protease inhibitors ( boceprevir , telaprevir ) to determine whether the higher svr they are able to produce translates into lower risk of hcc longterm . mammalian target of rapamycin ( mtor ) inhibitors , for example , sirolimus is an exception to this rule . mtor is overexpressed in approximately 2/3 of hcc making it an attractive therapeutic target . , there is little evidence on whether the immunosuppression regime should be completely changed to a mtor inhibitor or whether these agents should be added to the preexisting immunosuppressive regime when recurrent hcc presents post lt [ 35 , 78 , 79 ] . cni exposure should be minimised as there is evidence that this reduces the risk of tumor recurrence long term . hcc is an unique chemoresistant tumor and until 2007 no systemic drug was recommended for its management . in the early 1990s , a number of randomised controlled studies assessed the role of adjuvant chemotherapy but no benefit or efficacy was demonstrated . multiple agents have been assessed but doxorubicin , an anthracycline , has been most rigorously studied [ 81 , 82 ] . the main patient groups that should be considered for adjuvant chemotherapy are those being transplanted for extended criteria or have a high risk of recurrence based on the explant pathology . as patients selected for lt should be at low risk of recurrence the majority will not gain any benefit from routine adjuvant chemotherapy . since 2007 sorafenib , an oral tyrosine kinase inhibitor , has become the standard of care . based upon the sharp study that demonstrated sorafenib improved median survival from 7.9 months to 10.7 months and slowed time to progression from 2.8 to 5.5 months . a number of ongoing studies are establishing sorafenib 's adjuvant role in resection , local ablation ( sorafenib as adjuvant treatment in the prevention of recurrence of hepatocellular carcinoma ( storm ) ) , and tace . additionally , a phase 1 study is being undertaken in high - risk patients post - lt that on explant are outside milan criteria with microvascular or macrovascular invasion or histologically poorly differentiated hcc . at present there is no evidence of increased toxicity in lt recipients and sorafenib can produce a response based on published case reports [ 85 , 86 ] . management of hcc continues to evolve and interventional radiology in the form of tace rfa increasingly dominates management either as a bridge to lt or to downstage facilitating lt or resection . criteria for lt listing need to become more sophisticated by incorporating tumor biology in decision making , presently inferred from clinical behaviour but in the future by the use of molecular markers . ultimately , the aim of lt , irrespective of disease etiology is to give the maximum benefit from a limited organ pool .

benign prostatic hyperplasia ( bph ) is the most common urological disease among aged men , with incidence over 50% at age 60 years and over 90% at age 80 years . the development and progression of bph is a long - term process . in recent years accumulating evidence recent studies suggest that inflammation and abnormal immunoregulation may contribute to cytokine production by inflammatory cells driving local growth factor production and angiogenesis in the prostatic tissue , and that this proinflammatory microenvironment is closely related to bph stromal hyperproliferation and tissue remodeling . heat shock proteins ( hsps ) are a family of proteins that are produced by cells in response to exposure to stressful conditions . hsps are crucial for the maintenance of cell integrity during both normal cell growth and pathophysiological conditions [ 57 ] . by controlling binding and release , hsps function mainly as molecular chaperones , which participate in the folding and assembly of nascent and unfolding proteins and hsps are classified into 4 major families according to their biological activities : hsp90 , hsp70 , hsp60 , and small hsps . hsp27 is a widely expressed 27 kda protein as a member of the small hsp family . it has been implicated in different diseases , playing both protective and counter - protective roles , such as renal injury and fibrosis , cancer , and neuro - degenerative and cardiovascular diseases , highlighting its role as a potential biomarker and therapeutic target . recently , it has been reported that hsp27 presents in the prostate cancer cell line and its expression is related to the prostate cancer malignancy level . accumulating evidence now suggests that bph and prostate cancer share important anatomic , pathologic , and genetic links in addition to the well - established epidemiologic association . recent publications support the hypothesis that bph and prostate cancer are part of metabolic syndrome , and inflammation as a major contributor to the development of both bph and prostate cancer . however , the expression of hsp27 in bph and its correlation with inflammation level are unclear . in this study , to investigate the role of hsp27 in inflammatory bph , we evaluated hsp27 expression and its relation to conditions of prostatic inflammation associated with bph , correlating it with the levels of inflammatory factors such as tumor tnf- , il-6 , and cd3 . this study was approved by the ethics committee of the third affiliated hospital of hebei medical university . written informed consent was obtained from all subjects . we recruited 60 bph patients who received transurethral electroresection of prostate ( turp ) because of urinary retention or lower urinary tract symptoms ( luts ) at the third affiliated hospital of hebei medical university between april 2013 and october 2013 into this cross - sectional study . finasteride and receptor blockers were the main drugs taken by participants for the treatment of bph before the surgery . before the surgery , all the patients received tests for blood routine , urine routine , liver and renal function , chest radiograph , electrocardiogram , abdominal and urethral system ultrasonography , prostate - specific antigen ( psa ) , and c reactive protein ( crp ) . international prostate symptom score ( ipss ) and quality of life ( qol ) patients with the following conditions were excluded : history of acute prostatitis or chronic bacteriogenic prostatitis , urinary tract infection , a history of chronic pelvic pain , incidentally discovered prostatic cancer ( idpc ) or endothelial hyperplasia of prostate , cerebrovascular disease or spinal cord disease , and oral antibiotics a week before the surgery . prostate tissues were serially cryo - sectioned at a thickness of 4 m after 10% neutral formalin fixing for 24 h and paraffin embedding . sections were dewaxed in xylene and rehydrated through graded ethanol to water followed by hematoxylin and eosin staining . all the sections were evaluated by 2 experienced pathologist for hyperplasia of prostate and histological inflammation . histological inflammation was certified when inflammatory cell infiltration and accumulation were found in the prostate gland . among the 60 bph patients , there were 15 cases of non - inflammatory bph ( ni ) and 45 cases of inflammatory bph . the inflammatory bph group was further divided into 3 different degrees by the number of inflammatory cells , the extent of the damage to prostate gland , and the emergence of lymph nodules : mild - inflammatory bph ( mi , n=12 ) with sporadic inflammatory cell infiltration and no damage to the glandular epithelium ; moderate - inflammatory bph ( moi , n=25 ) with inflammatory cell accumulation but no damage to the glandular epithelium basement , or lymph nodules ; and severe - inflammatory bph ( si , n=8 ) with inflammatory cell accumulation , damage to the glandular epithelium basement , or emergence of lymph nodules . basic demographic information and clinical data were collected , including age , height , weight , the history of urinary retention , complications , international prostate symptom score ( ipss ) , quality of life ( qol ) , prostate volume , residual urine volume ( ruv ) , maximum flow rate ( qmax ) , prostate specific antigen ( psa ) , c - reactive protein ( crp ) , leucocyte counts in blood test and urine test , and body mass index ( bmi ) . psa in the blood was measured with a roche cobas e601 ( roche , germany ) . crp in the blood was measured with latex particle - enhanced immunoturbidimetric assay using a hitachi 7170a biochemistry analyzer ( hitachi , japan ) . urodynamic tests were performed for each patient , and patients with urine retention were tested a week after catheter setting . the indexes observed included postvoid residual urine , maximum flow rate , detrusor pressure , and bladder capacity . formalin - fixed , paraffin - embedded sections ( 4 m ) of prostate tissues were immunostained for hsp27 ( rabbit polyclonal antibody bs1177 , bioworld technology , usa ) , tnf- ( rabbit polyclonal antibody bs6000 , bioworld technology , usa ) , il-6 ( rabbit polyclonal antibody bs6419 , bioworld technology , usa ) and cd3 ( rabbit polyclonal antibody bs6280 , bioworld technology , usa ) as follows . after being dewaxed and rehydrated , endogenous peroxidase activity was blocked using 3% hydrogen peroxide in methanol . sections underwent antigen retrieval in 0.01 m citric acid buffer , ph 6.0 , by boiling water bath for 15 min . after passive cooling to room temperature , sections were then pre - incubated with goat serum to reduce nonspecific staining and incubated with primary antibodies at 4c overnight . after washing with pbs 3 times , sections were incubated with biotinylated secondary antibodies for 15 min at 37c and then washed in pbs before incubating with streptavidin - horseradish peroxidase ( sa - hrp ) conjugate for 15 min at 37c . then sections were again washed in pbs and the antibody - hrp complex was visualized by incubation with diaminobenzidine ( dab ) for 5 min . mean optical density ( mod ) of each section was analyzed with an image - pro plus 6.0 ( media cybernetics , ca , usa ) . preference settings were : select positive color under his mode , h ( chromaticity)=30 , i ( grayscale)=230 , s ( saturability)=255 . measuring items include : area , mean density and integrated optical density ( iod ) ; for area measurement , positive color ratio < 50% were excluded . batch processing was applied by macroprocessor pathology 6 ( mediacybemetics ) and mod values were obtained for each visual field . for each section , concentrations of hsp27 , tnf- and il-6 in serums were measured using commercially available enzyme - linked immunoassay ( elisa ) kits ( uscnlife , shanghai , china ) according to the manufacturer s instructions . the minimum and maximum detectable values were 0.312 ng / ml and 20 ng / ml . all samples were assayed in duplicate . correlation between hsp27 and tnf- and il-6 expression was analyzed using the spearman rank correlation test . data were analyzed using sas 16.0 ( sas institute inc . , cary , nc , usa ) . this study was approved by the ethics committee of the third affiliated hospital of hebei medical university . written informed consent was obtained from all subjects . we recruited 60 bph patients who received transurethral electroresection of prostate ( turp ) because of urinary retention or lower urinary tract symptoms ( luts ) at the third affiliated hospital of hebei medical university between april 2013 and october 2013 into this cross - sectional study . finasteride and receptor blockers were the main drugs taken by participants for the treatment of bph before the surgery . before the surgery , all the patients received tests for blood routine , urine routine , liver and renal function , chest radiograph , electrocardiogram , abdominal and urethral system ultrasonography , prostate - specific antigen ( psa ) , and c reactive protein ( crp ) . international prostate symptom score ( ipss ) and quality of life ( qol ) patients with the following conditions were excluded : history of acute prostatitis or chronic bacteriogenic prostatitis , urinary tract infection , a history of chronic pelvic pain , incidentally discovered prostatic cancer ( idpc ) or endothelial hyperplasia of prostate , cerebrovascular disease or spinal cord disease , and oral antibiotics a week before the surgery . prostate tissues were serially cryo - sectioned at a thickness of 4 m after 10% neutral formalin fixing for 24 h and paraffin embedding . sections were dewaxed in xylene and rehydrated through graded ethanol to water followed by hematoxylin and eosin staining . all the sections were evaluated by 2 experienced pathologist for hyperplasia of prostate and histological inflammation . histological inflammation was certified when inflammatory cell infiltration and accumulation were found in the prostate gland . among the 60 bph patients , there were 15 cases of non - inflammatory bph ( ni ) and 45 cases of inflammatory bph . the inflammatory bph group was further divided into 3 different degrees by the number of inflammatory cells , the extent of the damage to prostate gland , and the emergence of lymph nodules : mild - inflammatory bph ( mi , n=12 ) with sporadic inflammatory cell infiltration and no damage to the glandular epithelium ; moderate - inflammatory bph ( moi , n=25 ) with inflammatory cell accumulation but no damage to the glandular epithelium basement , or lymph nodules ; and severe - inflammatory bph ( si , n=8 ) with inflammatory cell accumulation , damage to the glandular epithelium basement , or emergence of lymph nodules . basic demographic information and clinical data were collected , including age , height , weight , the history of urinary retention , complications , international prostate symptom score ( ipss ) , quality of life ( qol ) , prostate volume , residual urine volume ( ruv ) , maximum flow rate ( qmax ) , prostate specific antigen ( psa ) , c - reactive protein ( crp ) , leucocyte counts in blood test and urine test , and body mass index ( bmi ) . psa in the blood was measured with a roche cobas e601 ( roche , germany ) . crp in the blood was measured with latex particle - enhanced immunoturbidimetric assay using a hitachi 7170a biochemistry analyzer ( hitachi , japan ) . urodynamic tests were performed for each patient , and patients with urine retention were tested a week after catheter setting . the indexes observed included postvoid residual urine , maximum flow rate , detrusor pressure , and bladder capacity . formalin - fixed , paraffin - embedded sections ( 4 m ) of prostate tissues were immunostained for hsp27 ( rabbit polyclonal antibody bs1177 , bioworld technology , usa ) , tnf- ( rabbit polyclonal antibody bs6000 , bioworld technology , usa ) , il-6 ( rabbit polyclonal antibody bs6419 , bioworld technology , usa ) and cd3 ( rabbit polyclonal antibody bs6280 , bioworld technology , usa ) as follows . after being dewaxed and rehydrated , endogenous peroxidase activity was blocked using 3% hydrogen peroxide in methanol . sections underwent antigen retrieval in 0.01 m citric acid buffer , ph 6.0 , by boiling water bath for 15 min . after passive cooling to room temperature , sections were then pre - incubated with goat serum to reduce nonspecific staining and incubated with primary antibodies at 4c overnight . after washing with pbs 3 times , sections were incubated with biotinylated secondary antibodies for 15 min at 37c and then washed in pbs before incubating with streptavidin - horseradish peroxidase ( sa - hrp ) conjugate for 15 min at 37c . then sections were again washed in pbs and the antibody - hrp complex was visualized by incubation with diaminobenzidine ( dab ) for 5 min . mean optical density ( mod ) of each section was analyzed with an image - pro plus 6.0 ( media cybernetics , ca , usa ) . preference settings were : select positive color under his mode , h ( chromaticity)=30 , i ( grayscale)=230 , s ( saturability)=255 . measuring items include : area , mean density and integrated optical density ( iod ) ; for area measurement , positive color ratio < 50% were excluded . batch processing was applied by macroprocessor pathology 6 ( mediacybemetics ) and mod values were obtained for each visual field . for each section , 5 visual fields were selected and the mean mod was calculated . concentrations of hsp27 , tnf- and il-6 in serums were measured using commercially available enzyme - linked immunoassay ( elisa ) kits ( uscnlife , shanghai , china ) according to the manufacturer s instructions . the minimum and maximum detectable values were 0.312 ng / ml and 20 ng / ml . all samples were assayed in duplicate . correlation between hsp27 and tnf- and il-6 expression was analyzed using the spearman rank correlation test . hyperplasia of prostate and tissue inflammation were evaluated via hematoxylin - eosin staining by 2 experienced pathologists . glandular epithelial cells were larger and columnar with inconspicuous nucleoli ; interstitial tissues mainly consisted of myofibroblasts , which had pink cytoplasm and short spindle nuclei without clear cell boundaries , arranged around the blood vessels in bundles or whorls to form thick - walled vessels . all 60 patients were diagnosed as having bph . histological inflammation was certified when inflammatory cell infiltration and accumulation were found in the prostate gland . mild - inflammatory bph was judged as diffused distribution of lymphocytes around the gland ; moderate - inflammatory bph was judged as increase and aggregation of lymphocyte , glandular atrophy , and saccular enlargement of the glandular lumens ; and severe - inflammatory bph was judged as accumulation and infiltration of inflammatory cells , damage of glandular epithelium basement or emergence of lymph nodules ( figure 1 ) . among the 60 bph cases , there were 15 non - inflammatory bph ( ni ) , 12 mild - inflammatory bph ( mi ) , 25 moderate - inflammatory bph ( moi ) , and 8 severe - inflammatory bph ( si ) . general data of bph patients in different groups are listed in table 1 . we found that , compared with bph groups , patients in inflammatory bph groups had higher ipss , more ruv , bigger prostates , and higher expression of psa and crp . the value of ipss , ruv , prostate volume , psa , and crp were also significantly different ( p<0.05 ) between different inflammatory bph groups . the expression of hsp27 and inflammatory cytokines tnf- , il-6 , cd3 were detected by immunohistochemistry . results showed that hsp27 was mainly expressed in glandular epithelium and were hardly expressed in mesenchymal cells ( figure 2 ) . hsp27 expression was higher in inflammatory bph groups than in the non - inflammatory bph group and increased along with the degree of inflammation . mod values of hsp27 expression in each group were : ni bph , 0.04270.0062 ; mi bph , 0.03710.012 ; moi bph , 0.04850.014 ; si bph , 0.05650.009 ( figure 3 ) . . there were similar expression patterns of tnf- , il-6 , cd3 among the 4 groups . expressions of tnf- , il-6 and cd3 were significantly increased in inflammatory bph groups and were much higher in the si bph group ( figures 46 ) . correlation between hsp27 and tnf- , il-6 , cd3 expression in the ni bph group and inflammatory bph group was also analyzed in this study ( table 2 ) . in the ni bph group spearman correlation coefficients between the mod value of hsp27 and tnf- , il-6 , cd3 were 0.556 , 0.651 , and 0.571 ( p < 0.01 ) , respectively . in inflammatory bph groups , spearman correlation coefficients between the mod value of hsp27 and tnf- , il-6 , cd3 were 0.673 , 0.582 , and 0.632 ( p < 0.01 ) , respectively . the results suggested a significant correlation between the expression of hsp27 and tnf- , il-6 , and cd3 . expressions of hsp27 , tnf- , and il-6 in serum were significantly higher in inflammatory bph groups compared with the ni bph group ( p<0.05 ) and increased along with the degree of inflammation ( figure 7 ) . the expression of hsp27 and inflammatory cytokines tnf- , il-6 , cd3 were detected by immunohistochemistry . results showed that hsp27 was mainly expressed in glandular epithelium and were hardly expressed in mesenchymal cells ( figure 2 ) . hsp27 expression was higher in inflammatory bph groups than in the non - inflammatory bph group and increased along with the degree of inflammation . mod values of hsp27 expression in each group were : ni bph , 0.04270.0062 ; mi bph , 0.03710.012 ; moi bph , 0.04850.014 ; si bph , 0.05650.009 ( figure 3 ) . the differences of hsp27 expression between 2 groups were all statistically significant ( p<0.01 ) . there were similar expression patterns of tnf- , il-6 , cd3 among the 4 groups . expressions of tnf- , il-6 and cd3 were significantly increased in inflammatory bph groups and were much higher in the si bph group ( figures 46 ) . correlation between hsp27 and tnf- , il-6 , cd3 expression in the ni bph group and inflammatory bph group was also analyzed in this study ( table 2 ) . in the ni bph group spearman correlation coefficients between the mod value of hsp27 and tnf- , il-6 , cd3 were 0.556 , 0.651 , and 0.571 ( p < 0.01 ) , respectively . in inflammatory bph groups , spearman correlation coefficients between the mod value of hsp27 and tnf- , il-6 , cd3 were 0.673 , 0.582 , and 0.632 ( p the results suggested a significant correlation between the expression of hsp27 and tnf- , il-6 , and cd3 . expressions of hsp27 , tnf- , and il-6 in serum were significantly higher in inflammatory bph groups compared with the ni bph group ( p<0.05 ) and increased along with the degree of inflammation ( figure 7 ) . certain differences exist in the detection rate of histological inflammation in bph patients reported by chinese studies and international reports , which are mainly because of the different diagnostic standards . according to the national institutes of health classification system for prostatitis , a standardized histopathological classification system for chronic prostatitis was established by the north american chronic prostatitis collaborative research network and the international prostatitis collaborative network , which provided a common standard for the basic and clinical research on prostatic inflammation associated with bph . but in china , the diagnostic criteria of histopathological prostatic inflammation associated with bph is not only the presence of inflammatory cells around the glands , but also the infiltration of inflammatory cells into the glandular epithelium and lumen , and destruction of the basement membrane , which is equivalent to grade 3 ( severe ) inflammation according to the international standard . in this study , prostate samples from 60 bph patients were assessed and classified according to the international standard . among all the 60 bph cases , 75% of patients presented chronic inflammation with 20% mild - inflammatory bph ( mi , n=12 ) , 41.7% moderate - inflammatory bph ( moi , n=25 ) , and 13.3% severe - inflammatory bph ( si , n=8 ) , which further suggested the role of chronic prostatic inflammation in the pathogenesis and progression of bph . heat shock proteins ( hsps ) are a group of evolutionarily highly conserved proteins , which can be divided into several major families according to their molecular weight , such as hsp110 , hsp90 , hsp70 , hsp60 , hsp27 , and ubiquitin . hsp27 ( hspb1 ) , a member of the small hsp family , is a key players in many signaling pathways contributing to tumorigenicity , treatment resistance , apoptosis inhibition , and inflammation . it has long been known that hsp27 is a component of the p38 mitogen - activated protein kinase ( mapk ) signaling pathway , which is important in the inflammatory response and other important functions in integrating physiological and pathological stimuli . alford et al . further demonstrated the role of hsp27 in pro - inflammatory cell signaling and the expression of pro - inflammatory genes by using sirnas to suppress hsp27 expression in hela cells and fibroblasts . they found that hsp27 was needed for the activation of tak1 and downstream signaling by p38 mapk , jnk , and their activators . hsp27 was also required for il-1-induced expression of the pro - inflammatory mediators il-6 and il-8 . in this work we also detected the expression of il-6 and found that , along with the high expression level of hsp27 in severe - inflammatory bph patients , the expression of il-6 relevantly increased , suggesting the actived pro - inflammatory cell signaling by hsp27 , which might be mediated by the p38 mapk signaling pathway , but further verification is needed . overexpression of hsp27 has also been linked to the development of some cancers , such as pancreatic cancer , non - small cell lung cancer , colorectal cancer and prostate cancer , which led to its use as a prognostic marker for these cancers . liu et al . studied the expression of hsp27 in prostatic hyperplasia , prostatic intraepithelial neoplasm , and adenocarcinoma of the prostate by immunohistochemistry , showing that there was a significant increase in hsp27 expression between bph tissues and cancerous tissues . the positive rate of hsp27 was 6.67% in prostatic hyperplasia , 26.67% in prostatic intraepithelial neoplasm , and 60.00% in adenocarcinoma of the prostate . they also found the expression of hsp27 in poorly differentiated group was higher than that of the well - moderately differentiated group , and hsp27 expression was higher in the bony metastasis group than in the no bony metastasis group , which suggested a potential prognostic role of hsp27 in the occurrence and development of prostate cancer . our preliminary results also suggested the use of hsp27 as a potential prognostic biomarker for inflammatory bph . epidemiological , histopathological , and molecular pathological studies have been providing emerging evidence implicating inflammation in the pathogenesis of both bph and prostate cancer [ 2426 ] . de marzo proposed that exposure to environmental factors such as infectious agents and dietary carcinogens , as well as hormonal imbalances , could lead to injury of the prostate and to the development of chronic inflammation and proliferative inflammatory atrophy ( pia ) , which might finally make the transition to adenocarcinoma . based on the potential role of histological inflammation in prostatic hyperplasia and prostate cancer , and the role of hsps in inflammation , hsp27 might be important in prevention and as a treatment target for inflammatory bph and prostate cancer . more studies are needed to elucidate the detailed mechanisms underlying the relevance of hsp27 to development and pathological process of bph with chronic inflammation . in this study , the expression of hsp27 was detected in the cytoplasm of prostate epithelial cells , without presence in mesenchymal cells , which is consistent with its reported role in emt in prostate cancer . with more inflammation , the expression of hsp27 increased , suggesting that elevated inflammatory stimulation induces hsp27 expression and promotes progression of clinical symptoms of prostate hyperplasia , resulting in urinary retention or severe urinary tract symptoms in patients . in this study we also found that cd3 expression was positively correlated with hsp27 expression and proinflammatory cytokines tnf alpha and il-6 expression , which suggests that inflammatory stimuli activates the immune system and stress response , which at the same time participates jointly in the development of prostatic inflammation associated with bph . our work suggests the potential role of hsp27 as a prognostic biomarker and therapeutic target for inflammatory bph .

backgroundheat shock protein 27 ( hsp 27 ) is known as a mediator in immune response and has been recently found to be expressed in prostate cancer . this study aimed to investigate the role of hsp27 in inflammatory bph.material/methodshospitalized bph patients who received turp were divided into 4 groups by the presence and degrees of chronic inflammation : non - inflammatory bph ( ni bph ) , mild - inflammatory bph ( mi bph ) , moderate - inflammatory bph ( moi bph ) , and severe - inflammatory bph ( si bph ) . expressions of hsp 27 , tnf- , il-6 , and cd3 in prostate tissues and serum of patients were detected by immunohistochemistry and elisa.resultsexpression of hsp27 in bph with histological inflammation was significantly higher than in non - inflammatory bph . in inflammatory bph groups , hsp27 expression gradually increased along with increasing inflammation . there was a significant correlation between the expression of tnf- , il-6 , cd3 and hsp27 among different inflammatory bph groups.conclusionshsp27 expression level is associated with the degree of chronic inflammation in bph and may participate in the pathological process in inflammatory bph .

Background Material and Methods Ethics statement Subjects Groups Demographic and clinical data Immunohistochemistry Enzyme-linked immuno sorbent assay Statistical analysis Results Expression HSP27, TNF-, IL-6 and CD3 in prostate tissue Expression of HSP27, TNF- and IL-6 in serum Discussion Conclusions

benign prostatic hyperplasia ( bph ) is the most common urological disease among aged men , with incidence over 50% at age 60 years and over 90% at age 80 years . in recent years accumulating evidence recent studies suggest that inflammation and abnormal immunoregulation may contribute to cytokine production by inflammatory cells driving local growth factor production and angiogenesis in the prostatic tissue , and that this proinflammatory microenvironment is closely related to bph stromal hyperproliferation and tissue remodeling . by controlling binding and release , hsps function mainly as molecular chaperones , which participate in the folding and assembly of nascent and unfolding proteins and hsps are classified into 4 major families according to their biological activities : hsp90 , hsp70 , hsp60 , and small hsps . it has been implicated in different diseases , playing both protective and counter - protective roles , such as renal injury and fibrosis , cancer , and neuro - degenerative and cardiovascular diseases , highlighting its role as a potential biomarker and therapeutic target . recently , it has been reported that hsp27 presents in the prostate cancer cell line and its expression is related to the prostate cancer malignancy level . accumulating evidence now suggests that bph and prostate cancer share important anatomic , pathologic , and genetic links in addition to the well - established epidemiologic association . recent publications support the hypothesis that bph and prostate cancer are part of metabolic syndrome , and inflammation as a major contributor to the development of both bph and prostate cancer . however , the expression of hsp27 in bph and its correlation with inflammation level are unclear . in this study , to investigate the role of hsp27 in inflammatory bph , we evaluated hsp27 expression and its relation to conditions of prostatic inflammation associated with bph , correlating it with the levels of inflammatory factors such as tumor tnf- , il-6 , and cd3 . this study was approved by the ethics committee of the third affiliated hospital of hebei medical university . we recruited 60 bph patients who received transurethral electroresection of prostate ( turp ) because of urinary retention or lower urinary tract symptoms ( luts ) at the third affiliated hospital of hebei medical university between april 2013 and october 2013 into this cross - sectional study . before the surgery , all the patients received tests for blood routine , urine routine , liver and renal function , chest radiograph , electrocardiogram , abdominal and urethral system ultrasonography , prostate - specific antigen ( psa ) , and c reactive protein ( crp ) . international prostate symptom score ( ipss ) and quality of life ( qol ) patients with the following conditions were excluded : history of acute prostatitis or chronic bacteriogenic prostatitis , urinary tract infection , a history of chronic pelvic pain , incidentally discovered prostatic cancer ( idpc ) or endothelial hyperplasia of prostate , cerebrovascular disease or spinal cord disease , and oral antibiotics a week before the surgery . histological inflammation was certified when inflammatory cell infiltration and accumulation were found in the prostate gland . among the 60 bph patients , there were 15 cases of non - inflammatory bph ( ni ) and 45 cases of inflammatory bph . the inflammatory bph group was further divided into 3 different degrees by the number of inflammatory cells , the extent of the damage to prostate gland , and the emergence of lymph nodules : mild - inflammatory bph ( mi , n=12 ) with sporadic inflammatory cell infiltration and no damage to the glandular epithelium ; moderate - inflammatory bph ( moi , n=25 ) with inflammatory cell accumulation but no damage to the glandular epithelium basement , or lymph nodules ; and severe - inflammatory bph ( si , n=8 ) with inflammatory cell accumulation , damage to the glandular epithelium basement , or emergence of lymph nodules . basic demographic information and clinical data were collected , including age , height , weight , the history of urinary retention , complications , international prostate symptom score ( ipss ) , quality of life ( qol ) , prostate volume , residual urine volume ( ruv ) , maximum flow rate ( qmax ) , prostate specific antigen ( psa ) , c - reactive protein ( crp ) , leucocyte counts in blood test and urine test , and body mass index ( bmi ) . urodynamic tests were performed for each patient , and patients with urine retention were tested a week after catheter setting . formalin - fixed , paraffin - embedded sections ( 4 m ) of prostate tissues were immunostained for hsp27 ( rabbit polyclonal antibody bs1177 , bioworld technology , usa ) , tnf- ( rabbit polyclonal antibody bs6000 , bioworld technology , usa ) , il-6 ( rabbit polyclonal antibody bs6419 , bioworld technology , usa ) and cd3 ( rabbit polyclonal antibody bs6280 , bioworld technology , usa ) as follows . for each section , concentrations of hsp27 , tnf- and il-6 in serums were measured using commercially available enzyme - linked immunoassay ( elisa ) kits ( uscnlife , shanghai , china ) according to the manufacturer s instructions . this study was approved by the ethics committee of the third affiliated hospital of hebei medical university . we recruited 60 bph patients who received transurethral electroresection of prostate ( turp ) because of urinary retention or lower urinary tract symptoms ( luts ) at the third affiliated hospital of hebei medical university between april 2013 and october 2013 into this cross - sectional study . before the surgery , all the patients received tests for blood routine , urine routine , liver and renal function , chest radiograph , electrocardiogram , abdominal and urethral system ultrasonography , prostate - specific antigen ( psa ) , and c reactive protein ( crp ) . international prostate symptom score ( ipss ) and quality of life ( qol ) patients with the following conditions were excluded : history of acute prostatitis or chronic bacteriogenic prostatitis , urinary tract infection , a history of chronic pelvic pain , incidentally discovered prostatic cancer ( idpc ) or endothelial hyperplasia of prostate , cerebrovascular disease or spinal cord disease , and oral antibiotics a week before the surgery . histological inflammation was certified when inflammatory cell infiltration and accumulation were found in the prostate gland . among the 60 bph patients , there were 15 cases of non - inflammatory bph ( ni ) and 45 cases of inflammatory bph . the inflammatory bph group was further divided into 3 different degrees by the number of inflammatory cells , the extent of the damage to prostate gland , and the emergence of lymph nodules : mild - inflammatory bph ( mi , n=12 ) with sporadic inflammatory cell infiltration and no damage to the glandular epithelium ; moderate - inflammatory bph ( moi , n=25 ) with inflammatory cell accumulation but no damage to the glandular epithelium basement , or lymph nodules ; and severe - inflammatory bph ( si , n=8 ) with inflammatory cell accumulation , damage to the glandular epithelium basement , or emergence of lymph nodules . basic demographic information and clinical data were collected , including age , height , weight , the history of urinary retention , complications , international prostate symptom score ( ipss ) , quality of life ( qol ) , prostate volume , residual urine volume ( ruv ) , maximum flow rate ( qmax ) , prostate specific antigen ( psa ) , c - reactive protein ( crp ) , leucocyte counts in blood test and urine test , and body mass index ( bmi ) . formalin - fixed , paraffin - embedded sections ( 4 m ) of prostate tissues were immunostained for hsp27 ( rabbit polyclonal antibody bs1177 , bioworld technology , usa ) , tnf- ( rabbit polyclonal antibody bs6000 , bioworld technology , usa ) , il-6 ( rabbit polyclonal antibody bs6419 , bioworld technology , usa ) and cd3 ( rabbit polyclonal antibody bs6280 , bioworld technology , usa ) as follows . concentrations of hsp27 , tnf- and il-6 in serums were measured using commercially available enzyme - linked immunoassay ( elisa ) kits ( uscnlife , shanghai , china ) according to the manufacturer s instructions . histological inflammation was certified when inflammatory cell infiltration and accumulation were found in the prostate gland . mild - inflammatory bph was judged as diffused distribution of lymphocytes around the gland ; moderate - inflammatory bph was judged as increase and aggregation of lymphocyte , glandular atrophy , and saccular enlargement of the glandular lumens ; and severe - inflammatory bph was judged as accumulation and infiltration of inflammatory cells , damage of glandular epithelium basement or emergence of lymph nodules ( figure 1 ) . among the 60 bph cases , there were 15 non - inflammatory bph ( ni ) , 12 mild - inflammatory bph ( mi ) , 25 moderate - inflammatory bph ( moi ) , and 8 severe - inflammatory bph ( si ) . we found that , compared with bph groups , patients in inflammatory bph groups had higher ipss , more ruv , bigger prostates , and higher expression of psa and crp . the value of ipss , ruv , prostate volume , psa , and crp were also significantly different ( p<0.05 ) between different inflammatory bph groups . the expression of hsp27 and inflammatory cytokines tnf- , il-6 , cd3 were detected by immunohistochemistry . hsp27 expression was higher in inflammatory bph groups than in the non - inflammatory bph group and increased along with the degree of inflammation . mod values of hsp27 expression in each group were : ni bph , 0.04270.0062 ; mi bph , 0.03710.012 ; moi bph , 0.04850.014 ; si bph , 0.05650.009 ( figure 3 ) . there were similar expression patterns of tnf- , il-6 , cd3 among the 4 groups . expressions of tnf- , il-6 and cd3 were significantly increased in inflammatory bph groups and were much higher in the si bph group ( figures 46 ) . correlation between hsp27 and tnf- , il-6 , cd3 expression in the ni bph group and inflammatory bph group was also analyzed in this study ( table 2 ) . in the ni bph group spearman correlation coefficients between the mod value of hsp27 and tnf- , il-6 , cd3 were 0.556 , 0.651 , and 0.571 ( p < 0.01 ) , respectively . in inflammatory bph groups , spearman correlation coefficients between the mod value of hsp27 and tnf- , il-6 , cd3 were 0.673 , 0.582 , and 0.632 ( p < 0.01 ) , respectively . the results suggested a significant correlation between the expression of hsp27 and tnf- , il-6 , and cd3 . expressions of hsp27 , tnf- , and il-6 in serum were significantly higher in inflammatory bph groups compared with the ni bph group ( p<0.05 ) and increased along with the degree of inflammation ( figure 7 ) . the expression of hsp27 and inflammatory cytokines tnf- , il-6 , cd3 were detected by immunohistochemistry . results showed that hsp27 was mainly expressed in glandular epithelium and were hardly expressed in mesenchymal cells ( figure 2 ) . hsp27 expression was higher in inflammatory bph groups than in the non - inflammatory bph group and increased along with the degree of inflammation . mod values of hsp27 expression in each group were : ni bph , 0.04270.0062 ; mi bph , 0.03710.012 ; moi bph , 0.04850.014 ; si bph , 0.05650.009 ( figure 3 ) . the differences of hsp27 expression between 2 groups were all statistically significant ( p<0.01 ) . there were similar expression patterns of tnf- , il-6 , cd3 among the 4 groups . expressions of tnf- , il-6 and cd3 were significantly increased in inflammatory bph groups and were much higher in the si bph group ( figures 46 ) . correlation between hsp27 and tnf- , il-6 , cd3 expression in the ni bph group and inflammatory bph group was also analyzed in this study ( table 2 ) . in the ni bph group spearman correlation coefficients between the mod value of hsp27 and tnf- , il-6 , cd3 were 0.556 , 0.651 , and 0.571 ( p < 0.01 ) , respectively . in inflammatory bph groups , spearman correlation coefficients between the mod value of hsp27 and tnf- , il-6 , cd3 were 0.673 , 0.582 , and 0.632 ( p the results suggested a significant correlation between the expression of hsp27 and tnf- , il-6 , and cd3 . expressions of hsp27 , tnf- , and il-6 in serum were significantly higher in inflammatory bph groups compared with the ni bph group ( p<0.05 ) and increased along with the degree of inflammation ( figure 7 ) . certain differences exist in the detection rate of histological inflammation in bph patients reported by chinese studies and international reports , which are mainly because of the different diagnostic standards . according to the national institutes of health classification system for prostatitis , a standardized histopathological classification system for chronic prostatitis was established by the north american chronic prostatitis collaborative research network and the international prostatitis collaborative network , which provided a common standard for the basic and clinical research on prostatic inflammation associated with bph . but in china , the diagnostic criteria of histopathological prostatic inflammation associated with bph is not only the presence of inflammatory cells around the glands , but also the infiltration of inflammatory cells into the glandular epithelium and lumen , and destruction of the basement membrane , which is equivalent to grade 3 ( severe ) inflammation according to the international standard . in this study , prostate samples from 60 bph patients were assessed and classified according to the international standard . among all the 60 bph cases , 75% of patients presented chronic inflammation with 20% mild - inflammatory bph ( mi , n=12 ) , 41.7% moderate - inflammatory bph ( moi , n=25 ) , and 13.3% severe - inflammatory bph ( si , n=8 ) , which further suggested the role of chronic prostatic inflammation in the pathogenesis and progression of bph . heat shock proteins ( hsps ) are a group of evolutionarily highly conserved proteins , which can be divided into several major families according to their molecular weight , such as hsp110 , hsp90 , hsp70 , hsp60 , hsp27 , and ubiquitin . hsp27 ( hspb1 ) , a member of the small hsp family , is a key players in many signaling pathways contributing to tumorigenicity , treatment resistance , apoptosis inhibition , and inflammation . it has long been known that hsp27 is a component of the p38 mitogen - activated protein kinase ( mapk ) signaling pathway , which is important in the inflammatory response and other important functions in integrating physiological and pathological stimuli . further demonstrated the role of hsp27 in pro - inflammatory cell signaling and the expression of pro - inflammatory genes by using sirnas to suppress hsp27 expression in hela cells and fibroblasts . hsp27 was also required for il-1-induced expression of the pro - inflammatory mediators il-6 and il-8 . in this work we also detected the expression of il-6 and found that , along with the high expression level of hsp27 in severe - inflammatory bph patients , the expression of il-6 relevantly increased , suggesting the actived pro - inflammatory cell signaling by hsp27 , which might be mediated by the p38 mapk signaling pathway , but further verification is needed . overexpression of hsp27 has also been linked to the development of some cancers , such as pancreatic cancer , non - small cell lung cancer , colorectal cancer and prostate cancer , which led to its use as a prognostic marker for these cancers . studied the expression of hsp27 in prostatic hyperplasia , prostatic intraepithelial neoplasm , and adenocarcinoma of the prostate by immunohistochemistry , showing that there was a significant increase in hsp27 expression between bph tissues and cancerous tissues . the positive rate of hsp27 was 6.67% in prostatic hyperplasia , 26.67% in prostatic intraepithelial neoplasm , and 60.00% in adenocarcinoma of the prostate . they also found the expression of hsp27 in poorly differentiated group was higher than that of the well - moderately differentiated group , and hsp27 expression was higher in the bony metastasis group than in the no bony metastasis group , which suggested a potential prognostic role of hsp27 in the occurrence and development of prostate cancer . our preliminary results also suggested the use of hsp27 as a potential prognostic biomarker for inflammatory bph . epidemiological , histopathological , and molecular pathological studies have been providing emerging evidence implicating inflammation in the pathogenesis of both bph and prostate cancer [ 2426 ] . de marzo proposed that exposure to environmental factors such as infectious agents and dietary carcinogens , as well as hormonal imbalances , could lead to injury of the prostate and to the development of chronic inflammation and proliferative inflammatory atrophy ( pia ) , which might finally make the transition to adenocarcinoma . based on the potential role of histological inflammation in prostatic hyperplasia and prostate cancer , and the role of hsps in inflammation , hsp27 might be important in prevention and as a treatment target for inflammatory bph and prostate cancer . more studies are needed to elucidate the detailed mechanisms underlying the relevance of hsp27 to development and pathological process of bph with chronic inflammation . in this study , the expression of hsp27 was detected in the cytoplasm of prostate epithelial cells , without presence in mesenchymal cells , which is consistent with its reported role in emt in prostate cancer . with more inflammation , the expression of hsp27 increased , suggesting that elevated inflammatory stimulation induces hsp27 expression and promotes progression of clinical symptoms of prostate hyperplasia , resulting in urinary retention or severe urinary tract symptoms in patients . in this study we also found that cd3 expression was positively correlated with hsp27 expression and proinflammatory cytokines tnf alpha and il-6 expression , which suggests that inflammatory stimuli activates the immune system and stress response , which at the same time participates jointly in the development of prostatic inflammation associated with bph . our work suggests the potential role of hsp27 as a prognostic biomarker and therapeutic target for inflammatory bph .

studies have shown cognitive deficits ( in particular deficits in information processing speed , concentration , and working memory ) to be present in the early stages of ms [ 24 ] . cognitive impairment has a negative impact on quality of life ( qol ) independent of physical symptoms [ 5 , 6 ] . there exists mounting evidence for neuroplasticity as a mechanism to compensate for accumulating pathology in ms and some tentative evidence that cognitive rehabilitation may be effective in preserving or improving cognitive function in patients with ms [ 79 ] . computer - assisted cognitive rehabilitation has the potential to provide a structured and standardised approach to rehabilitation . rehacom is one particular type of software designed and utilised for treatment of cognitive impairment in a number of disease states such as stroke , brain injury , and psychiatric disorders [ 10 , 11 ] . it has been used in a growing number of trials of cognitive rehabilitation in ms as a more standardised intervention [ 8 , 9 , 12 , 13 ] . the difficulty level of the computerised tasks adapts to an individual 's performance , only increasing in difficulty in response to improving performance . few studies have examined the structural basis of cognitive rehabilitation and longitudinal studies are relatively lacking [ 8 , 14 ] . animal data suggest that myelination is , at least in part , regulated by neuronal activity . it is therefore conceivable that techniques , such as magnetisation transfer ( mt ) , which is sensitive to myelin content , might be sensitive to structural plasticity in ms . in this study we combined neuropsychological assessment , functional mri ( fmri ) , and quantitative magnetisation transfer ( qmt ) imaging to explore whether home - based , computerised cognitive rehabilitation is an effective means of promoting cognitive rehabilitation and whether the structural basis for rehabilitation can be better defined . the primary outcome of the study was measured as any improvement in cognition after the training , while the secondary outcomes included changes in fmri , fatigue , and quality of life assessments . participants . thirty - eight patients with objective evidence of cognitive impairment were invited to participate in this study between february 2014 and february 2015 . all participants signed informed written consent before undergoing testing . inclusion criteria were as follows : ( a ) age between 18 and 65 , ( b ) clinically definite ms , according to the mcdonald criteria , ( c ) expanded disability status scale ( edss ) 6.5 , and ( d ) cognitive impairment defined as scores below the 5th percentile for normative data adjusted for age , sex , and years of formal education on one or more of the bicams tests . patients were excluded if they had a history of significant psychiatric disorders , alcohol or substance abuse , visual acuity less than 6/18 corrected , oscillopsia , or diplopia that would interfere with testing . patients were also excluded if they had a ms relapse , received corticosteroids , or changes made to psychoactive medications within the previous month . the study was approved by the northern ireland research ethics committee . study design . neuropsychological and mri data were obtained at baseline ( time 1 ) , immediately following a 6-week intervention period ( time 2 ) and after an additional 12-week follow - up period ( time 3 ) , during which no additional intervention was administered ( supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4292585 ) . it was not possible for the cognitive assessments to be completed by a blinded assessor . the mri analysis was conducted by a researcher blind to the patients ' group allocation . cognitive and behavioural assessments . at entry all participants underwent a detailed clinical neurological assessment including edss conducted by an experienced neurologist . patients were screened for cognitive impairment using the brief international cognitive assessment for ms ( bicams ) . bicams is a brief ( 15-minute ) screening tool to identify cognitive impairment in patients with ms and comprises the first five learning trials of the california verbal learning test ii ( cvlt - ii ) , the first three recall trials of brief visuospatial memory test revised ( bvmt - r ) , and the symbol digits modalities test ( sdmt ) . the bicams assessment was conducted by a neurology clinical fellow with almost ten years of clinical experience ( j.c ) . the assessing neurologist was trained in bicams assessing methods by an experienced neuropsychologist ( d.l ) . at baseline participants also completed a number of behavioural and qol assessments including euroqol five - dimension questionnaire ( eq-5d ) , a generic health - related quality of life scale , functional assessment of ms ( fams ) ( a ms specific quality of life scale ) , patient activation measure ( pam-13 ) ( a 13-item generic scale for chronic illness management ) , a measure of patient empowerment in ms , unidimensional self - efficacy scale for ms ( use - ms ) , the hospital anxiety and depression scale ( hads ) , multiple sclerosis neuropsychological questionnaire ( msnq ) self - report ( a patient self - reported measure of cognitive function ) , and the fatigue severity scale . at each subsequent time point participants underwent repeat cognitive assessment using bicams ( same test forms ) as well as repeat behavioural and qol assessments . randomisation was performed using a random number generator and allocations were placed inside sealed folders . the treatment group underwent six weeks of home - based , computer - assisted cognitive rehabilitation using rehacom software ( https://www.fixxl.co.uk/ ) . the control group were asked to watch a series of natural history dvds of corresponding duration and frequency to the rehabilitation sessions performed by the treatment group for six weeks . the need to evaluate mri parameters in studies with active control conditions has been highlighted . treatment sessions consisted of training in three specific modules involving working memory , visuospatial memory , and divided attention . in all tasks the level of difficulty is tailored to the individuals performance and increases automatically but only in line with satisfactory progress . real - time data pertaining to performance , progress , and compliance is transmitted to the investigator over the internet during the intervention period . multiple distractions must be navigated and the speed and direction of the vehicle altered according to road conditions . as the complexity of the task increases , more distractors are introduced with increased multitasking skills required . working memory the working memory task consists of remembering a series of playing cards presented briefly on screen . the participant is then asked to select which cards were presented from a longer series of options including distractor cards . as the complexity of the task increases , participants are asked to remember only cards of a particular value or suit and the number of items to remember increases . higher levels involve having to remember the cards in reverse order . visuospatial memory is a similar task involving various objects presented briefly on screen with the patient asked to remember the object as well as its position in the sequence . as the complexity of the task increases , the following sequences were acquired in an order designed to minimise the potential for fatigue on the fmri task : ( 1 ) dual - echo turbo spin - echo for lesion identification ; ( 2 ) high - resolution t1-weighted magnetisation - prepared rapid - acquisition gradient echo ( mprage ) ; ( 3 ) functional mri with echo - planar imaging ( epi ) acquired during a n - visually presented back task ; ( 4 ) quantitative magnetisation transfer ( qmt ) with balanced steady - state free precession ( bssfp ) . t2 lesion volume was measured at baseline for each participant using the software package jim ( version 3.0 , xinapse systems ltd . , this was adapted from sweet et al . and involved three conditions : 0-back , 1-back , and 2-back tasks . the 0-back condition was designed to act as the baseline condition and would provide the baseline activation for comparison in fmri analysis . the 1-back and 2-back conditions provided increasing working memory demands . the n - back task did not constitute part of the cognitive rehabilitation . all participants were allowed to briefly practice the n - back task under supervision for five minutes prior to the mri scan to ensure comprehension of the task and allow familiarity with it . stimuli were projected onto a mirrored screen inside the mri scanner 45 cm from a participant 's nose . this involved of a series of pseudo - randomised consonants in both upper and lower cases . the stimulus duration was 1000 ms with a between stimulus interval of 2000 ms . 0-back , 1-back , and 2-back tasks were presented in a randomised manner resulting in six blocks per nine - minute run . each block consisted of 126 stimuli , one - third of which were targets . twice as many 0-back tasks were presented as 1-back or 2-back . the primary outcome was cognitive performance as measured by improvement in sdmt , bvmt , and cvlt between groups compared to baseline . the means of continuous variables were compared using the independent samples t - test or the mann all tests were two - tailed ; p - values less than 0.05 were considered significant . outcomes were compared between the two groups using independent samples t - test to compare gain scores for cognitive data between groups . to compare differences between groups for other behavioural and qol data , a 2 3 repeat measures analysis of variance ( anova ) was used with time as the within - subject factor and treatment as the between factor ( active rehabilitation versus control ) . analyses were performed using spss version 21 ( armonk , ny : ibm corp ) . fmri data were analysed using spm8 ( wellcome department of cognitive neurology , ucl , london , http://www.fil.ion.ucl.ac.uk/spm/ ) . for each time series individual epis were then realigned to the first remaining image of the series by rigid - body transformation to correct for involuntary head movements during acquisition before normalisation into a standard anatomical space ( montreal neurological institute [ mni ] ) using linear and nonlinear transformations . finally , images were smoothed with an 8 mm full - width - at - half - maximum ( fwhm ) 3d gaussian kernel . first - level analysis . for each participant , the difference in blood oxygen level - dependent ( bold ) response between the 0-back , 1-back , and 2-back conditions was estimated at every voxel across the whole brain using the general linear model ( glm ) . this produced a series of contrasts representing mean activation during each n - back condition minus the 0-back condition . each contrast obtained at the first - level was entered into a second - level glm to generate summary statistical parametric maps ( spms ) . for between - group analysis of difference between the time points , we used a 3 2 anova flexible factorial design with group ( between - subject ) and time ( within - subject ) as separate factors to examine the main effects on group ( treatment versus control ) , time and the interaction between them to evaluate areas of relative change in activity after cognitive training versus control . the threshold for significance was set at alpha of 0.05 corrected for multiple comparisons ( family - wise error ( fwe ) corrected ) . , the location of local maxima of signal intensity increase is expressed as x , y , and z coordinates in mni space . mt and t1 mapping data from all three sessions were first realigned to subject specific mprage structural images using the spm8 rigid - body registration function . the mprage were then segmented into white matter , grey matter , and csf to yield a parenchymal mask . a t1 map was calculated for all datasets by fitting the theoretical spoiled gradient echo as a function of the flip angle to the signal measured by the 3d flash sequences . mt parameters were obtained by performing a voxelwise nonlinear least squares fitting ( levenberg - marquardt ) to a binary spin bath model for bssfp . the statistical analysis was performed voxelwise in spm8 on the resulting warped and smoothed mt maps . the same glms used for the second - level fmri analysis and described in the previous section were used for estimating the main effects of time and group and the interaction between these two factors . patients ( 70.3% ) had rrms , and 11 patients ( 29.7% ) had spms . the duration of ms from diagnosis to enrolment ranged from 12 months to 40 years ( mean 11.61 years , sd 8.2 yrs ) . 20 patients ( 52.6% ) were on disease modifying therapy at enrolment ( natalizumab n = 6 , beta - interferon n = 7 , fingolimod n = 6 , and teriflunomide n = 1 ) . after randomisation to either computer - assisted cognitive training ( treatment group , n = 19 ) or the active control condition ( n = 19 ) , there were no significant differences in terms of baseline demographics ( table 1 ) or quality of life measures ( supplementary table 3 ) between the two groups . the treatment group had higher baseline cognitive scores on the bicams battery ; however , these did not differ significantly from the control group . the most frequently failed component of the bicams test battery was the sdmt with 33 ( 86.8% ) of participants scoring below the 5th centile , 18 ( 47.4% ) failing the cvlt - ii , and 13 ( 34.2% ) failing the bvmt - r . overall 21 ( 55.2% ) failed one test , 10 ( 26.3% ) failed two tests , and 7 ( 18.4% ) failed all three tests of the bicams test battery . this level of impairment is consistent with other published ms samples on bicams [ 3133 ] . overall , 88.9% of patients ( 16/18 ) in the intervention group completed at least 75% of the prescribed sessions with 66.7% ( 12/18 ) completing all the prescribed sessions . , the treatment group showed a significantly greater improvement in gain scores between baseline and early follow - up ( time 2 ) compared to the control group on the sdmt ( treatment 3.94 ( sd 5.08 ) , controls 0.63 ( sd 3.30 ) , 1.47 to 7.66 , ( 95% ci 1.47 to 7.66 ) , p = 0.005 ) illustrated in figure 1 . similar gain scores in the cvlt and bvmt - r were not significantly different between the groups although the bvmt - r gain scores did approach significance ( p = 0.098 ) . overall , there was an improvement in bicams performance across participants at follow - up . the gain scores between the groups at time 3 compared to baseline were , however , not statistically significantly different . there were no significant differences in qol outcome measures , measures of self - efficacy , or subjective cognitive performance between the two groups ( supplementary table 4 ) . the baseline error rate between the treatment and control groups was low ( 8.64% versus 9.48% , p = 0.814 ) . no significant differences were observed in the error rate during the n - back task between the groups at baseline or at follow - up ( supplementary table 5 ) . the 1-back task was associated with activations involving the dorsolateral prefrontal cortex bilaterally as well as bilateral inferior parietal lobule and insular and cerebellar regions relative to the 0-back contrast . the same regions were activated in the 2-back condition but the spatial extent and magnitude of the responses were greater , particularly over the frontoparietal regions ( supplementary figure 2 and supplementary table 2 ) . time 2 versus time 1 . at time 2 , increased activation was seen in the right temporoparietal regions ( right supramarginal and angular gyri ( p < 0.005fwe corrected at cluster level ( k = 228 ) ) ) in the 1-back in the treatment group relative to controls ( group - by - time interaction ) . 3 significant increases in activation were seen in both the 1-back and 2-back conditions in the treatment group relative to controls . in the 1-back task , increased activation was seen in the left frontal ( p < 0.001fwe corrected at cluster level ( k = 294 ) ) and right temporoparietal regions ( p < 0.012fwe corrected at cluster level ( k = 187 ) ) . in the 2-back task , increases in activation were seen in bilateral prefrontal ( p < 0.013fwe corrected at cluster level ( k = 206 ) ) and right temporoparietal regions ( p < 0.024fwe corrected at cluster level ( k = 178 ) ) ( figure 2 ) . quantitative magnetisation transfer . no significant between - group changes were seen in the qmt at time 2 or time 3 , with respect to time 1 . overall qmt measures showed stability across all participants over the course of the study in measures of all indices . in line with previous work , the main outcome of this study was that 6 weeks of computerised cognitive rehabilitation was associated with improvement in cognitive performance as measured on the sdmt . significant alterations in brain fmri activations during the n - back task were also seen at follow - up . the sdmt improvement in the treatment group was , however , not maintained after cessation of cognitive rehabilitation ( time 3 assessments ) although the functional mri changes were seen to persist at follow - up . the sdmt is among the most sensitive tests of slowed information processing speed in ms [ 34 , 35 ] and may also be a proxy for general cognitive impairment . compared to time 1 , the treatment group showed a significantly greater improvement in gain scores between baseline and early follow - up ( time 2 ) compared to the control group on the sdmt ( p = 0.005 ) . however , the gain scores between the groups at time 3 compared to baseline were not statistically significantly different . . it may be the case that cognitive rehabilitation does indeed result in improved cognitive performance but that maintenance of such improvement requires some form of ongoing intervention in the longer term . only one version of the bicams test battery has been validated in ms and thus was used in this study . reported test - retest coefficients on the bicams tests are excellent , suggesting that practice effects are negligible [ 31 , 32 ] . in addition , the experimental design was to compare two groups with identical testing schedules ; therefore the impact of practice effects on the results is likely to be minimal . qol is a complex construct influenced by a multitude of factors such as employment status , social networks , and perceptions of self - worth and self - efficacy . it is possible that cognitive rehabilitation has a positive impact on a number of these factors but such changes in such factors may take time to manifest as improvements in qol . further longitudinal analysis may be required to investigate this . in order to minimise practice effects associated with repeat testing , participants were not directly trained in the n - back task ; rather it was utilised as an outcome measure of working memory . it was anticipated that if cognitive rehabilitation was effective at improving working memory and attention , then the effects would be reflected on the performance on the n - back task . no differences were seen in the error rate between the groups during the n - back task at follow - up ; however , the error rate was low at baseline in both groups . the n - back fmri paradigm in our study cohort was , however , associated with robust baseline cortical activations ( in particular within the dlpfc and posterior parietal cortex ) in keeping with known working memory networks . a significant group - by - time interaction was seen with the treatment group exhibiting increased activation in the bilateral prefrontal cortex and right temporoparietal regions relative to control group at time 3 ( p < 0.05fwecorr ) . changes in functional activation within these regions within the treatment group are felt to be functionally relevant with respect to cognitive rehabilitation . it has been shown that the prefrontal cortex is critical in the executive control of working memory and has a role in response inhibition [ 38 , 39 ] . effective organisation of working memory may attenuate task difficulty resulting in improved working memory performance . a right hemisphere dominant ventral attentional network consisting of the temporoparietal junction , ventral prefrontal cortex , and anterior insula previous work in ms has indicated that attention may be one of the domains most amenable to rehabilitation . many of the computer - training tasks involve sustained attention and it might be postulated that the increased activation seen in the right temporoparietal region at follow - up in the treatment group is as a result of improved efficiency of this network . interestingly , the evolution of much activity on fmri developed after cessation of the active intervention phase . this solidification of neural networks may extend to areas / networks outside those directly trained and may explain why working memory centres such as the prefrontal cortex were seen to be persistently active after cessation of formal training . debate remains however as to the possible interplay between adaptive and maladaptive responses during functional brain reorganisation . the discrepancy between the apparent lack of clinical difference between the groups at time 3 and the sustained fmri effect at time 3 may reflect the fact that bicams does not adequately measure working memory which is primarily domain utilised during the n - back fmri paradigm . some studies have identified structural changes on diffusion tensor imaging as a result of rehabilitation in the context of physiotherapy [ 42 , 43 ] . we did not detect any structural change on qmt after training . due to the short duration of follow - up , this is not entirely surprising . functional alterations in cortical activity may subsequently modulate brain structure at the microstructural level but such changes in structural brain architecture might only be detectable over the longer term . in contrast to many previous studies , which often rely on one - to - one or outpatient administered cognitive rehabilitation , this study sought to explore whether a home - based approach to cognitive rehabilitation was feasible . a home - based approach to cognitive rehabilitation is significantly less resource intensive and may pave the way to greater access for a greater number of patients to such interventions in the future firstly , the groups were relatively small and there was a dropout of patients mainly in the control group between time 2 and time 3 . there was heterogeneity with regard to the cognitive domains that showed deficits among participants in the study . it is likely therefore that they may not have benefited from the rehabilitation in the same way . as the study was largely exploratory in nature , it utilised an open design and is therefore subject to a number of limitations inherent to this type of design . for pragmatic reasons blinding of the investigating neurologist was not established due to the potential need for interaction between patient and investigator . this does present the potential for observer bias , particularly where repeat testing is required . spm analysis of mri data offers objective , largely automated measures , which are independent of measurement bias . investigator blinding was maintained for any methods such as assessment of white matter lesion volumes that involved manual interpretation . in many respects , the sdmt may provide a proxy for overall cognitive functioning but a more detailed cognitive assessment of the domains directly trained may have provided additional insight into effectiveness of cognitive training . bicams is primarily designed as a screening tool for cognitive impairment in ms assessing a limited number of domains . however , strong ecological validity has been demonstrated in relation to everyday task performance and employment , suggesting that the three domains are strongly predictive of comprehensive real - world performance [ 45 , 46 ] . bicams may not necessarily be sensitive to change over the short - term , although the reported test - retest coefficients are excellent which would suggest sensitivity over this period [ 31 , 32 , 47 ] . the lack of significant change in qmt measurements , however , suggests that the microstructural changes thought to underpin adaptive responses may , at present , be beyond the resolution of even the most advanced mri techniques or not manifest within the timescale of this study . additional follow - up of this cohort is planned to determine what , if any , changes are observed in terms of both cortical activation as measured by fmri and structural changes measurable with qmt . longer - term studies may also provide insight into the true functional impact of cognitive rehabilitation such as maintenance of employment .

aim . to explore the efficacy of home - based , computerised , cognitive rehabilitation in patients with multiple sclerosis using neuropsychological assessment and advanced structural and functional magnetic resonance imaging ( fmri ) . methods . 38 patients with ms and cognitive impairment on the brief international cognitive assessment for ms ( bicams ) were enrolled . patients were randomised to undergo 45 minutes of computerised cognitive rehabilitation using rehacom software ( n = 19 ) three times weekly for six weeks or to a control condition ( n = 19 ) . neuropsychological and mri data were obtained at baseline ( time 1 ) , following the 6-week intervention ( time 2 ) , and after a further twelve weeks ( time 3 ) . cortical activations were explored using fmri and microstructural changes were explored using quantitative magnetisation transfer ( qmt ) imaging . results . the treatment group showed a greater improvement in sdmt gain scores between baseline and time 2 compared to the control group ( p = 0.005 ) . the treatment group exhibited increased activation in the bilateral prefrontal cortex and right temporoparietal regions relative to control group at time 3 ( p < 0.05fwe corrected ) . no significant changes were observed on qmt . conclusion . this study supports the hypothesis that home - based , computerised , cognitive rehabilitation may be effective in improving cognitive performance in patients with ms . clinical trials registration is isrctn54901925 .

1. Introduction 2. Subjects and Method 3. Results 4. Discussion

studies have shown cognitive deficits ( in particular deficits in information processing speed , concentration , and working memory ) to be present in the early stages of ms [ 24 ] . cognitive impairment has a negative impact on quality of life ( qol ) independent of physical symptoms [ 5 , 6 ] . there exists mounting evidence for neuroplasticity as a mechanism to compensate for accumulating pathology in ms and some tentative evidence that cognitive rehabilitation may be effective in preserving or improving cognitive function in patients with ms [ 79 ] . computer - assisted cognitive rehabilitation has the potential to provide a structured and standardised approach to rehabilitation . rehacom is one particular type of software designed and utilised for treatment of cognitive impairment in a number of disease states such as stroke , brain injury , and psychiatric disorders [ 10 , 11 ] . it has been used in a growing number of trials of cognitive rehabilitation in ms as a more standardised intervention [ 8 , 9 , 12 , 13 ] . few studies have examined the structural basis of cognitive rehabilitation and longitudinal studies are relatively lacking [ 8 , 14 ] . it is therefore conceivable that techniques , such as magnetisation transfer ( mt ) , which is sensitive to myelin content , might be sensitive to structural plasticity in ms . in this study we combined neuropsychological assessment , functional mri ( fmri ) , and quantitative magnetisation transfer ( qmt ) imaging to explore whether home - based , computerised cognitive rehabilitation is an effective means of promoting cognitive rehabilitation and whether the structural basis for rehabilitation can be better defined . the primary outcome of the study was measured as any improvement in cognition after the training , while the secondary outcomes included changes in fmri , fatigue , and quality of life assessments . thirty - eight patients with objective evidence of cognitive impairment were invited to participate in this study between february 2014 and february 2015 . inclusion criteria were as follows : ( a ) age between 18 and 65 , ( b ) clinically definite ms , according to the mcdonald criteria , ( c ) expanded disability status scale ( edss ) 6.5 , and ( d ) cognitive impairment defined as scores below the 5th percentile for normative data adjusted for age , sex , and years of formal education on one or more of the bicams tests . patients were excluded if they had a history of significant psychiatric disorders , alcohol or substance abuse , visual acuity less than 6/18 corrected , oscillopsia , or diplopia that would interfere with testing . patients were also excluded if they had a ms relapse , received corticosteroids , or changes made to psychoactive medications within the previous month . study design . neuropsychological and mri data were obtained at baseline ( time 1 ) , immediately following a 6-week intervention period ( time 2 ) and after an additional 12-week follow - up period ( time 3 ) , during which no additional intervention was administered ( supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4292585 ) . the mri analysis was conducted by a researcher blind to the patients ' group allocation . patients were screened for cognitive impairment using the brief international cognitive assessment for ms ( bicams ) . bicams is a brief ( 15-minute ) screening tool to identify cognitive impairment in patients with ms and comprises the first five learning trials of the california verbal learning test ii ( cvlt - ii ) , the first three recall trials of brief visuospatial memory test revised ( bvmt - r ) , and the symbol digits modalities test ( sdmt ) . at baseline participants also completed a number of behavioural and qol assessments including euroqol five - dimension questionnaire ( eq-5d ) , a generic health - related quality of life scale , functional assessment of ms ( fams ) ( a ms specific quality of life scale ) , patient activation measure ( pam-13 ) ( a 13-item generic scale for chronic illness management ) , a measure of patient empowerment in ms , unidimensional self - efficacy scale for ms ( use - ms ) , the hospital anxiety and depression scale ( hads ) , multiple sclerosis neuropsychological questionnaire ( msnq ) self - report ( a patient self - reported measure of cognitive function ) , and the fatigue severity scale . at each subsequent time point participants underwent repeat cognitive assessment using bicams ( same test forms ) as well as repeat behavioural and qol assessments . the treatment group underwent six weeks of home - based , computer - assisted cognitive rehabilitation using rehacom software ( https://www.fixxl.co.uk/ ) . the control group were asked to watch a series of natural history dvds of corresponding duration and frequency to the rehabilitation sessions performed by the treatment group for six weeks . the need to evaluate mri parameters in studies with active control conditions has been highlighted . treatment sessions consisted of training in three specific modules involving working memory , visuospatial memory , and divided attention . in all tasks the level of difficulty is tailored to the individuals performance and increases automatically but only in line with satisfactory progress . real - time data pertaining to performance , progress , and compliance is transmitted to the investigator over the internet during the intervention period . higher levels involve having to remember the cards in reverse order . visuospatial memory is a similar task involving various objects presented briefly on screen with the patient asked to remember the object as well as its position in the sequence . as the complexity of the task increases , the following sequences were acquired in an order designed to minimise the potential for fatigue on the fmri task : ( 1 ) dual - echo turbo spin - echo for lesion identification ; ( 2 ) high - resolution t1-weighted magnetisation - prepared rapid - acquisition gradient echo ( mprage ) ; ( 3 ) functional mri with echo - planar imaging ( epi ) acquired during a n - visually presented back task ; ( 4 ) quantitative magnetisation transfer ( qmt ) with balanced steady - state free precession ( bssfp ) . t2 lesion volume was measured at baseline for each participant using the software package jim ( version 3.0 , xinapse systems ltd . and involved three conditions : 0-back , 1-back , and 2-back tasks . the n - back task did not constitute part of the cognitive rehabilitation . all participants were allowed to briefly practice the n - back task under supervision for five minutes prior to the mri scan to ensure comprehension of the task and allow familiarity with it . 0-back , 1-back , and 2-back tasks were presented in a randomised manner resulting in six blocks per nine - minute run . the primary outcome was cognitive performance as measured by improvement in sdmt , bvmt , and cvlt between groups compared to baseline . outcomes were compared between the two groups using independent samples t - test to compare gain scores for cognitive data between groups . fmri data were analysed using spm8 ( wellcome department of cognitive neurology , ucl , london , http://www.fil.ion.ucl.ac.uk/spm/ ) . for each time series individual epis were then realigned to the first remaining image of the series by rigid - body transformation to correct for involuntary head movements during acquisition before normalisation into a standard anatomical space ( montreal neurological institute [ mni ] ) using linear and nonlinear transformations . for each participant , the difference in blood oxygen level - dependent ( bold ) response between the 0-back , 1-back , and 2-back conditions was estimated at every voxel across the whole brain using the general linear model ( glm ) . each contrast obtained at the first - level was entered into a second - level glm to generate summary statistical parametric maps ( spms ) . for between - group analysis of difference between the time points , we used a 3 2 anova flexible factorial design with group ( between - subject ) and time ( within - subject ) as separate factors to examine the main effects on group ( treatment versus control ) , time and the interaction between them to evaluate areas of relative change in activity after cognitive training versus control . the threshold for significance was set at alpha of 0.05 corrected for multiple comparisons ( family - wise error ( fwe ) corrected ) . , the location of local maxima of signal intensity increase is expressed as x , y , and z coordinates in mni space . the mprage were then segmented into white matter , grey matter , and csf to yield a parenchymal mask . a t1 map was calculated for all datasets by fitting the theoretical spoiled gradient echo as a function of the flip angle to the signal measured by the 3d flash sequences . mt parameters were obtained by performing a voxelwise nonlinear least squares fitting ( levenberg - marquardt ) to a binary spin bath model for bssfp . the statistical analysis was performed voxelwise in spm8 on the resulting warped and smoothed mt maps . the same glms used for the second - level fmri analysis and described in the previous section were used for estimating the main effects of time and group and the interaction between these two factors . patients ( 70.3% ) had rrms , and 11 patients ( 29.7% ) had spms . 20 patients ( 52.6% ) were on disease modifying therapy at enrolment ( natalizumab n = 6 , beta - interferon n = 7 , fingolimod n = 6 , and teriflunomide n = 1 ) . after randomisation to either computer - assisted cognitive training ( treatment group , n = 19 ) or the active control condition ( n = 19 ) , there were no significant differences in terms of baseline demographics ( table 1 ) or quality of life measures ( supplementary table 3 ) between the two groups . the treatment group had higher baseline cognitive scores on the bicams battery ; however , these did not differ significantly from the control group . the most frequently failed component of the bicams test battery was the sdmt with 33 ( 86.8% ) of participants scoring below the 5th centile , 18 ( 47.4% ) failing the cvlt - ii , and 13 ( 34.2% ) failing the bvmt - r . overall 21 ( 55.2% ) failed one test , 10 ( 26.3% ) failed two tests , and 7 ( 18.4% ) failed all three tests of the bicams test battery . overall , 88.9% of patients ( 16/18 ) in the intervention group completed at least 75% of the prescribed sessions with 66.7% ( 12/18 ) completing all the prescribed sessions . , the treatment group showed a significantly greater improvement in gain scores between baseline and early follow - up ( time 2 ) compared to the control group on the sdmt ( treatment 3.94 ( sd 5.08 ) , controls 0.63 ( sd 3.30 ) , 1.47 to 7.66 , ( 95% ci 1.47 to 7.66 ) , p = 0.005 ) illustrated in figure 1 . similar gain scores in the cvlt and bvmt - r were not significantly different between the groups although the bvmt - r gain scores did approach significance ( p = 0.098 ) . overall , there was an improvement in bicams performance across participants at follow - up . the gain scores between the groups at time 3 compared to baseline were , however , not statistically significantly different . there were no significant differences in qol outcome measures , measures of self - efficacy , or subjective cognitive performance between the two groups ( supplementary table 4 ) . the baseline error rate between the treatment and control groups was low ( 8.64% versus 9.48% , p = 0.814 ) . no significant differences were observed in the error rate during the n - back task between the groups at baseline or at follow - up ( supplementary table 5 ) . the 1-back task was associated with activations involving the dorsolateral prefrontal cortex bilaterally as well as bilateral inferior parietal lobule and insular and cerebellar regions relative to the 0-back contrast . the same regions were activated in the 2-back condition but the spatial extent and magnitude of the responses were greater , particularly over the frontoparietal regions ( supplementary figure 2 and supplementary table 2 ) . time 2 versus time 1 . at time 2 , increased activation was seen in the right temporoparietal regions ( right supramarginal and angular gyri ( p < 0.005fwe corrected at cluster level ( k = 228 ) ) ) in the 1-back in the treatment group relative to controls ( group - by - time interaction ) . 3 significant increases in activation were seen in both the 1-back and 2-back conditions in the treatment group relative to controls . in the 1-back task , increased activation was seen in the left frontal ( p < 0.001fwe corrected at cluster level ( k = 294 ) ) and right temporoparietal regions ( p < 0.012fwe corrected at cluster level ( k = 187 ) ) . in the 2-back task , increases in activation were seen in bilateral prefrontal ( p < 0.013fwe corrected at cluster level ( k = 206 ) ) and right temporoparietal regions ( p < 0.024fwe corrected at cluster level ( k = 178 ) ) ( figure 2 ) . quantitative magnetisation transfer . no significant between - group changes were seen in the qmt at time 2 or time 3 , with respect to time 1 . in line with previous work , the main outcome of this study was that 6 weeks of computerised cognitive rehabilitation was associated with improvement in cognitive performance as measured on the sdmt . the sdmt improvement in the treatment group was , however , not maintained after cessation of cognitive rehabilitation ( time 3 assessments ) although the functional mri changes were seen to persist at follow - up . the sdmt is among the most sensitive tests of slowed information processing speed in ms [ 34 , 35 ] and may also be a proxy for general cognitive impairment . compared to time 1 , the treatment group showed a significantly greater improvement in gain scores between baseline and early follow - up ( time 2 ) compared to the control group on the sdmt ( p = 0.005 ) . however , the gain scores between the groups at time 3 compared to baseline were not statistically significantly different . it may be the case that cognitive rehabilitation does indeed result in improved cognitive performance but that maintenance of such improvement requires some form of ongoing intervention in the longer term . only one version of the bicams test battery has been validated in ms and thus was used in this study . reported test - retest coefficients on the bicams tests are excellent , suggesting that practice effects are negligible [ 31 , 32 ] . in addition , the experimental design was to compare two groups with identical testing schedules ; therefore the impact of practice effects on the results is likely to be minimal . qol is a complex construct influenced by a multitude of factors such as employment status , social networks , and perceptions of self - worth and self - efficacy . it is possible that cognitive rehabilitation has a positive impact on a number of these factors but such changes in such factors may take time to manifest as improvements in qol . further longitudinal analysis may be required to investigate this . in order to minimise practice effects associated with repeat testing , participants were not directly trained in the n - back task ; rather it was utilised as an outcome measure of working memory . it was anticipated that if cognitive rehabilitation was effective at improving working memory and attention , then the effects would be reflected on the performance on the n - back task . no differences were seen in the error rate between the groups during the n - back task at follow - up ; however , the error rate was low at baseline in both groups . the n - back fmri paradigm in our study cohort was , however , associated with robust baseline cortical activations ( in particular within the dlpfc and posterior parietal cortex ) in keeping with known working memory networks . a significant group - by - time interaction was seen with the treatment group exhibiting increased activation in the bilateral prefrontal cortex and right temporoparietal regions relative to control group at time 3 ( p < 0.05fwecorr ) . changes in functional activation within these regions within the treatment group are felt to be functionally relevant with respect to cognitive rehabilitation . it has been shown that the prefrontal cortex is critical in the executive control of working memory and has a role in response inhibition [ 38 , 39 ] . a right hemisphere dominant ventral attentional network consisting of the temporoparietal junction , ventral prefrontal cortex , and anterior insula previous work in ms has indicated that attention may be one of the domains most amenable to rehabilitation . many of the computer - training tasks involve sustained attention and it might be postulated that the increased activation seen in the right temporoparietal region at follow - up in the treatment group is as a result of improved efficiency of this network . this solidification of neural networks may extend to areas / networks outside those directly trained and may explain why working memory centres such as the prefrontal cortex were seen to be persistently active after cessation of formal training . debate remains however as to the possible interplay between adaptive and maladaptive responses during functional brain reorganisation . the discrepancy between the apparent lack of clinical difference between the groups at time 3 and the sustained fmri effect at time 3 may reflect the fact that bicams does not adequately measure working memory which is primarily domain utilised during the n - back fmri paradigm . some studies have identified structural changes on diffusion tensor imaging as a result of rehabilitation in the context of physiotherapy [ 42 , 43 ] . we did not detect any structural change on qmt after training . due to the short duration of follow - up , this is not entirely surprising . in contrast to many previous studies , which often rely on one - to - one or outpatient administered cognitive rehabilitation , this study sought to explore whether a home - based approach to cognitive rehabilitation was feasible . a home - based approach to cognitive rehabilitation is significantly less resource intensive and may pave the way to greater access for a greater number of patients to such interventions in the future firstly , the groups were relatively small and there was a dropout of patients mainly in the control group between time 2 and time 3 . there was heterogeneity with regard to the cognitive domains that showed deficits among participants in the study . it is likely therefore that they may not have benefited from the rehabilitation in the same way . as the study was largely exploratory in nature , it utilised an open design and is therefore subject to a number of limitations inherent to this type of design . for pragmatic reasons blinding of the investigating neurologist was not established due to the potential need for interaction between patient and investigator . spm analysis of mri data offers objective , largely automated measures , which are independent of measurement bias . in many respects , the sdmt may provide a proxy for overall cognitive functioning but a more detailed cognitive assessment of the domains directly trained may have provided additional insight into effectiveness of cognitive training . bicams is primarily designed as a screening tool for cognitive impairment in ms assessing a limited number of domains . the lack of significant change in qmt measurements , however , suggests that the microstructural changes thought to underpin adaptive responses may , at present , be beyond the resolution of even the most advanced mri techniques or not manifest within the timescale of this study . additional follow - up of this cohort is planned to determine what , if any , changes are observed in terms of both cortical activation as measured by fmri and structural changes measurable with qmt . longer - term studies may also provide insight into the true functional impact of cognitive rehabilitation such as maintenance of employment .

the human ventral stream contains macroscopic regions that respond selectively to certain categories , including faces and places ( epstein and kanwisher , 1998 , kanwisher et al . , 1997 ) . domain - specific computational mechanisms ( kanwisher , 2000 ) might be required to meet the difficult computational challenge of visual object recognition . the particular category preferences found appear broadly consistent with the behavioural importance of the ability to recognize faces and places . however , we do not yet understand the computations performed in these category - selective regions or their intrinsic spatial organisation . in addition , a longstanding debate has yet to be resolved about the question whether these regions form visual areas ( felleman & van essen , 1991 ; van essen criteria for visual areas ) and functional modules ( kanwisher , 2000 ) or merely peaks of selectivity within a single more comprehensive object - form topography ( haxby et al . , 2001 ) . a prominent theory of the global layout of the ventral stream states that regions selective for faces and places start out in development as a retinotopic protomap ( hasson et al . , 2003 , hasson et al . , 2002 , levy et al . , 2001 ) . through experience , each patch of cortex develops selectivity for the visual shapes that most often appear in the retinal region it represents . faces often appear at the fovea , because we tend to fixate them and because their retinal size is only a few degrees visual angle when viewed at typical distances . the central part of the retinotopic protomap , according to the theory , therefore turns into the fusiform face area ( ffa ; kanwisher et al . , 1997 , 1996 ) and the occipital face area ( ofa ; gauthier et al . , 2000 ) . places and scenes , by contrast , are more physically extended and typically occupy a wide visual angle . the parahippocampal place area ( aguirre et al . , 1996 , epstein and kanwisher , 1998 ) therefore develops in the peripheral part of the protomap , according to the theory . here we investigate the hypothesis that the same principle explains the intrinsic spatial organisation of face - selective regions . let 's start with an oversimplification and imagine faces always appeared frontally at the same distance and in the same retinal location ( e.g. , fixated centrally ) . a retinotopic protomap whose receptive fields cover face parts would then be expected to acquire selectivities corresponding to face parts , including the eyes , nose , and mouth . moreover , the spatial organization of the parts would resemble the spatial layout of a face , with the nose represented in a cortical patch that lies somewhere in between the patches representing the eyes and the mouth . we refer to this kind of cortical map of face - feature detectors as faciotopic . whereas a retinotopic map is a cortical representation whose topology resembles that of the retina , a faciotopic map is a cortical representation whose topology resembles that of a face . the scenario sketched above was an oversimplification . in natural experience , faces are viewed at a variety of distances , and they are not always fixated centrally . in order to test whether the faciotopy hypothesis is even plausible when we consider more natural viewing conditions , we used a simple simulation ( fig . 1 ) . for each face feature , we estimated the spatial distribution of retinal exposures when viewing conditions were drawn randomly from realistic distributions of viewing distances and fixation points . this gave us the spatial distribution on the retina of mouth exposures , for example , and a similar distribution for each other face feature . despite the variability in viewing conditions , the peaks of the retinal feature exposure this suggests that a retinotopic protomap with a receptive field size roughly corresponding to face parts might develop into a faciotopic map if its patches acquire selectivity for the features they are most frequently exposed to even when viewing conditions are quite variable . although our simulation did not include variations of viewing angle , it models a substantial part of our visual experience with faces and convinced us that the faciotopy hypothesis is plausible and merits an empirical investigation with functional magnetic resonance imaging ( fmri ) . the ffa and ofa respond to faces even when they are presented peripherally ( hasson et al . , 2003 ) . even if selectivities to certain types of natural shapes develop from a retinotopic protomap , the resulting shape detectors might acquire substantial tolerance to retinal position . we hypothesized that within a faciotopic map , similarly , each feature detector will respond to its preferred feature with some level of tolerance to the precise retinal position . in this study , we tested the faciotopy hypothesis by presenting images of isolated face features to subjects during fmri scanning . to avoid confounding faciotopy with retinotopy , results suggest that ofa , and to a lesser extent also ffa , is organized into a faciotopic map . thirteen healthy volunteers ( 6 females , age range 2045 ) with normal or corrected - to - normal vision took part in this study . data from one subject had to be excluded from the analysis due to technical difficulties during data acquisition ( scanner failure ) . ethical approval for the research was obtained from cambridge psychology research ethics committee ( cprec ) . the stimuli were images of face features which had been sampled from high - resolution frontal face photographs of 92 individuals . the faces in the photographs were first aligned using matlab by manually marking the midpoints of the eyes and the mouth in each image , then finding the rigid spatial transformation between these points and applying the transformations to the images . from the aligned face images , the following twelve face features were sampled using equal - sized , non - overlapping windows ( fig . 2a ) : left and right eye , the space between the eyes , nose , mouth , left and right hairline , left and right ear , left and right jaw line , and chin . the vertical positions of the sampling windows for the ears needed to be manually adjusted to match the individual variability in the position of the ears but all other features were sampled using the same windows for each face . 2b ) : one small feature ( image diameter : 3 ) presented at the centre of the screen , one large feature ( image diameter : 6 ) presented at the centre of the screen , and nine small features ( all the same ) presented in parallel . the subjects fixated a black cross at the centre of the screen throughout the experiment . the stimuli were shown in a blocked fmri design , where during one 16-sec stimulation - block , 16 different exemplars of the same face feature were presented ( e.g. , 16 noses sampled from different individual faces ) . each image was shown for 750 ms with a 250 ms fixation baseline between the different feature exemplars . each experimental run consisted of two blocks for each of the face features , and every fifth block was a baseline block with the fixation cross presented alone . subjects attended two measurement sessions with two experimental runs for each stimulus layout within each session . the presentation order of the face features within each run and the order of the spatial - layout runs within a session were pseudorandomized and balanced across the subjects and between the two measurement sessions for each subject . the stimuli were created with matlab , and their timing was controlled with presentation ( neurobehavioral systems ) . the stimuli were projected with a christie video projector to a semitransparent screen , which the subjects viewed via a mirror . the subjects were familiarized to the stimuli and task before the experiments . to direct subjects ' attention to the stimuli during the experiment , they performed a task on the stimuli . the sampling window of the face feature was displaced by half of its width 13 times within a stimulus block ( e.g. , nose not shown at the centre of the visual field but shifted to the left from the fixation cross ; the position and size of the stimulus images remained the same however ) , and the subjects were instructed to press a button when detecting these displacements of the features . the primary visual cortex ( v1 ) was localized in each individual based on the cortical folds via a surface - based atlas alignment approach developed by hinds et al . peripheral v1 was excluded from the rois based on the spatial extent of the overall fmri response to the face - feature stimuli . ofa and ffa were localized based on independent functional localizer data . during the functional localizer run , the subjects were presented with blocks of images of faces ( different from the faces used for sampling the face features ) , scenes , objects , and phase - randomized textures . functional and anatomical mri data were acquired using a 3 t siemens tim trio mri scanner equipped with a 32-channel head coil . during each main experimental run , 252 functional volumes were acquired using an epi sequence with imaging parameters : repetition time 2.18 sec , 35 slices with 2 mm slice thickness ( no gap ) , field of view 192 mm 192 mm , imaging matrix 96 96 , echo time 30 ms , and flip angle 78. each subject attended two measurement sessions with six main experimental runs in each ( two runs for each stimulus layout , fig . two high - resolution structural images were acquired in the beginning of the first measurement session using an mprage sequence , from which the white and gray matter borders were segmented and reconstructed using freesurfer software package ( dale et al . , 1999 , fischl et al . , 1999 ) . one structural image was acquired in the beginning of the second measurement session to co - register the data between the two sessions . functional data were pre - processed with spm8 ( wellcome department of imaging neuroscience ) matlab toolbox . the first four functional images from each run were excluded from the analysis to reach stable magnetization . the data from the second measurement session were co - registered and re - sampled to the same space with the first measurement session data . for the linear discriminant analysis , the data were also spatially smoothed using a 4 mm gaussian smoothing kernel . we estimated the responses for the face - feature stimuli using general linear model ( glm ) analysis as implemented in spm8 . the onsets and durations of the stimulus blocks were entered as regressors - of - interest to the glm , and convolved with the canonical hemodynamic response model . additional regressors included the timings of the task images and the six head - motion - parameters . during the parameter estimation , the data were high - pass filtered with 300-sec cut - off , and serial autocorrelations were estimated with restricted maximum likelihood algorithm using a first - order autoregressive model . for representational similarity analysis , , 2007 , nili et al . , 2014 ) to study the discriminability of the response patterns evoked by the different face - feature stimuli . the data were first divided into two independent sets based on the measurement session . for each pair of face - feature stimuli , fisher linear discriminant analysis was applied to find the weights for the voxels that discriminated between the response patterns and then the weights were applied to the independent data to calculate the linear - discriminant t - value , reflecting the discriminability between the response patterns evoked by two different face - features . the analyses were done on individual data , and the linear - discriminant t - values were pooled across the twelve subjects and converted to p - values . all pairwise comparisons of the face - features were collected to matrices ; multiple testing ( 66 pairwise comparisons of 12 face features ) was accounted for by controlling the false - discovery rate . to test for size - tolerance of the face - feature representations , the fisher linear discriminant was fit to the response patterns evoked by the small face - feature images and tested on the response patterns evoked by the large face - feature images . to characterize the face - feature representations in each roi , we computed the dissimilarities between the response patterns evoked by the face - feature stimuli and compared them with model predictions of the representational distances ( kriegeskorte et al . , 2008 , nili et al . , 2014 ) . for each roi , the dissimilarities between the response patterns were assembled in a representational dissimilarity matrix ( rdm ; a brain rdm ) , where each value reflects the representational distance between two face - feature stimuli . our measure of response - pattern dissimilarity was correlation distance ( 1 pearson linear correlation ) . for each individual , the rdms were calculated separately from the response patterns for each stimulus layout ( fig . for the comparison of the brain representation to the model representations , the rdms for the small and large face - features from the two measurement sessions were averaged . the face - feature representations in v1 , ofa and ffa were compared to three predictions of the representational distances between the face features : 1 ) gabor wavelet pyramid ( gwp ) model , 2 ) physical distances between the face features in a face ( physical distance reference , fig . 2a ) , and 3 ) physical distances between the face features when symmetric face features are represented in same locations ( symmetric reference ) . the gwp model captures the low - level image similarities between the face - feature stimuli , and was adopted from kay , naselaris , prenger , and gallant ( 2008 ) . 2a shows the physical distance reference matrix , where the values are the distances between the face - feature sampling windows . the symmetric reference was otherwise identical to the matrix shown in fig . 2a , but the distances between the symmetric features ( eyes , ears , hairlines , jaw lines ) were 0 and the distances from the two symmetric features to the other features were the same . we tested the relatedness between the model and brain rdms by comparing the rank orders of the dissimilarities using kendall 's tau - a rank correlation ( for details , please see nili et al . the relatedness of each of the model rdms ( gwp , physical distance reference , symmetric reference ) to a brain rdm was tested using one - sided signed - rank test across the single subject rdm correlations . to evaluate differences between the relatedness of the model rdms to a brain rdm , the difference between the rdm correlations of two models in each subject was calculated and tested using a two - sided signed - rank test across the subjects . this was repeated for each pair of models and the multiple testing was accounted for by controlling the false - discovery rate . a noise ceiling of the expected rdm correlation was estimated for each brain region as described by nili et al . the relationships of the model and brain rdms were visualized using multidimensional scaling ( nili et al . , 2014 ) . the first step is to build a matrix of the pairwise correlations ( 1 kendall 's tau - a rank correlation ) between all brain and model rdms . to avoid the contribution of intrinsic fluctuations inflating the representational similarity between two brain regions ( henriksson , khaligh - razavi , kay , & kriegeskorte , 2015 ) , the rdms of the visual areas were compared between rdms constructed from response patterns from different measurement sessions . the multidimensional scaling arrangement of the ( dis)similarity matrix of the rdms provided a visualization of the relatedness of the face - feature representations in different visual areas , and between the visual areas and models . finally , we tested whether the face - feature representations reflect faciotopy , that is , whether the cortical distances between the representations of different face features were explainable by the physical distances between the features in a face . within each roi ( left and right v1 , left and right ofa , left and right ffa ) , we estimated a single location for each face feature using the following approach . for each voxel , we determined which feature was preferred ( highest t value ) over the other features at that voxel . we then considered the local spherical neighbourhood around each voxel in an roi like ofa , and assigned the voxel the feature that was most frequently the preferred feature in the neighbourhood . we then looked for the voxel with the highest feature preference ( defined as the number of times the feature was preferred in the local neighbourhood ) and assigned that voxel together with its local neighbourhood to that feature . this procedure was repeated until all features had a cluster of voxels or all above - threshold voxels had been assigned to features . the size of the neighborhood ( radius of a sphere ) and the t - value threshold were optimized by evaluating the replicability of the distance matrix across the two measurement sessions ( no assumption of faciotopy , only for replicable distance matrix between the face - feature locations ) . the feature - preference clusters were searched for in 3d space ( voxel coordinates ) and assigned to the cortical surface of the individual . all pair - wise distances between the cortical patches assigned to the face features were calculated along the cortical surface and assembled in a matrix similar to the distance matrix shown in fig . the patch - distance matrix was compared to the matrix of the physical distances between the features in a face . this analysis was identical to the representational similarity analysis of the response - pattern dissimilarity matrices and model rdms . we measured fmri responses to visual presentations of 12 isolated face - features ( fig . 2a ) : left and right eye , the space between the eyes , nose , mouth , left and right hairline , left and right ear , left and right jaw line , and chin . the features were extracted from frontal face photographs using equal - sized , non - overlapping sampling and were presented in a blocked fmri design with three conditions ( fig 2b ) : one small feature shown at the centre of the screen , one large feature shown at the centre of the screen , and nine small features presented in parallel . the small stimulus ( 3 diameter ) was selected to roughly correspond to the size of the face features at a viewing distance of 1 m , whereas the simultaneous presentation of nine small features could be an optimal stimulus for a feature detector . two face - selective regions - of - interest ( ofa , ffa ) and the primary visual cortex ( v1 ) were defined in each hemisphere in each subject based on independent localizer data . 3 shows the mean fmri response strengths for the face - feature stimuli in v1 , ofa and ffa . the different conditions ( small features , large features , 9 parallel features ) are shown in different shades of gray . the v1-roi covered eccentricities up - to the size of the 9-parallel - features stimulus , and thus it is expected that in v1 the small stimulus evokes the smallest response and the largest stimulus ( 9 parallel features ) evokes the largest mean response ( fig . more interestingly , in ofa and ffa , this retinotopic effect was largely abolished and the three sizes of the face - feature stimuli evoked approximately equal - sized responses . the only exception is the mouth stimulus that evoked a larger response both in ofa ( p = .027 ; signed - rank tests ) and in ffa ( p = .016 ; signed - rank tests ) when presented in the nine parallel feature configuration compared to the one small feature presented at the centre of the screen . overall , each face - feature stimulus evoked a clear response in all regions - of - interest . we have now shown that v1 , ofa and ffa respond to isolated face - features ( fig . 3 ) , but do they also discriminate between the face features ( e.g. , an eye from a mouth ) ? 4a shows the results from linear discriminant analysis ( nili et al . , 2014 ) : the discriminability of each pair of face - feature stimuli was evaluated by fitting a fisher linear discriminant to the response patterns from the first fmri session and by testing the performance on the response patterns from the second fmri session ( same subject , different day , different stimulus presentation order , all stimulus layouts ) . the analyses were done on individual data and the results were pooled across the twelve subjects . the left column in fig . 4a shows the linear - discriminant t - values , reflecting the discrimability of each pair of face - feature stimuli from the response patterns , and the right column shows the corresponding p - values . in v1 , the response patterns discriminated each pair of face - feature stimuli , except the two hairlines from each other and the mouth from the chin ( fig . , each pair of the face - feature stimuli could be discriminated from the response patterns ( fig . 4a , middle row ) . in addition , there appears to be a distinction between the inner ( first five elements in the linear discriminant t - value and p - value matrices ; e.g. , the eyes ) and outer face - features ( elements 612 in the matrices ; e.g. , the ears ) , that is , the t - values are high for the discriminability of these stimulus pairs in ofa , and also in ffa . moreover , in ffa , the symmetric face - features ( the eyes , the hairlines , the ears , the jaw lines ) evoked indistinguishable response patterns ( bottom row in fig . the use of both small and large features as stimuli enabled us to study the size - tolerance of the face - feature representations in v1 , ofa and ffa . in general , a true higher - level representation of an object category should show tolerance to identity - preserving image transformations , such as scaling the image size . 4b shows the results from fisher linear discriminant analysis when the classifier was trained to distinguish the response patterns for the small face - feature stimuli and the testing was done on the response patterns for the large stimuli most importantly , ofa response - patterns discriminated between each pair of face - feature stimuli ( middle row in fig . in v1 , the classifier 's performance was much worse than with the same - sized stimulus images ( cf . was also impaired by the use of different sized stimulus images for training and testing . in ffa , however , the distinction between the inner and outer face features was preserved . results on the generalization from the small features to the nine parallel features , and from the large features to the nine parallel features are shown in supplementary fig . 1 . next we characterized the face - feature representations in v1 , ofa and ffa using representational dissimilarity matrices ( rdms ; kriegeskorte et al . 2014 ) , which compare the response patterns elicited by the stimuli here each value in an rdm reflects the representational distance between two face - feature stimuli . rdms can be directly compared between two brain regions by computing the rank - correlation between their rdms ; if two brain regions represent the stimuli identically , the ordering from the most similar stimulus - pair to the least similar stimulus - pair is the same ( high rank correlation ) . this comparison should , however , be done on independent trials to avoid the contribution of intrinsic cortical dynamics inflating the representational similarity between brain regions ( henriksson et al . , 2015 ) . moreover , a brain rdm can directly be compared to an rdm constructed based on model predictions of the similarity of the representations between the stimuli . we compared the brain rdms to three models : physical distances between the face features in a face ( physical distance reference ; fig . 2a ) , physical distances between the face features when symmetric face - features are represented in the same locations ( symmetric reference ) , and gwp model . 2a shows the physical distance reference matrix , where each value in the matrix reflects the distance between two features in a face . 2a , but the distance between the symmetric face features was zero and the distances from two symmetric face features to other features was identical . the gwp - model captures the low - level image properties of the stimuli ( edges , for example , at the same locations in the stimulus images would be predicted to elicit a similar response in low - level visual areas ) . 5a shows the results from the comparison of the brain rdms to the three model rdms . the brain rdms were constructed from the response patterns to the small and large stimulus images ( see methods for details ) ; the results are shown separately for the different stimulus layouts in supplementary fig . 2 . the v1 rdm of the face - feature stimuli was best explained by the gwp model ( p < .001 ; one - sided signed - rank test ) , reflecting the low - level image properties of the stimulus images . the gwp model explained the v1 representation better than the face - feature physical distance matrix or the symmetric distance matrix ( fdr q .05 ; two - sided signed - rank tests ; fdr - corrected for multiple comparisons ) . in ofa , both the face - feature physical distance matrix and the symmetric distance matrix explained variance in the representation ( p < .01 ; one - sided signed - rank tests ) . in addition , the physical distances between the face - features explained the representation better than the low - level image properties captured by the gwp model ( fdr q .05 ; two - sided signed - rank test ; fdr - corrected for multiple comparisons ) . a similar trend was observed in ffa , where the physical distance matrix and the symmetric distance matrix both explained variance in the representation . 5b shows a multidimensional - scaling visualization of the relationships between the three models and the v1 , ofa and ffa representations , as captured by the response - pattern dissimilarity matrices . the distances reflect the correlation distance between the rdms ; that is , how similar the representations are . the ofa and ffa representations are more similar to each other than to the v1 representation . the v1 representation was most similar to the gwp model whereas the ofa representation was more similar to the face - feature physical distance models . thus far we have shown that especially in ofa the response - pattern dissimilarities do reflect the physical distances between the face features ( fig . 5 a b ) . for the underlying representation to be truly faciotopic , the distances between cortical locations with preference for a specific feature would also reflect the topology of the face features in a face . to test for a faciotopic representation , we estimated for each face feature a single location on the cortex within each roi and calculated the distances between these face - feature preference patches along the cortical surface . all pair - wise distances between the face - feature preference patches were collected to a matrix similar to the reference matrix shown in fig . 5c shows the results how well the distances between the face - feature preference patches along the cortical surface were explained by the physical distances between the face - features in a face ( blue bars ) or by the symmetric map where symmetric face - features have overlapping representations ( red bars ) . the results are consistent with the response - pattern dissimilarity results shown in fig . were better explained by the low - level image properties between the stimulus images as captured by the gwp model than by the physical distances between the face - features ( fdr q .05 , two - tailed signed - rank test ; fdr - corrected for multiple comparisons ) . the opposite was true for ofa , where the physical distances between the face features best explained the distances between the face - feature preference patches ( fdr q .05 , two - tailed signed - rank test ; fdr - corrected for multiple comparisons ) . 5d shows a multidimensional - scaling visualization of the relationships , where the distances reflect the similarity of the representations . the physical distances between the face features in a face best explain the ofa representation , as reflected in the cortical distances between the face - feature - preference patches , suggesting faciotopy in ofa . our hypothesis was that face - selective regions in human ventral cortex might be organized into faciotopic maps , in which face feature detectors form a map whose topology matches that of a face . faces , and especially the eye region , are frequently fixated from an early age ( farroni et al . , 2002 , goren et al . , 1975 ) , and a retinotopic protomap ( hasson et al . , 2003 , levy et al . , 2001 ) could develop into a faciotopic map if patches acquired selectivity for the face features that they are most frequently exposed to . we first performed a simple simulation to support the faciotopy hypothesis , and then measured fmri responses to isolated face features , all presented foveally to prevent the results from being driven by retinotopy . first , we reported that v1 , ofa , and ffa respond to isolated face features and their response patterns distinguish the different face - feature stimuli . both ofa and ffa emphasized the distinction between the inner ( e.g. , eyes , nose , mouth ) and outer face features ( e.g. , ears , chin , hairline ) in their representations . furthermore , the face - feature discrimination was tolerant to a change of feature size in ofa . we then tested for each region how well the observed response - pattern dissimilarities could be explained by each of three models : a physical - distance model ( fig . 2a ) , a mirror - symmetric physical - distance model , and a gabor wavelet pyramid model . the first two models reflect natural face topology ; the latter captures the low - level image properties of the stimuli . our results indicate that the response - pattern dissimilarity structure in ofa is better explained by the physical distances between the face features than by low - level image properties , whereas the opposite is true for v1 . results for ffa were similar to ofa , but the difference between the models was weaker . finally , a true faciotopic organization requires more than a match between pattern dissimilarities and physical face - feature distances : it requires that the map of cortical locations which preferentially respond to each face feature reflects the topology of a face . to test this , we computed cortical distances between feature - preference locations , and compared them to our three models . the distances between the cortical feature - preference patches in ofa were indeed best explained by the physical distances between the features in a face , supporting the existence of a faciotopic map in ofa . the function of ofa has also previously been associated with processing of face features ( e.g. , haxby et al . , 2000 , liu et al . , 2010 , pitcher et al . , 2007 ) previous studies have shown that transcranial magnetic stimulation ( tms ) at right ofa disrupts face - feature discrimination ( pitcher et al . , 2007 ) and that ofa is activated more by a face with real inner face - features present than a face with the features replaced by black ovals ( liu et al . , 2010 ) . moreover , the face - selective neurons in the macaque posterior lateral face patch ( pl ) , the likely homologue of human ofa , are driven by a single eye , especially when presented in the contralateral upper visual hemifield ( issa & dicarlo , 2012 ) . in the present study , we did not find any special role for the eyes over the other face features . this could be explained by the temporal resolution of fmri ; issa & dicarlo , 2012 reported the eye - preference mainly for the early response ( 60100 ms ) , and our fmri responses reflect a mixture of early and late responses . in a later time - window ( > 100 ms ) , the macaque pl neurons also respond to other face features ( issa & dicarlo , 2012 ) . previous research suggests that ofa is less sensitive than ffa to the correct configuration of the face features within a face ( liu et al . , 2010 , pitcher et al . , 2007 this would be consistent with ofa containing a map of somewhat independently operating face - feature detectors . results from monkey electrophysiology support a functional distinction between the two regions : the macaque posterior face patch ( the putative homologue of ofa ) seems to linearly integrate features ( whole = sum of the parts ) , at least in the early response phase ( issa & dicarlo , 2012 ) , while the majority of neurons in the middle macaque face patch ( the putative homologue of ffa ) shows interactions between the face features ( freiwald , tsao , & livingstone , 2009 ) . this is consistent also with a human magnetoencephalography ( meg ) study showing that the early face - selective meg response ( peaking at a latency of 100 ms ) reflects the presence of real face parts more strongly than the naturalness of their configuration , whereas the later response ( 170 ms ) shows the opposite sensitivity ( liu , harris , & kanwisher , 2002 ) . if faciotopy and retinotopy coexisted in ofa , we would expect face features presented in their typical locations to elicit the largest response . this would imply a preference of the region as a whole for a natural configuration of the features . we would also expect that the features elicit the strongest response when presented in the canonical upright position , as done here . in addition , a faciotopic map would be expected to exhibit non - linear interactions to some degree when multiple face features are presented together . this would be analogous to early retinotopic cortex , which exhibits non - linear spatial interactions when multiple visual - field regions are stimulated at the same time ( pihlaja , henriksson , james , & vanni , 2008 ) . in visual field eccentricity maps ( hasson et al . , 2002 ) , ofa shows a preference for the central part of the visual field . in addition , human ofa has been shown to prefer contralateral stimuli ( hemond , kanwisher , & op de beeck , 2007 ) and to support both position - invariant linear readout of category information and category - invariant linear readout of position information ( schwarzlose , swisher , dang , & kanwisher , 2008 ) . although a more detailed retinotopic organization has not yet been demonstrated in human ofa , there is evidence for retinotopy in subregions of the macaque face patches ( rajimehr , bilenko , vanduffel , & tootell , 2014 ) . the reported preference for an eye - like feature in its natural visual - field position relative to fixation in monkey pl ( issa & dicarlo , 2012 ) also suggests that retinotopy might play a role , and that the conjunction of the feature and its retinal location might determine the response . in the present results , the stimulus layout with the nine parallel mouths was more effective than the single small mouth for both ofa and ffa this could be explained by one of the nine mouths landing in the natural location below the fixation . the optimal fixation point across different face recognition tasks has been reported to be , on average , just below the eyes ( peterson & eckstein , 2012 ) , which could define the center of the faciotopic map . however , recent studies also report that face - fixation patterns , although stable within an individual , differ across individuals ( mehoudar et al . , 2014 , peterson and eckstein , 2013 ) . if faciotopic maps arise from retinal face - feature exposure , they might similarly exhibit individual variability reflecting differences in individual 's preferred fixation locations . sensitivity to retinal position is theoretically compatible with faciotopy and expected if a faciotopic map developed from a retinotopic protomap . this is analogous to the coarser scale , where preferences for faces and places in ffa and ppa co - exist with retinotopic biases . it is all the more striking , then , that the face - feature map can be driven by centrally - presented face features independent of retinotopy . this suggests that , despite residual retinotopic biases , the face feature detectors respond with some level of position tolerance . future studies should investigate how retinotopy and faciotopy combine in ofa and other face regions . future studies could determine the relative contributions of retinotopy and faciotopy by systematically varying the retinal position of the presented features . several pieces of evidence suggest a more global topographic representation of the human body within human occipitotemporal cortex ( bernstein et al . , 2014 , chan et al . , 2010 , orlov et al . , 2010 , song et al . , visual representations are sensitive to the configuration in which a face and a body are arranged in an image , with stronger responses for the typical configuration of face and body ( bernstein et al . , 2014 , song et al . , 2013 ) and for the typical configuration of the left and right halves of the body ( right side of the body in the left visual field ; chan et al . , 2010 ) . a topographic representation of the body would be expected to devote larger areas to more informative body parts , which would include faces and perhaps also hands . the higher - level visual representations may , thus , exhibit cortical magnification of the most informative features , analogous to the early retinotopic visual areas , with their enlarged representation of the central visual field ( duncan and boynton , 2003 , engel et al . , 1994 ) and to the somatosensory homunculus with its enlarged representation of the parts of the organism 's own body that provide richer tactile and proprioceptive input . selective magnification of the most informative parts might also be a feature of a faciotopic representation . this question might be addressed in future studies that sample the face locations with higher spatial resolution in the stimulus domain and also image ofa response patterns with higher resolution , for example using high - field fmri . the nature of the magnified representation might turn out to be quite different in body - part and face - part maps : whereas face perception is tightly coupled with social communication and ultimately relies on holistic perception of the face , visual representations of individual body parts may play an important role in tasks such as action understanding . the spatial organisation of a cortical representation is likely to reflect not only real - world spatial regularities of the stimuli , but also functional relationships . it has been proposed that functional relationships explain the spatial organisation of motor cortex ( aflalo and graziano , 2006 , graziano , 2006 ) . for higher - level visual cortex , a close relationship between body - part and tool - use representations has recently been established ( bracci & peelen , 2013 ) . it remains to be explored whether there are also other areas with cortical maps reflecting topology of external objects . as an object category , faces are also typically perceived in a canonical upright orientation and are fixated at particular locations . these properties make faces an ideal candidate for the development of a cortical feature map . if faciotopy develops from the central part of a retinotopic protomap ( hasson et al . , 2002 , hasson et al . , 2003 , levy et al . , 2001 ) , the peripheral part of the protomap might develop a topological map of the local environment in the occipital and/or parahippocampal place area . overall , the idea of topographic cortical representations of face and body is consistent with an eccentricity - based protomap within the occipitotemporal cortex that develops into more specialized category representations ( hasson et al . , 2002 , levy et al . although the state of development of the cortical face - processing network in newborn infants remains to be determined , there is converging evidence that a subcortical route is responsible for the early tendency of newborns to orient towards face - like stimuli ( johnson , 2005 ) . the innate subcortical route may be responsible for bringing faces to central visual field in newborns and might promote the development of faciotopy . consistent with this hypothesis , visual experience during the first months after birth is necessary for normal development of configural processing of faces and , in particular , the processing of information about the spacing between the face features ( le grand , mondloch , maurer , & brent , 2001 ) . an innate neural mechanism that triggers central fixation of faces would spatially align the repeated face exposures of a retinotopic protomap , and could explain the development of faciotopy .

the occipital face area ( ofa ) and fusiform face area ( ffa ) are brain regions thought to be specialized for face perception . however , their intrinsic functional organization and status as cortical areas with well - defined boundaries remains unclear . here we test these regions for faciotopy , a particular hypothesis about their intrinsic functional organisation . a faciotopic area would contain a face - feature map on the cortical surface , where cortical patches represent face features and neighbouring patches represent features that are physically neighbouring in a face . the faciotopy hypothesis is motivated by the idea that face regions might develop from a retinotopic protomap and acquire their selectivity for face features through natural visual experience . faces have a prototypical configuration of features , are usually perceived in a canonical upright orientation , and are frequently fixated in particular locations . to test the faciotopy hypothesis , we presented images of isolated face features at fixation to subjects during functional magnetic resonance imaging . the responses in v1 were best explained by low - level image properties of the stimuli . ofa , and to a lesser degree ffa , showed evidence for faciotopic organization . when a single patch of cortex was estimated for each face feature , the cortical distances between the feature patches reflected the physical distance between the features in a face . faciotopy would be the first example , to our knowledge , of a cortical map reflecting the topology , not of a part of the organism itself ( its retina in retinotopy , its body in somatotopy ) , but of an external object of particular perceptual significance .

Introduction Materials and methods Results Discussion

in addition , a longstanding debate has yet to be resolved about the question whether these regions form visual areas ( felleman & van essen , 1991 ; van essen criteria for visual areas ) and functional modules ( kanwisher , 2000 ) or merely peaks of selectivity within a single more comprehensive object - form topography ( haxby et al . the central part of the retinotopic protomap , according to the theory , therefore turns into the fusiform face area ( ffa ; kanwisher et al . , 1997 , 1996 ) and the occipital face area ( ofa ; gauthier et al . moreover , the spatial organization of the parts would resemble the spatial layout of a face , with the nose represented in a cortical patch that lies somewhere in between the patches representing the eyes and the mouth . whereas a retinotopic map is a cortical representation whose topology resembles that of the retina , a faciotopic map is a cortical representation whose topology resembles that of a face . in order to test whether the faciotopy hypothesis is even plausible when we consider more natural viewing conditions , we used a simple simulation ( fig . for each face feature , we estimated the spatial distribution of retinal exposures when viewing conditions were drawn randomly from realistic distributions of viewing distances and fixation points . this gave us the spatial distribution on the retina of mouth exposures , for example , and a similar distribution for each other face feature . despite the variability in viewing conditions , the peaks of the retinal feature exposure this suggests that a retinotopic protomap with a receptive field size roughly corresponding to face parts might develop into a faciotopic map if its patches acquire selectivity for the features they are most frequently exposed to even when viewing conditions are quite variable . although our simulation did not include variations of viewing angle , it models a substantial part of our visual experience with faces and convinced us that the faciotopy hypothesis is plausible and merits an empirical investigation with functional magnetic resonance imaging ( fmri ) . even if selectivities to certain types of natural shapes develop from a retinotopic protomap , the resulting shape detectors might acquire substantial tolerance to retinal position . in this study , we tested the faciotopy hypothesis by presenting images of isolated face features to subjects during fmri scanning . to avoid confounding faciotopy with retinotopy , results suggest that ofa , and to a lesser extent also ffa , is organized into a faciotopic map . the vertical positions of the sampling windows for the ears needed to be manually adjusted to match the individual variability in the position of the ears but all other features were sampled using the same windows for each face . the stimuli were shown in a blocked fmri design , where during one 16-sec stimulation - block , 16 different exemplars of the same face feature were presented ( e.g. each experimental run consisted of two blocks for each of the face features , and every fifth block was a baseline block with the fixation cross presented alone . the presentation order of the face features within each run and the order of the spatial - layout runs within a session were pseudorandomized and balanced across the subjects and between the two measurement sessions for each subject . , nose not shown at the centre of the visual field but shifted to the left from the fixation cross ; the position and size of the stimulus images remained the same however ) , and the subjects were instructed to press a button when detecting these displacements of the features . peripheral v1 was excluded from the rois based on the spatial extent of the overall fmri response to the face - feature stimuli . during the functional localizer run , the subjects were presented with blocks of images of faces ( different from the faces used for sampling the face features ) , scenes , objects , and phase - randomized textures . , 2014 ) to study the discriminability of the response patterns evoked by the different face - feature stimuli . for each pair of face - feature stimuli , fisher linear discriminant analysis was applied to find the weights for the voxels that discriminated between the response patterns and then the weights were applied to the independent data to calculate the linear - discriminant t - value , reflecting the discriminability between the response patterns evoked by two different face - features . to test for size - tolerance of the face - feature representations , the fisher linear discriminant was fit to the response patterns evoked by the small face - feature images and tested on the response patterns evoked by the large face - feature images . to characterize the face - feature representations in each roi , we computed the dissimilarities between the response patterns evoked by the face - feature stimuli and compared them with model predictions of the representational distances ( kriegeskorte et al . for each roi , the dissimilarities between the response patterns were assembled in a representational dissimilarity matrix ( rdm ; a brain rdm ) , where each value reflects the representational distance between two face - feature stimuli . the face - feature representations in v1 , ofa and ffa were compared to three predictions of the representational distances between the face features : 1 ) gabor wavelet pyramid ( gwp ) model , 2 ) physical distances between the face features in a face ( physical distance reference , fig . 2a ) , and 3 ) physical distances between the face features when symmetric face features are represented in same locations ( symmetric reference ) . the gwp model captures the low - level image similarities between the face - feature stimuli , and was adopted from kay , naselaris , prenger , and gallant ( 2008 ) . 2a shows the physical distance reference matrix , where the values are the distances between the face - feature sampling windows . to evaluate differences between the relatedness of the model rdms to a brain rdm , the difference between the rdm correlations of two models in each subject was calculated and tested using a two - sided signed - rank test across the subjects . a noise ceiling of the expected rdm correlation was estimated for each brain region as described by nili et al . to avoid the contribution of intrinsic fluctuations inflating the representational similarity between two brain regions ( henriksson , khaligh - razavi , kay , & kriegeskorte , 2015 ) , the rdms of the visual areas were compared between rdms constructed from response patterns from different measurement sessions . the multidimensional scaling arrangement of the ( dis)similarity matrix of the rdms provided a visualization of the relatedness of the face - feature representations in different visual areas , and between the visual areas and models . finally , we tested whether the face - feature representations reflect faciotopy , that is , whether the cortical distances between the representations of different face features were explainable by the physical distances between the features in a face . within each roi ( left and right v1 , left and right ofa , left and right ffa ) , we estimated a single location for each face feature using the following approach . the size of the neighborhood ( radius of a sphere ) and the t - value threshold were optimized by evaluating the replicability of the distance matrix across the two measurement sessions ( no assumption of faciotopy , only for replicable distance matrix between the face - feature locations ) . the feature - preference clusters were searched for in 3d space ( voxel coordinates ) and assigned to the cortical surface of the individual . all pair - wise distances between the cortical patches assigned to the face features were calculated along the cortical surface and assembled in a matrix similar to the distance matrix shown in fig . the patch - distance matrix was compared to the matrix of the physical distances between the features in a face . the features were extracted from frontal face photographs using equal - sized , non - overlapping sampling and were presented in a blocked fmri design with three conditions ( fig 2b ) : one small feature shown at the centre of the screen , one large feature shown at the centre of the screen , and nine small features presented in parallel . two face - selective regions - of - interest ( ofa , ffa ) and the primary visual cortex ( v1 ) were defined in each hemisphere in each subject based on independent localizer data . more interestingly , in ofa and ffa , this retinotopic effect was largely abolished and the three sizes of the face - feature stimuli evoked approximately equal - sized responses . 3 ) , but do they also discriminate between the face features ( e.g. , 2014 ) : the discriminability of each pair of face - feature stimuli was evaluated by fitting a fisher linear discriminant to the response patterns from the first fmri session and by testing the performance on the response patterns from the second fmri session ( same subject , different day , different stimulus presentation order , all stimulus layouts ) . 4a shows the linear - discriminant t - values , reflecting the discrimability of each pair of face - feature stimuli from the response patterns , and the right column shows the corresponding p - values . in v1 , the response patterns discriminated each pair of face - feature stimuli , except the two hairlines from each other and the mouth from the chin ( fig . , the eyes ) and outer face - features ( elements 612 in the matrices ; e.g. , the ears ) , that is , the t - values are high for the discriminability of these stimulus pairs in ofa , and also in ffa . moreover , in ffa , the symmetric face - features ( the eyes , the hairlines , the ears , the jaw lines ) evoked indistinguishable response patterns ( bottom row in fig . the use of both small and large features as stimuli enabled us to study the size - tolerance of the face - feature representations in v1 , ofa and ffa . in ffa , however , the distinction between the inner and outer face features was preserved . 2014 ) , which compare the response patterns elicited by the stimuli here each value in an rdm reflects the representational distance between two face - feature stimuli . moreover , a brain rdm can directly be compared to an rdm constructed based on model predictions of the similarity of the representations between the stimuli . we compared the brain rdms to three models : physical distances between the face features in a face ( physical distance reference ; fig . 2a ) , physical distances between the face features when symmetric face - features are represented in the same locations ( symmetric reference ) , and gwp model . 2a shows the physical distance reference matrix , where each value in the matrix reflects the distance between two features in a face . 2a , but the distance between the symmetric face features was zero and the distances from two symmetric face features to other features was identical . the gwp - model captures the low - level image properties of the stimuli ( edges , for example , at the same locations in the stimulus images would be predicted to elicit a similar response in low - level visual areas ) . the v1 rdm of the face - feature stimuli was best explained by the gwp model ( p < .001 ; one - sided signed - rank test ) , reflecting the low - level image properties of the stimulus images . in ofa , both the face - feature physical distance matrix and the symmetric distance matrix explained variance in the representation ( p < .01 ; one - sided signed - rank tests ) . in addition , the physical distances between the face - features explained the representation better than the low - level image properties captured by the gwp model ( fdr q .05 ; two - sided signed - rank test ; fdr - corrected for multiple comparisons ) . a similar trend was observed in ffa , where the physical distance matrix and the symmetric distance matrix both explained variance in the representation . thus far we have shown that especially in ofa the response - pattern dissimilarities do reflect the physical distances between the face features ( fig . for the underlying representation to be truly faciotopic , the distances between cortical locations with preference for a specific feature would also reflect the topology of the face features in a face . to test for a faciotopic representation , we estimated for each face feature a single location on the cortex within each roi and calculated the distances between these face - feature preference patches along the cortical surface . all pair - wise distances between the face - feature preference patches were collected to a matrix similar to the reference matrix shown in fig . 5c shows the results how well the distances between the face - feature preference patches along the cortical surface were explained by the physical distances between the face - features in a face ( blue bars ) or by the symmetric map where symmetric face - features have overlapping representations ( red bars ) . were better explained by the low - level image properties between the stimulus images as captured by the gwp model than by the physical distances between the face - features ( fdr q .05 , two - tailed signed - rank test ; fdr - corrected for multiple comparisons ) . the opposite was true for ofa , where the physical distances between the face features best explained the distances between the face - feature preference patches ( fdr q .05 , two - tailed signed - rank test ; fdr - corrected for multiple comparisons ) . the physical distances between the face features in a face best explain the ofa representation , as reflected in the cortical distances between the face - feature - preference patches , suggesting faciotopy in ofa . our hypothesis was that face - selective regions in human ventral cortex might be organized into faciotopic maps , in which face feature detectors form a map whose topology matches that of a face . faces , and especially the eye region , are frequently fixated from an early age ( farroni et al . , 1975 ) , and a retinotopic protomap ( hasson et al . , 2001 ) could develop into a faciotopic map if patches acquired selectivity for the face features that they are most frequently exposed to . we first performed a simple simulation to support the faciotopy hypothesis , and then measured fmri responses to isolated face features , all presented foveally to prevent the results from being driven by retinotopy . first , we reported that v1 , ofa , and ffa respond to isolated face features and their response patterns distinguish the different face - feature stimuli . furthermore , the face - feature discrimination was tolerant to a change of feature size in ofa . the first two models reflect natural face topology ; the latter captures the low - level image properties of the stimuli . our results indicate that the response - pattern dissimilarity structure in ofa is better explained by the physical distances between the face features than by low - level image properties , whereas the opposite is true for v1 . finally , a true faciotopic organization requires more than a match between pattern dissimilarities and physical face - feature distances : it requires that the map of cortical locations which preferentially respond to each face feature reflects the topology of a face . to test this , we computed cortical distances between feature - preference locations , and compared them to our three models . the distances between the cortical feature - preference patches in ofa were indeed best explained by the physical distances between the features in a face , supporting the existence of a faciotopic map in ofa . , 2007 ) and that ofa is activated more by a face with real inner face - features present than a face with the features replaced by black ovals ( liu et al . moreover , the face - selective neurons in the macaque posterior lateral face patch ( pl ) , the likely homologue of human ofa , are driven by a single eye , especially when presented in the contralateral upper visual hemifield ( issa & dicarlo , 2012 ) . this could be explained by the temporal resolution of fmri ; issa & dicarlo , 2012 reported the eye - preference mainly for the early response ( 60100 ms ) , and our fmri responses reflect a mixture of early and late responses . in a later time - window ( > 100 ms ) , the macaque pl neurons also respond to other face features ( issa & dicarlo , 2012 ) . previous research suggests that ofa is less sensitive than ffa to the correct configuration of the face features within a face ( liu et al . , 2007 this would be consistent with ofa containing a map of somewhat independently operating face - feature detectors . results from monkey electrophysiology support a functional distinction between the two regions : the macaque posterior face patch ( the putative homologue of ofa ) seems to linearly integrate features ( whole = sum of the parts ) , at least in the early response phase ( issa & dicarlo , 2012 ) , while the majority of neurons in the middle macaque face patch ( the putative homologue of ffa ) shows interactions between the face features ( freiwald , tsao , & livingstone , 2009 ) . if faciotopy and retinotopy coexisted in ofa , we would expect face features presented in their typical locations to elicit the largest response . in addition , a faciotopic map would be expected to exhibit non - linear interactions to some degree when multiple face features are presented together . the reported preference for an eye - like feature in its natural visual - field position relative to fixation in monkey pl ( issa & dicarlo , 2012 ) also suggests that retinotopy might play a role , and that the conjunction of the feature and its retinal location might determine the response . sensitivity to retinal position is theoretically compatible with faciotopy and expected if a faciotopic map developed from a retinotopic protomap . it is all the more striking , then , that the face - feature map can be driven by centrally - presented face features independent of retinotopy . , 2013 ) and for the typical configuration of the left and right halves of the body ( right side of the body in the left visual field ; chan et al . , 1994 ) and to the somatosensory homunculus with its enlarged representation of the parts of the organism 's own body that provide richer tactile and proprioceptive input . the nature of the magnified representation might turn out to be quite different in body - part and face - part maps : whereas face perception is tightly coupled with social communication and ultimately relies on holistic perception of the face , visual representations of individual body parts may play an important role in tasks such as action understanding . the spatial organisation of a cortical representation is likely to reflect not only real - world spatial regularities of the stimuli , but also functional relationships . as an object category , faces are also typically perceived in a canonical upright orientation and are fixated at particular locations . if faciotopy develops from the central part of a retinotopic protomap ( hasson et al . , 2001 ) , the peripheral part of the protomap might develop a topological map of the local environment in the occipital and/or parahippocampal place area . although the state of development of the cortical face - processing network in newborn infants remains to be determined , there is converging evidence that a subcortical route is responsible for the early tendency of newborns to orient towards face - like stimuli ( johnson , 2005 ) . consistent with this hypothesis , visual experience during the first months after birth is necessary for normal development of configural processing of faces and , in particular , the processing of information about the spacing between the face features ( le grand , mondloch , maurer , & brent , 2001 ) . an innate neural mechanism that triggers central fixation of faces would spatially align the repeated face exposures of a retinotopic protomap , and could explain the development of faciotopy .

dental implants facilitate the rehabilitation of edentulous patients , and markedly improve both function and esthetics . however , crestal bone loss on the order of 1.5 mm-1.6 mm has been observed radiographically around some dental implants within the first year of loading [ 1 , 2 ] . the connection of the abutment to the endosseous component in two - stage systems invariably results in a microgap ( 1050 m ) , which communicates with the much larger residual cavity created between the abutment screw and the internal implant wall . previous evidence has shown that gram - positive , and gram - negative bacteria found in the oral cavity can colonize the inner surfaces of implants and the microgap area [ 35 ] ; and cause inflammatory reactions in the peri - implant soft tissues . furthermore , in a fascinating study zipprich et al . have shown that cyclical loading of the implant / abutment interface can result in the pumping of the liquid contained in implant cavities into the peri - implant compartment . the existence of an inflammatory cell infiltrate at the level of the implant - abutment junction even around implants placed in areas of meticulous plaque control and clinically healthy , soft tissues has been described in a series of animal studies [ 710 ] . however , other pre - clinical studies have reported a significant reduction in both inflammatory cell infiltrate and bone loss in both one - piece implants ( compared to conventional two - piece implants ) and designs in which the abutment / implant interface has been changed [ 9 , 11 ] . thus , it has been suggested that the microgap in two - piece implants plays a key role in the generation of an inflammatory cell infiltrate , and crestal bone loss , through effects on hard and soft peri - implant tissues . demonstrated that in dogs , the major cell type constituting the inflammatory infiltrate around the microgap of submerged and nonsubmerged two - piece implants was the neutrophil . in their experiment , while there was no selective neutrophil accumulation and a significant reduction of mononuclear cells adjacent to one - piece implants , the population of mononuclear cells was significantly increased around two - piece implants . another study , in monkeys , showed an inflammatory cell infiltrate containing mostly lymphocytes and plasma cells in two - piece implants . the pathophysiological consequences of the implant - abutment interface position have clinically important implications , since esthetic demands encourage the placement of implants in a more apical position . such placement could promote inflammation and bone loss , gingival recession , and esthetic failure . relative to the original alveolar crest position , implants placed on the crestal or subcrestal level have demonstrated greater bone loss than implants placed supracrestally [ 13 , 14 , 16 ] . additionally , experiments with subcrestal implant interfaces showed a significantly greater density of neutrophils than did supracrestal interfaces , but the size of the microgap was not a contributing factor in crestal bone resorption [ 12 , 17 ] . a prospective clinical trial has identified a similar relationship between the location of the interface and the magnitude of bone loss , which ratifies the findings described above . in this clinical investigation , when the implant interface was positioned close to the original alveolar crest , greater bone loss occurred in comparison to implant interfaces placed more supracrestally . thus , the location of the interface is an important determinant of alveolar bone loss in humans , as has been described in initial observations and previous animal studies [ 20 , 21 ] . it is widely known that bone matrix is resorbed by osteoclasts , which are multinucleated giant cells that originate from hematopoietic progenitors of the monocyte / macrophage lineage . it has been reported that lipopolysaccharide ( lps ) can induce bone resorption [ 23 , 24 ] and osteoclast formation . lps supported the survival of preosteoclasts ( mononuclear osteoclasts ) through nf-b activation independent of host factors such as pge2 , rankl , il-1 , and tnf- . osteoclast formation can be modulated by il-1 and tnf- while enhancing the expression of rankl . lps derived from different sources has been employed to specify an effector mechanism relevant to dental implants . thus , while tesmer et al . have shown that certain dental implant designs contribute to the development of multiple colony forming units for both a. actinomycetemcomitans and p. gingivalis , koutouzis et al . employed e. coli bacterial culture solution to demonstrate that implant design affects invasion of oral microorganisms into the fixture abutment interface microgap under dynamic - loading conditions . thus , it has been shown that inflammatory cells , microbial cells and/or fluid [ 810 , 14 , 30 ] are associated with the implant - abutment interface . [ 10 , 12 , 14 , 16 ] nevertheless , the putative correlation between the microgap presence and crestal bone loss has remained refractory to mechanistic explanation . consequently , the purpose of the present study was to investigate the possible mechanisms of crestal bone loss around dental implants using a bacterial endotoxin model in vitro . we hypothesized that bacterial products originating from the microgap cause the upregulation of cytokines , in healthy peri - implant macrophages , mesenchymal stromal cells and gingival fibroblasts that result in the recruitment of resorptively active osteoclasts and , ultimately , bone resorption . the notion that healthy fibroblasts in the periodontium could play an important role in providing sustainable inflammatory mediators in the transition from gingivitis to periodontitis has also recently been discussed , although such cells would not be expected in the peri - implant compartment . thus , in order to address our question , we started with the premise that the peri - implant tissues surrounding the microgap are normal and healthy at the time of healing abutment connection . we further assumed that bacterial products would act on cells in the supra - bony soft tissue , and thus we investigated the effect of lps on cytokine expression and secretion in macrophages , stromal cells , and gingival fibroblasts . to qualify our lps model , we first undertook some comparative studies to assess the effects of laboratory produced , porphyromonas gingivalis ( p. gingivalis ) , prevotella intermedia ( p. intermedia ) , and commercially available , escherichia coli ( e. coli ) , lps on human gingival fibroblasts , since concern has recently been raised over the use of home made lps preparations . samples of gingival tissues were obtained , with informed consent , from 5 patients who showed no clinical signs of periodontal disease . the tissues were collected from different sites : ( 1 ) the palate , used for free connective tissue gingival grafting ; ( 2 ) the marginal gingivae , following extraction of third molars ; ( 3 ) the gingiva around dental implants during 2nd stage surgery . the collected tissue was washed 3 times with -mem supplemented with penicillin g ( 167 units / ml ) , gentamicin ( 50 g / ml ) , and 10% fbs , following which it was cut into small pieces and digested with a mixture of 3 mg / ml collagenase type 1 ( sigma - aldrich ) and 4 mg / ml trypsin in sm for 1 hour at 37 degrees . the digested tissue was centrifuged for 5 minutes at 1150 rpm , and the supernatant was discarded . then , 10 ml of sm was added to the tube containing the pellet , and the cell suspension was transferred to a t-75 culture flask . when cells reached 8090% confluence , they were passaged to new t-75 flasks and plated at a concentration of 1.0 10 cells / cm in 6-well plates at passage 3 . two healthy volunteers , a 35-year - old female and a 50-year - old male , donated blood for this experiment , after providing informed consent . venous blood ( 30 ml ) was collected from a brachial vein into heparinized tubes and immediately processed . peripheral blood mononuclear cells ( pbmc ) isolated pbmcs were incubated in rpmi ( roswell park memorial institute ) medium-1640 ( gibco ) with 10% fbs in 6 well - plates at a density of approximately 5 10 ^ 6 cells / cm in a final volume of 2 ml . the cells were allowed to adhere to plastic dishes for 90 minutes at 37 degrees , 5% co2 , in rpmi medium . the cells were vigorously washed with pbs three times , and the nonadherent cells ( mainly lymphocytes ) were removed . cell viability was 91% before cells were seeded and characterized by immunofluorescence analysis with human anti - cd14 , human mhc ii , and fitc - conjugated mouse igg ( all from bd scientific ) . hbmcs were obtained , following local research ethics board ( reb ) approval , from the collection kit filters ( baxter , deerfield , il , usa ) normally discarded following harvesting of bone marrow , from healthy donors , for transplantation . the cells were washed with pbs and then plated in 75 cm flasks in 10 ml of sm . when cells achieved 7080% confluency , they were trypsinized ( 0.25% trypsin in pbs at 37 degrees for 5 minutes ) , harvested , and expanded in new 75 cm flasks . cells were expanded to achieve 8085% confluency at passage 1 through passage 4 . at this stage , the hbmcs were suspended in sm and seeded at a concentration of 1.0 10 cells / cm in 6-well plates at passage 4 . we collected filters from six different donor aspirates and repeated the hbmcs culture procedures six times . the mouse macrophage cell line , raw 264.7 ( atcc , manassas , va , usa ) , was initially suspended in fresh supplemented medium ( sm ) composed of 10% fetal bovine serum ( fbs ) ( hyclone , logan , ut , usa ) , 80% dmem ( atcc ) and 10% antibiotics ( 167 units / ml penicillin g , 50 g / ml gentamicin ) , and 0.3 g / ml fungizone ( all sigma - aldrich , st . subsequently , the cells were plated in 75 cm ( t-75 ) polystyrene tissue culture flasks ( bd , franklin lakes , nj , usa ) , and incubated at 37 degrees with 5% co2 . the cells grew to 80% confluency , as observed by inverted phase microscopy , after which they were washed with pbs ( gibco , carlsbad , ca , usa ) and removed from the culture flask after the addition of 2.5 ml 0.25% trypsin ( gibco ) in pbs for 5 minutes at 37 degrees . to inactivate the trypsin , 5 ml sm was added , and the cell suspension was then centrifuged at 1150 rpm for 5 minutes . the total viable cell count was obtained from a vicell - xr automated cell counter ( beckman coulter , fullerton , ca , usa ) . this cell culture protocol was followed for cell expansion from passage 0 to passage 2 . on passage 3 , raw 264.7 cells were plated at a concentration of 2.0 10 cells / well in 6-well plates . the harvested femora were soaked and washed for 10 minutes three times with -mem medium ( gibco ) supplemented with 10% antibiotics ( as above ) . both epiphyses were cut off and the bone marrow was gently flushed out from both sides with a 10 ml syringe ( fitted with a 20-gauge needle ) filled with fully supplemented medium ( fsm : 10% fbs , 80% -mem and 10% antibiotics ) . the volume of bone marrow suspension was adjusted to 30 ml with fsm and divided in two aliquots of 15 ml each , which were distributed in two t-75 flasks . the first medium change was done at 24 hours and then three times per week subsequently . cells were grown for 5 to 6 days to 8085% confluence . at each passage , and up to passage 8 , 1.0 10 ^ 6 cells were collected and fixed with formalin in a 1.5 ml microcentrifuge tube for flow cytometry ( beckman coulter , miami , fl , usa ) analysis of the cd45 positive cell population ( cd45 , mouse anti - rat caltag laboratories , burlingame , ca , usa ) , for investigation of the presence of hematopoietic cells . aliquots of 2.0 10 ^ 6 cells / cm of the rsbmcs at passage 7 were seeded into 6-well plates . the passage 7 was chosen in this assay because passage 7 included only 0.36% of cd45 cells . stromal cells were isolated from bone marrow of two rats for quantification of cd45 cells . hgfs were stimulated with different types of lps : p. gingivalis , p. intermedia , and e. coli lps ( escherichia coli 055:b5 , sigma - aldrich ) . both lps of p. gingivals and p. intermedia were isolated from colonies of these bacterial cultures . the strains used in this study were p. intermedia atcc 25611 , p. gingivalis atcc 33277 . all anaerobes were maintained on brucella hk agar ( kyokuto , tokyo , japan ) supplemented with 5% laked sheep blood in an atmosphere of 80% n2 , 10% co2 , and 10% h2o in the aerobic chamber for 48 hours 37 degrees . lps was extracted by using an lps extraction kit ( intron biotechnology , kyunggi - do , korea ) , dissolved in 10 mm tris - hcl buffer , and freeze - dried . in separate experiments , hbmcs and hgfs were stimulated with e. coli lps ( 0 g / ml or 0.1 g / ml or 1 g / ml ) for 24 , 48 , or 96 hours , and they were stimulated with p. gingivalis lps or p. intermedia lps ( 1 g / ml ) for 2 or 24 hours ( 37 degrees , 5% co2 ) . raw 264.7 cells were stimulated with e. coli lps ( escherichia coli 055:b5 , sigma - aldrich ) ( 0 g / ml or 1 g / ml ) for 12 , 24 , 36 , 48 , and 60 hours ; hmo were stimulated with lps ( 0 g / ml or 1 g / ml ) for 24 hours ; rsbmcs were stimulated with e. coli lps ( 0 g / ml or 0.1 g / ml or 1 g / ml ) for 6 , 24 , 48 and 96 hours ; hbmcs and hgfs were stimulated with e. coli lps ( 0 g / ml or 0.1 g / ml or 1 g / ml ) for 24 , 48 , and 96 hours . in each case , lps was added to the culture medium when cells reached 7580% confluence , which was approximately at day 3 for mmo and day 7 for rsbmcs , hgfs , and hbmcs . cell culture supernatants of hbmcs and hgfs were harvested and stored at 80 degrees prior to enzyme - linked immunosorbent assay ( elisa , thermo fisher scientific , waltham , ma , usa ) . rna was isolated from both lps stimulated cells and cell , lysate after the culture medium was removed ( see below ) . total cellular rna was extracted from hbmcs , hgfs , , hmo , mmo and rsbmcs . with 1 ml/10 cm culture dish area of tri reagent ( ambion , austin , tx , usa ) . cdna templates of these cells were produced using a reverse transcriptase kit ( quanti tect reverse transcription kit , qiagen , mississauga , on , usa ) . gapdh , a house - keeping gene , was used for all species of human , rat , and mouse . specific primers of il-1 , il-1 , tnf- , il-6 , rank , rankl , trl-2 , trl-4 , cd14 and md-2 were designed for human or rat , and mouse individually using an electronic gene database ( blast , national library of medicine ) . the specific primers ( table 1 ) were added to each cdna sample to be amplified using a taq polymerase kit ( platinum taq dna polymerase , invitrogen , carisbad , ca , usa ) . expression of cdna was assessed by running 25 to 35 cycles at 55 degrees to 65 degrees on reverse transcriptase - polymerase chain reaction ( rt - pcr ) amplification . amplified products were analyzed by 1.5% agarose gel electrophoresis and visualized with ethidium bromide ( sigma - aldrich ) staining . culture supernatants from each cell type were removed and stored at 80 degrees prior to elisa analysis . elisa kits were used to quantify il-1 , il-1 , tnf- , and il-6 concentration in cell - free culture supernatants according to the manufacturer 's protocols . hbmcs and hgfs were extracted with a lysis buffer ( cell signaling technology , beverly , ms , usa ) and the protein lysates were stored in the same manner . elisa kits were used to quantify il-1 , il-1 , and tnf- concentration in cell lysates according to the manufacturer 's protocols . the absorbance at 450 nm was read with wavelength correction set at 550 nm . the sensitivities of the commercial elisa kits were < 2 pg / ml for il-1 , < 1 pg / ml for il-1 , < 2 pg / ml for tnf- , and < 1 pg / ml for il-6 . the number of samples of cell - free culture supernatant samples , the cell lysate samples , and the times of repeated analysis of each sample are shown in table 2 . the concentration of il-1 , il-1 , il-6 , and tnf- was analyzed in cell - free culture supernatants and the cell lysate for hbmcs and hgfs . in hmo , we analyzed only the concentration of il-1 and il-1. mmo ( raw 264.7 ) were washed with pbs and fixed with 10% formalin at room temperature for 5 minutes . then , they were washed with distilled water and incubated in michaelis veronal acetate buffer ( ph 5.0 ) containing naphtol as - bi phosphate as substrate , pararosanilin - hcl as coupler , and tartaric acid for detection of trap activity , for 40 minutes at 37 degrees . they were analyzed and counted in micrographs taken with a digital camera attached to the light microscope . mmo cells in a 96-well plate were washed with pbs and add 100 l / well of the assay solution : 1 ml of 0.1 m pnpp ( disodium p - nitrophenylphophate hexahydrate ) , 1 ml of 10 buffer ( 1 m sodium acetate and 0.1 m sodium tartrate ) , 1 ml of 0.1% triton - x , and fill up to 10 ml with distilled water . incubate the plate for 10 minutes at room temperature and add 50 l / well of 0.2 m naoh as a stop solution . then , measure the absorbance at 405 nm . the osteoclastic bone resorption assay was performed using the commercially available osteologic culture system ( biocoat osteologic bone cell culture system , bd ) , in which the substrate is coated with a thin layer of osteoclast resorbable calcium phosphate ceramic . raw 264.7 cells ( 1.0 10 ^ 4 cells / well ) were cultured on the osteologic plates for 1 day . then , lps ( 1 g / ml ) , or rankl ( receptor activator of nuclear factor b ligand ) ( 200 ng / ml ) was added to the cell culture medium , and cells were stimulated with either lps or rankl for 7 days . after 7 days , the cell culture medium was removed and cells were fixed with 10% formalin . we investigated the effects of different types of lps on pro - inflammatory and osteoclastogenic cytokine gene expression following exposure to lps for 2 hours in hgf . all three lps types ( derived from e.coli , p. gingivalis , p. intermedia . ) showed similar upregulation of il-1 , il-6 , and rankl gene expression ( figures 1(b ) , 1(c ) , and 1(e ) ) . tnf- and rankl expression was detected in both non - lps and lps conditions ( figures 1(d ) and 1(f ) ) . we next examined the effects of lps on the trl 2 and trl 4 lps receptors and their signaling cascade factors ( figure 2(a ) ) . without lps , this was also the case for cd-14 gene expression , although md-2 was expressed with or without lps stimulation ( figure 2(b ) ) . therefore , for convenience , we selected e. coli lps for subsequent experiments as it is commercially available . however , our experimental purpose was to investigate the cellular influence on osteoclastogenesis in the surrounding tissue cells . therefore , we also examined the effect of macrophages and bone marrow cells , which could be expected to exist around dental implant fixtures , from human , mouse , and rat . hmo expressed il-1 and tnf- gene in the presence or absence of lps for 24 hours at 35 cycles of rt - pcr ( figure 3 ) . both il-1 and il-6 expression was increased by lps stimulation in human macrophages at 30 cycles of rt - pcr ( figure 3 ) . mmo cultures showed that the expression of il-1 , il-1 , and il-6 genes was upregulated with lps stimulation at 30 cycles of rt - pcr ( figure 4 ) . however , gene expression of tnf- was observed irrespective of lps stimulation at all time points , except for 60 hours ( figure 4 ) . the il-1 and il-6 gene expression decreased at 48 hours , while il-1 gene expression was decreased at 60 hours after lps stimulation ( figure 4 ) . hbmcs showed a similar trend for gene expression of cytokines as that found with rsbmcs ( figure 5 ) . the gene expression of il-1 , il-1 , and il-6 was stimulated by the presence of lps . however , the expression of il-1 and il-1 gene was reduced at 96 hours in hbmcs culture . tnf- gene expression also occurred in the presence of lps at 24 hours and 48 hours ( figure 5 ) . nevertheless , the tnf- expression was very weak at each lps concentration and time point . the lps - stimulated genes for il-1 and il-1 were observed by rt - pcr at 30 cycles , and for il-6 gene at 25 cycles . lps upregulated gene expression of il-1 , il-1 , and il-6 during the rsbmcs poststimulation period ( 696 hours , figure 6 ) , showing the same trend of mmo cultures . the expression of tnf- gene in the presence of lps at 6 and 48 hours at 30 cycles was very weak ( figure 6 ) . running cycles for rt - pcr were same as hbmcs . although we used hgf for estimating the effect of different lps in this study , those experiments were carried out with 2 hours lps exposure . we also investigated the lps effects against hgf with longer time periods , 24 , 48 , and 96 hours , in a subsequent experiment . all genes , for il-1 , il-1 , tnf- , and il-6 , were expressed in the presence of lps at all time points in hgf cultures . however , expression of il-1 was not expressed at 96 hours in spite of increased ( + 5 ) cycles ; the expression of il-1 , il-1 , and tnf- was observed at 35 cycles . interestingly , all gene expression was stronger at 0.1 g / ml than at 1 g / ml lps ( figure 7 ) . lps - stimulated il-1 and il-1 gene expression was reduced in a time - dependent manner , however , il-6 gene expression increased especially with 1 g / ml concentration of lps ( figure 7 ) . elisa was used to assess levels of protein secretion ( il-1 , il-1 , tnf- , and il-6 ) in cultures of hmos , hbmcs , and hgfs . significant secretion of il-1 and il-1 was found in the cell - free culture supernatant after stimulation with lps for 24 hours ( table 3(a ) ) . il-1 and il-1 concentrations were approximately 470 times and 12.5 times higher in the presence of lps . the analysis of cell lysate by elisa revealed that lps - stimulated hbmcs secreted il-1 at 24 hours and il-1 at 24 , 48 , and 96 hours ( table 3(b ) ) but these protein levels were very low . secretions of tnf- from protein lysate were detected both with and without lps at all time points ( table 3(b ) ) . thus , the secretion of il-1 , il-1 , and tnf- was meaningless in hbmcs cultures . to the contrary , the level of il-6 secretion was extremely high in the presence of lps in hbmcs ( figure 8(a ) ) . il-6 concentration was approximately 40000 times higher at 96 hours in the presence of lps as maximum compared to absence of lps . the secretions of il-6 from cell - free culture supernatants were detected from only with lps at all time points ( figure 8(a ) ) . therefore , the secretion of il-6 protein was induced by lps stimulation in a time - dependent manner , but not an lps dose - dependent manner . secretion of tnf- was not specific to the presence of lps stimulation at 24 , 48 , and 96 hours ( table 3(c ) ) . the levels of il-6 protein were clearly increased with lps in hgfs ( figure 8(b ) ) . the secretions of il-6 were detected from cell - free culture supernatants at all time points : 24 , 48 and 96 hours . secretion of il-6 by hgfs in the presence of lps stimulation was found to be time- and dose - dependent hgfs ( figure 8(b ) ) . mmo ( raw 264.7 cells ) were cultured in medium with 300 ng / ml gst - rankl for 3 days demonstrated the highest trap positive reaction compared to negative controls , without rankl , and lps stimulated mmo ( figure 9 ) . mmo were cultured in medium containing 1 g / ml lps for 5 to 7 days and observed under phase contrast for the presence of multinucleated cells ( mncs ) ( figure 10(b ) ) . the mncs were enlarged the size , but the amount of mncs was decreased at day 7 . mmo did not show morphological changes in the absence of lps ( figure 10(a ) ) . osteoclast formation was examined by trap staining , which is a selectable staining to identify osteoclasts and activated macrophages . trap - positive mononuclear cells and mncs were observed in mmo cultures in the presence of lps ( figure 10(e ) ) and in the presence of rankl ( figure 10(h ) ) . mmo cultures without the addition of lps or rankl were used as negative control and did not show positive reaction to trap staining ( figure 10(c ) ) . mmo cultures stimulated with rankl were used as a positive control ( figure 10(h ) ) . pit forming activity was analyzed using the commercially available biocoat osteologic bone cell culture system ( osteologic ) , which consists of quartz plates with a thin film surface coating of calcium phosphate ( cap ) . pit formation on the cap substrates was observed in mouse macrophage cultures in the presence of lps ( figure 10(f ) ) and rankl ( figure 10(g ) ) . although gingival and periodontal pathologies are known to cause the upregulation and secretion of pro - inflammatory cytokines , [ 3436 ] our studies were aimed at elucidating a mechanism whereby dental implants , placed in healthy tissue , and could cause the genesis of osteoclasts that would be the effectors of peri - implant crestal bone loss . since the latter is a local , not systemic phenomenon , thus , we hypothesized that bacterial products , originating from bacteria colonizing implant / abutment connections , could cause the upregulation of pro - inflammatory cytokines in the cells of healthy peri - implant tissues that would , in turn , stimulate the recruitment of resorptively active osteoclasts . indeed , our results show that e. coli lps , as a model bacterial endotoxin , can selectively stimulate the upregulation of pro - inflammatory cytokines in healthy candidate resident cells , which can cause the genesis of osteoclasts that are resorptively active . despite differences in implant / abutment designs , implant / abutment connections will invariably form a microgap , that under function ( cyclic opening / closing ) , will allow passage of organic fluids containing bacteria and endotoxin from and to the implant cavities . furthermore , the bacterial colonization of the internal cavities of two piece dental implants has been repeatedly demonstrated through both microscopical observations and in vitro studies [ 35 ] . in fact , it is likely that these cavities provide anaerobic chambers for pathogens that could induce an immune response in the host . the intensity of such response would depend on : the immunologic profile of each individual ( as is known to be the case in periodontal disease ) ; the amount and types of periodontal pathogens present in the mouth ; possibly , the position ( in relation to the bone crest ) of the implant / abutment interface [ 11 , 13 ] . indeed , there seems to be a delicate balance between cytokines that trigger immunological response ( pro - inflammatory ) and those that will modulate the immunological response . interestingly , clinical studies of implant / abutment self - locking connections [ 39 , 40 ] , which are more stable and reduce fluid percolation ( with respect to the less stable flat - to - flat nonself - locking components ) , have shown a reduction in the overall loss of crestal bone . our results show that lps upregulated il-1 and il-6 gene expression in all the examined cells types ; while il- , and tnf- were only upregulated in cultures of hbmcs , and hgfs . on the contrary , analysis of protein secretion showed very low or undetectable levels il-1 , il-1 and tnf- in cultures of hm , hbmcs and hgfs . however , there was a clear increase in il-6 secretion , with lps treatment , for both hbmcs and hgfs , a finding that corresponds to previous reports [ 41 , 42 ] . indeed , il-6 has been shown to stimulate osteoclast activity through enhancement of the expression of rankl with soluble il-6 receptors ( sil-6r ) in both calvarial bones and osteoblasts , an effect reliant on the synergistic effect of il-6 and sil-6r . furthermore , il-6 can stimulate osteoclast - like formation in long - term human bone marrow cultures by inducing il-1 release . thus , il-6 might be responsible for osteoclast differentiation and activation through enhancement of rankl expression [ 4345 ] . gingival tissues obtained from diseased periodontal sites contain higher levels of il-6 as compared to healthy sites and crevicular fluid from periodontally diseased sites contain more il-6 than healthy sites . while il- , il-10 , il-12 , il-8 , il-6 , and tnf- have all been found to be present in peri - implant crevicular fluid , recorded levels tended to be higher than those observed around teeth , and il-6 expression is stimulated by il-1 and tnf- in hgfs osteoblasts have also been shown to produce il-6 in response to local bone - resorbing agents . thus , it is likely that il-6 , in cooperation with other cytokines , plays a role in the bone destruction and tissue damage . in fact , increases in inflammatory cytokines are associated with bone loss and progressive failure of dental implants [ 50 , 51 ] . since cytokines are small molecules of short life span and short acting distance which can produce responses in very small concentrations and in a paracrine fashion this may explain why the recent introduction of platform switching in implant design [ 53 , 54 ] contains the pro - inflammatory cytokine production to the area between the abutment and platform ; thereby shielding the surrounding bone from their deleterious effects . in comparison to the specific expression and abundant secretion of il-6 by hbmcs and hgfs , it is noteworthy that low amounts of il-1 , il-1 , and tnf- were also produced and secreted . the il-1 and il-1 have been shown to enhance rankl expression in both osteoblasts and stromal cells [ 44 , 45 ] , while il-1 promotes the survival of osteoclasts through nf - kb activation which results in induction of osteoclast activation . tnf- stimulates the differentiation and survival of osteoclasts , but not the function of osteoclasts . thus , the differentiation and activation of osteoclasts could be stimulated by lps through il-1 , il-1 , and tnf-. our results show that lps activated macrophages can form resorptively active osteoclast - like trap positive cells and suggest that lps may have a direct action on resident macrophages in addition to its known ability to act indirectly on preosteoclasts . taken together , our results support the hypothesis that bacterial endotoxins may upregulate pro - inflammatory genes in a number of resident cells found in the healthy peri - implant compartment , and , that the local synergistic action of cytokines secreted by such cells result in the genesis of resorptively active osteoclasts .

purpose . the immunological mechanisms of peri - implant crestal bone loss have , hitherto , not been elucidated . we hypothesized that bacterial products from the microgap cause upregulation of cytokines in otherwise healthy peri - implant cells , which results in osteoclast formation and , ultimately , in bone resorption . materials and methods . we used rt - pcr and elisa to assay mediators of osteoclastogenesis in rat and human macrophages ( r - and hmo ) ; bone marrow derived stromal cells ( r - and hbmcs ) ; and human gingival fibroblasts ( hgf)with or without stimulation by lps . trap positive multinucleate cells were assessed for their resorptive ability . results . we show that il-1 , il-1 , and il-6 were expressed by all examined cell types , and tnf- was upregulated in hgf . secretion of il-1 and il-1 proteins was stimulated in hmo by lps , and il-6 protein secretion was highly stimulated in hbmcs and hgf . both lps and rankl stimulated macrophages to form osteoclast - like trap positive cells , which resorbed calcium phosphate substrates . conclusion . taken together , the results of our study support the hypothesis that bacterial endotoxins upregulate enhanced mediators of osteoclastogenesis in resident cells found in the healthy peri - implant compartment and that the local synergistic action of cytokines secreted by such cells results in the genesis of resorptively active osteoclasts .

1. Introduction 2. Material and Methods 3. Results 4. Effect of LPS on Protein Secretion 5. Effect of LPS on the Differentiation of Mouse Macrophages into Osteoclasts 6. Discussion 7. Conclusion

however , crestal bone loss on the order of 1.5 mm-1.6 mm has been observed radiographically around some dental implants within the first year of loading [ 1 , 2 ] . the connection of the abutment to the endosseous component in two - stage systems invariably results in a microgap ( 1050 m ) , which communicates with the much larger residual cavity created between the abutment screw and the internal implant wall . previous evidence has shown that gram - positive , and gram - negative bacteria found in the oral cavity can colonize the inner surfaces of implants and the microgap area [ 35 ] ; and cause inflammatory reactions in the peri - implant soft tissues . have shown that cyclical loading of the implant / abutment interface can result in the pumping of the liquid contained in implant cavities into the peri - implant compartment . thus , it has been suggested that the microgap in two - piece implants plays a key role in the generation of an inflammatory cell infiltrate , and crestal bone loss , through effects on hard and soft peri - implant tissues . demonstrated that in dogs , the major cell type constituting the inflammatory infiltrate around the microgap of submerged and nonsubmerged two - piece implants was the neutrophil . such placement could promote inflammation and bone loss , gingival recession , and esthetic failure . additionally , experiments with subcrestal implant interfaces showed a significantly greater density of neutrophils than did supracrestal interfaces , but the size of the microgap was not a contributing factor in crestal bone resorption [ 12 , 17 ] . a prospective clinical trial has identified a similar relationship between the location of the interface and the magnitude of bone loss , which ratifies the findings described above . thus , the location of the interface is an important determinant of alveolar bone loss in humans , as has been described in initial observations and previous animal studies [ 20 , 21 ] . it has been reported that lipopolysaccharide ( lps ) can induce bone resorption [ 23 , 24 ] and osteoclast formation . lps supported the survival of preosteoclasts ( mononuclear osteoclasts ) through nf-b activation independent of host factors such as pge2 , rankl , il-1 , and tnf- . osteoclast formation can be modulated by il-1 and tnf- while enhancing the expression of rankl . [ 10 , 12 , 14 , 16 ] nevertheless , the putative correlation between the microgap presence and crestal bone loss has remained refractory to mechanistic explanation . consequently , the purpose of the present study was to investigate the possible mechanisms of crestal bone loss around dental implants using a bacterial endotoxin model in vitro . we hypothesized that bacterial products originating from the microgap cause the upregulation of cytokines , in healthy peri - implant macrophages , mesenchymal stromal cells and gingival fibroblasts that result in the recruitment of resorptively active osteoclasts and , ultimately , bone resorption . the notion that healthy fibroblasts in the periodontium could play an important role in providing sustainable inflammatory mediators in the transition from gingivitis to periodontitis has also recently been discussed , although such cells would not be expected in the peri - implant compartment . thus , in order to address our question , we started with the premise that the peri - implant tissues surrounding the microgap are normal and healthy at the time of healing abutment connection . we further assumed that bacterial products would act on cells in the supra - bony soft tissue , and thus we investigated the effect of lps on cytokine expression and secretion in macrophages , stromal cells , and gingival fibroblasts . to qualify our lps model , we first undertook some comparative studies to assess the effects of laboratory produced , porphyromonas gingivalis ( p. gingivalis ) , prevotella intermedia ( p. intermedia ) , and commercially available , escherichia coli ( e. coli ) , lps on human gingival fibroblasts , since concern has recently been raised over the use of home made lps preparations . the cells were allowed to adhere to plastic dishes for 90 minutes at 37 degrees , 5% co2 , in rpmi medium . the cells were vigorously washed with pbs three times , and the nonadherent cells ( mainly lymphocytes ) were removed . cell viability was 91% before cells were seeded and characterized by immunofluorescence analysis with human anti - cd14 , human mhc ii , and fitc - conjugated mouse igg ( all from bd scientific ) . hbmcs were obtained , following local research ethics board ( reb ) approval , from the collection kit filters ( baxter , deerfield , il , usa ) normally discarded following harvesting of bone marrow , from healthy donors , for transplantation . subsequently , the cells were plated in 75 cm ( t-75 ) polystyrene tissue culture flasks ( bd , franklin lakes , nj , usa ) , and incubated at 37 degrees with 5% co2 . the volume of bone marrow suspension was adjusted to 30 ml with fsm and divided in two aliquots of 15 ml each , which were distributed in two t-75 flasks . at each passage , and up to passage 8 , 1.0 10 ^ 6 cells were collected and fixed with formalin in a 1.5 ml microcentrifuge tube for flow cytometry ( beckman coulter , miami , fl , usa ) analysis of the cd45 positive cell population ( cd45 , mouse anti - rat caltag laboratories , burlingame , ca , usa ) , for investigation of the presence of hematopoietic cells . stromal cells were isolated from bone marrow of two rats for quantification of cd45 cells . all anaerobes were maintained on brucella hk agar ( kyokuto , tokyo , japan ) supplemented with 5% laked sheep blood in an atmosphere of 80% n2 , 10% co2 , and 10% h2o in the aerobic chamber for 48 hours 37 degrees . in separate experiments , hbmcs and hgfs were stimulated with e. coli lps ( 0 g / ml or 0.1 g / ml or 1 g / ml ) for 24 , 48 , or 96 hours , and they were stimulated with p. gingivalis lps or p. intermedia lps ( 1 g / ml ) for 2 or 24 hours ( 37 degrees , 5% co2 ) . raw 264.7 cells were stimulated with e. coli lps ( escherichia coli 055:b5 , sigma - aldrich ) ( 0 g / ml or 1 g / ml ) for 12 , 24 , 36 , 48 , and 60 hours ; hmo were stimulated with lps ( 0 g / ml or 1 g / ml ) for 24 hours ; rsbmcs were stimulated with e. coli lps ( 0 g / ml or 0.1 g / ml or 1 g / ml ) for 6 , 24 , 48 and 96 hours ; hbmcs and hgfs were stimulated with e. coli lps ( 0 g / ml or 0.1 g / ml or 1 g / ml ) for 24 , 48 , and 96 hours . in each case , lps was added to the culture medium when cells reached 7580% confluence , which was approximately at day 3 for mmo and day 7 for rsbmcs , hgfs , and hbmcs . specific primers of il-1 , il-1 , tnf- , il-6 , rank , rankl , trl-2 , trl-4 , cd14 and md-2 were designed for human or rat , and mouse individually using an electronic gene database ( blast , national library of medicine ) . expression of cdna was assessed by running 25 to 35 cycles at 55 degrees to 65 degrees on reverse transcriptase - polymerase chain reaction ( rt - pcr ) amplification . elisa kits were used to quantify il-1 , il-1 , tnf- , and il-6 concentration in cell - free culture supernatants according to the manufacturer 's protocols . hbmcs and hgfs were extracted with a lysis buffer ( cell signaling technology , beverly , ms , usa ) and the protein lysates were stored in the same manner . elisa kits were used to quantify il-1 , il-1 , and tnf- concentration in cell lysates according to the manufacturer 's protocols . the sensitivities of the commercial elisa kits were < 2 pg / ml for il-1 , < 1 pg / ml for il-1 , < 2 pg / ml for tnf- , and < 1 pg / ml for il-6 . the number of samples of cell - free culture supernatant samples , the cell lysate samples , and the times of repeated analysis of each sample are shown in table 2 . the concentration of il-1 , il-1 , il-6 , and tnf- was analyzed in cell - free culture supernatants and the cell lysate for hbmcs and hgfs . in hmo , we analyzed only the concentration of il-1 and il-1. the osteoclastic bone resorption assay was performed using the commercially available osteologic culture system ( biocoat osteologic bone cell culture system , bd ) , in which the substrate is coated with a thin layer of osteoclast resorbable calcium phosphate ceramic . then , lps ( 1 g / ml ) , or rankl ( receptor activator of nuclear factor b ligand ) ( 200 ng / ml ) was added to the cell culture medium , and cells were stimulated with either lps or rankl for 7 days . after 7 days , the cell culture medium was removed and cells were fixed with 10% formalin . showed similar upregulation of il-1 , il-6 , and rankl gene expression ( figures 1(b ) , 1(c ) , and 1(e ) ) . tnf- and rankl expression was detected in both non - lps and lps conditions ( figures 1(d ) and 1(f ) ) . without lps , this was also the case for cd-14 gene expression , although md-2 was expressed with or without lps stimulation ( figure 2(b ) ) . however , our experimental purpose was to investigate the cellular influence on osteoclastogenesis in the surrounding tissue cells . therefore , we also examined the effect of macrophages and bone marrow cells , which could be expected to exist around dental implant fixtures , from human , mouse , and rat . hmo expressed il-1 and tnf- gene in the presence or absence of lps for 24 hours at 35 cycles of rt - pcr ( figure 3 ) . both il-1 and il-6 expression was increased by lps stimulation in human macrophages at 30 cycles of rt - pcr ( figure 3 ) . mmo cultures showed that the expression of il-1 , il-1 , and il-6 genes was upregulated with lps stimulation at 30 cycles of rt - pcr ( figure 4 ) . the il-1 and il-6 gene expression decreased at 48 hours , while il-1 gene expression was decreased at 60 hours after lps stimulation ( figure 4 ) . the gene expression of il-1 , il-1 , and il-6 was stimulated by the presence of lps . however , the expression of il-1 and il-1 gene was reduced at 96 hours in hbmcs culture . the lps - stimulated genes for il-1 and il-1 were observed by rt - pcr at 30 cycles , and for il-6 gene at 25 cycles . lps upregulated gene expression of il-1 , il-1 , and il-6 during the rsbmcs poststimulation period ( 696 hours , figure 6 ) , showing the same trend of mmo cultures . running cycles for rt - pcr were same as hbmcs . although we used hgf for estimating the effect of different lps in this study , those experiments were carried out with 2 hours lps exposure . we also investigated the lps effects against hgf with longer time periods , 24 , 48 , and 96 hours , in a subsequent experiment . all genes , for il-1 , il-1 , tnf- , and il-6 , were expressed in the presence of lps at all time points in hgf cultures . however , expression of il-1 was not expressed at 96 hours in spite of increased ( + 5 ) cycles ; the expression of il-1 , il-1 , and tnf- was observed at 35 cycles . lps - stimulated il-1 and il-1 gene expression was reduced in a time - dependent manner , however , il-6 gene expression increased especially with 1 g / ml concentration of lps ( figure 7 ) . elisa was used to assess levels of protein secretion ( il-1 , il-1 , tnf- , and il-6 ) in cultures of hmos , hbmcs , and hgfs . significant secretion of il-1 and il-1 was found in the cell - free culture supernatant after stimulation with lps for 24 hours ( table 3(a ) ) . il-1 and il-1 concentrations were approximately 470 times and 12.5 times higher in the presence of lps . the analysis of cell lysate by elisa revealed that lps - stimulated hbmcs secreted il-1 at 24 hours and il-1 at 24 , 48 , and 96 hours ( table 3(b ) ) but these protein levels were very low . thus , the secretion of il-1 , il-1 , and tnf- was meaningless in hbmcs cultures . to the contrary , the level of il-6 secretion was extremely high in the presence of lps in hbmcs ( figure 8(a ) ) . therefore , the secretion of il-6 protein was induced by lps stimulation in a time - dependent manner , but not an lps dose - dependent manner . secretion of tnf- was not specific to the presence of lps stimulation at 24 , 48 , and 96 hours ( table 3(c ) ) . secretion of il-6 by hgfs in the presence of lps stimulation was found to be time- and dose - dependent hgfs ( figure 8(b ) ) . mmo ( raw 264.7 cells ) were cultured in medium with 300 ng / ml gst - rankl for 3 days demonstrated the highest trap positive reaction compared to negative controls , without rankl , and lps stimulated mmo ( figure 9 ) . osteoclast formation was examined by trap staining , which is a selectable staining to identify osteoclasts and activated macrophages . pit forming activity was analyzed using the commercially available biocoat osteologic bone cell culture system ( osteologic ) , which consists of quartz plates with a thin film surface coating of calcium phosphate ( cap ) . pit formation on the cap substrates was observed in mouse macrophage cultures in the presence of lps ( figure 10(f ) ) and rankl ( figure 10(g ) ) . although gingival and periodontal pathologies are known to cause the upregulation and secretion of pro - inflammatory cytokines , [ 3436 ] our studies were aimed at elucidating a mechanism whereby dental implants , placed in healthy tissue , and could cause the genesis of osteoclasts that would be the effectors of peri - implant crestal bone loss . since the latter is a local , not systemic phenomenon , thus , we hypothesized that bacterial products , originating from bacteria colonizing implant / abutment connections , could cause the upregulation of pro - inflammatory cytokines in the cells of healthy peri - implant tissues that would , in turn , stimulate the recruitment of resorptively active osteoclasts . indeed , our results show that e. coli lps , as a model bacterial endotoxin , can selectively stimulate the upregulation of pro - inflammatory cytokines in healthy candidate resident cells , which can cause the genesis of osteoclasts that are resorptively active . the intensity of such response would depend on : the immunologic profile of each individual ( as is known to be the case in periodontal disease ) ; the amount and types of periodontal pathogens present in the mouth ; possibly , the position ( in relation to the bone crest ) of the implant / abutment interface [ 11 , 13 ] . interestingly , clinical studies of implant / abutment self - locking connections [ 39 , 40 ] , which are more stable and reduce fluid percolation ( with respect to the less stable flat - to - flat nonself - locking components ) , have shown a reduction in the overall loss of crestal bone . our results show that lps upregulated il-1 and il-6 gene expression in all the examined cells types ; while il- , and tnf- were only upregulated in cultures of hbmcs , and hgfs . on the contrary , analysis of protein secretion showed very low or undetectable levels il-1 , il-1 and tnf- in cultures of hm , hbmcs and hgfs . furthermore , il-6 can stimulate osteoclast - like formation in long - term human bone marrow cultures by inducing il-1 release . while il- , il-10 , il-12 , il-8 , il-6 , and tnf- have all been found to be present in peri - implant crevicular fluid , recorded levels tended to be higher than those observed around teeth , and il-6 expression is stimulated by il-1 and tnf- in hgfs osteoblasts have also been shown to produce il-6 in response to local bone - resorbing agents . thus , it is likely that il-6 , in cooperation with other cytokines , plays a role in the bone destruction and tissue damage . in comparison to the specific expression and abundant secretion of il-6 by hbmcs and hgfs , it is noteworthy that low amounts of il-1 , il-1 , and tnf- were also produced and secreted . the il-1 and il-1 have been shown to enhance rankl expression in both osteoblasts and stromal cells [ 44 , 45 ] , while il-1 promotes the survival of osteoclasts through nf - kb activation which results in induction of osteoclast activation . thus , the differentiation and activation of osteoclasts could be stimulated by lps through il-1 , il-1 , and tnf-. our results show that lps activated macrophages can form resorptively active osteoclast - like trap positive cells and suggest that lps may have a direct action on resident macrophages in addition to its known ability to act indirectly on preosteoclasts . taken together , our results support the hypothesis that bacterial endotoxins may upregulate pro - inflammatory genes in a number of resident cells found in the healthy peri - implant compartment , and , that the local synergistic action of cytokines secreted by such cells result in the genesis of resorptively active osteoclasts .

this study provides class iii evidence that shorter , more frequent early mobilization improves the chance of regaining independence after stroke . detailed methods are described elsewhere . in brief , avert , conducted in 56 stroke units in 5 countries , is a pragmatic , parallel - group , randomized controlled trial . patients with ischemic or hemorrhagic stroke , admitted within 24 hours of onset , were eligible . patients with early deterioration , another serious illness , unstable coronary conditions , not rousable to voice , or failing physiologic screening criteria were excluded . patients were randomized to receive either usual care or frequent out - of - bed activity ( mobilization ) in addition to usual care ( vem ) . vem patients commenced mobilization within 24 hours of stroke and trained physiotherapy and nursing staff helped them continue task - specific out - of - bed activity , targeting recovery of active sitting , standing , and walking activity , at a frequency and intensity ( amount ) guided by an intervention protocol . functional ability at baseline , monitored daily and adjusted with recovery , guided the intervention dose with 4 titrations specified . for example , in low arousal , dependent patients ( level 1 ) , active sitting with assistance was the mobilization target , with each session lasting a minimum of 10 and maximum of 30 minutes . with higher - functioning patients ( level 4 ) , standing and walking were likely targets , each session again lasting a minimum of 10 minutes with no restricted maximum ( patient - dependent ) . importantly , passive sitting ( resting in a chair ) was not classified as a vem mobilization activity and sitting for more than 50 minutes at one time was discouraged . physiotherapists and nurses , with separate intervention targets , worked together to deliver the intervention dose . our primary outcome was a favorable outcome on the modified rankin scale ( mrs 02 ) at 3 months poststroke . secondary outcomes were time ( days ) to achieve unassisted walking over 50 meters , the proportion of patients achieving unassisted walking by 3 months , death , and the number of serious adverse events ( saes ) at 3 months . immobility - related saes ( deep venous thrombosis , pulmonary emboli , pressure sores , chest infections , urinary tract infections ) and neurologic saes ( stroke progression , recurrent stroke ) were examined separately . the dose - response analyses examine the 3 main characteristics of dose : ( 1 ) time from stroke onset to first mobilization out of bed ( ttfm , hours ) , ( 2 ) median number of out - of - bed sessions per patient per day ( frequency ) , and ( 3 ) median minutes of out - of - bed activity per patient per day ( daily amount ) . total minutes of out - of - bed activity over the intervention period ( total amount ) accounts for varying lengths of hospital stay . nurses recorded type of activity and time of the day each activity began , but not minutes , as this was not routine practice . physiotherapists recorded activity type , time the activity began , and total out - of - bed activity time ( minutes ) , consistent with their routine practice . consequently , daily amount ( minutes ) and total amount ( minutes ) of out - of - bed activity reflect physiotherapy data alone , while ttfm and frequency of mobilizations is derived from both nurse and physiotherapist data . episodes of sitting , standing , or walking activity separated from another episode of activity by > 5 minutes of rest ( e.g. , in a chair ) constituted 2 separate mobilizations . active time ( minutes ) , e.g. , practicing sit - to - stand from the chair , was recorded by the physiotherapist . these dose - response analyses repeat our major primary and secondary analyses with dose characteristics ( ttfm , frequency , daily amount , total amount ) as independent variables , and were prespecified in our statistical analysis plan . to avoid excessive collinearity between daily amount and total amount , we tested 2 separate models , adjusted for age and baseline stroke severity ( nih stroke scale [ nihss ] ) , for all analyses , as follows : model 1 : ttfm , median daily number of out - of - bed sessions ( frequency ) , median daily out - of - bed session time ( in 5-minute increments).model 2 : ttfm , median daily number of out - of - bed sessions ( frequency ) , total minutes in out - of - bed activity over the intervention period ( in 5-minute increments ) . model 1 : ttfm , median daily number of out - of - bed sessions ( frequency ) , median daily out - of - bed session time ( in 5-minute increments ) . model 2 : ttfm , median daily number of out - of - bed sessions ( frequency ) , total minutes in out - of - bed activity over the intervention period ( in 5-minute increments ) . the primary analysis , with favorable outcome ( mrs 02 ) at 3 months as the dependent variable , was conducted using binary logistic regression models . the dose effect on the odds of achieving unassisted walking by 3 months was investigated using binary logistic regression analyses ( effect sizes : adjusted odds ratios [ ors ] and 95% confidence intervals [ cis ] ) while the time ( days ) to achieve unassisted walking ( censored at 3 months ) was assessed using cox regression analyses ( adjusted hazard ratios with 95% cis ) . we analyzed mortality outcomes using binary logistic regression with death at 3 months ( mrs 6 ) as the dependent variable ( effect sizes : adjusted ors with 95% cis ) . we investigated dose effect on counts of saes using negative binomial regression ( effect sizes : adjusted incidence rate ratios with 95% cis ) . we used classification and regression tree ( cart ) advanced analysis ( salford predictive modeler software suite version 7 , salford systems , san diego , ca ) to further investigate the complex interactions between patient and dose characteristics and favorable outcome . cart is a binary partitioning statistical method that starts with the total sample and , in a stepwise manner , splits the sample into subsamples that are homogenous with respect to a defined outcome . the input variable that achieves the most effective split is dichotomized by automated analysis at an optimal threshold , maximizing the homogeneity within , and separation between , resulting subgroups . to maximize model performance ( assessed by area under the receiver operating characteristic [ roc ] curve ) , a 10-fold internal cross - validation , where data are randomly divided into 10 groups with 9 used to build the model ( training ) and 1 used to validate ( testing ) , is performed . in addition to the classification tree , cart numerically ranks each input used to build the tree by relative importance . our cart1 ( figure 1 ) analysis included all prespecified subgroup variables ( age , nihss , stroke type , recombinant tissue plasminogen activator [ rtpa ] treatment ) , group allocation , and the 3 dose characteristics ( ttfm , frequency , and daily amount ) . we explored the relative importance of each variable to achieving a favorable outcome ( mrs 02 ) . cart2 ( figure e-1 on the neurology web site at neurology.org ) was used to investigate multidimensional relationships between dose characteristics alone and favorable outcome . time to first mobilization , median daily number of out - of - bed sessions per day ( frequency ) , median daily out - of - bed activity session time ( amount ) , age ( in years ) , and stroke severity ( nih stroke scale [ nihss ] ) . patients were randomized to receive either usual care or frequent out - of - bed activity ( mobilization ) in addition to usual care ( vem ) . vem patients commenced mobilization within 24 hours of stroke and trained physiotherapy and nursing staff helped them continue task - specific out - of - bed activity , targeting recovery of active sitting , standing , and walking activity , at a frequency and intensity ( amount ) guided by an intervention protocol . functional ability at baseline , monitored daily and adjusted with recovery , guided the intervention dose with 4 titrations specified . for example , in low arousal , dependent patients ( level 1 ) , active sitting with assistance was the mobilization target , with each session lasting a minimum of 10 and maximum of 30 minutes . with higher - functioning patients ( level 4 ) , standing and walking were likely targets , each session again lasting a minimum of 10 minutes with no restricted maximum ( patient - dependent ) . importantly , passive sitting ( resting in a chair ) was not classified as a vem mobilization activity and sitting for more than 50 minutes at one time was discouraged . intervention lasted 14 days or until discharge , whichever was sooner . physiotherapists and nurses , with separate intervention targets , worked together to deliver the intervention dose . our primary outcome was a favorable outcome on the modified rankin scale ( mrs 02 ) at 3 months poststroke . secondary outcomes were time ( days ) to achieve unassisted walking over 50 meters , the proportion of patients achieving unassisted walking by 3 months , death , and the number of serious adverse events ( saes ) at 3 months . immobility - related saes ( deep venous thrombosis , pulmonary emboli , pressure sores , chest infections , urinary tract infections ) and neurologic saes ( stroke progression , recurrent stroke ) were examined separately . the dose - response analyses examine the 3 main characteristics of dose : ( 1 ) time from stroke onset to first mobilization out of bed ( ttfm , hours ) , ( 2 ) median number of out - of - bed sessions per patient per day ( frequency ) , and ( 3 ) median minutes of out - of - bed activity per patient per day ( daily amount ) . total minutes of out - of - bed activity over the intervention period ( total amount ) accounts for varying lengths of hospital stay . nurses recorded type of activity and time of the day each activity began , but not minutes , as this was not routine practice . physiotherapists recorded activity type , time the activity began , and total out - of - bed activity time ( minutes ) , consistent with their routine practice . consequently , daily amount ( minutes ) and total amount ( minutes ) of out - of - bed activity reflect physiotherapy data alone , while ttfm and frequency of mobilizations is derived from both nurse and physiotherapist data . episodes of sitting , standing , or walking activity separated from another episode of activity by > 5 minutes of rest ( e.g. , in a chair ) constituted 2 separate mobilizations . active time ( minutes ) , e.g. , practicing sit - to - stand from the chair , was recorded by the physiotherapist . these dose - response analyses repeat our major primary and secondary analyses with dose characteristics ( ttfm , frequency , daily amount , total amount ) as independent variables , and were prespecified in our statistical analysis plan . to avoid excessive collinearity between daily amount and total amount , we tested 2 separate models , adjusted for age and baseline stroke severity ( nih stroke scale [ nihss ] ) , for all analyses , as follows : model 1 : ttfm , median daily number of out - of - bed sessions ( frequency ) , median daily out - of - bed session time ( in 5-minute increments).model 2 : ttfm , median daily number of out - of - bed sessions ( frequency ) , total minutes in out - of - bed activity over the intervention period ( in 5-minute increments ) . model 1 : ttfm , median daily number of out - of - bed sessions ( frequency ) , median daily out - of - bed session time ( in 5-minute increments ) . model 2 : ttfm , median daily number of out - of - bed sessions ( frequency ) , total minutes in out - of - bed activity over the intervention period ( in 5-minute increments ) . the primary analysis , with favorable outcome ( mrs 02 ) at 3 months as the dependent variable , was conducted using binary logistic regression models . the dose effect on the odds of achieving unassisted walking by 3 months was investigated using binary logistic regression analyses ( effect sizes : adjusted odds ratios [ ors ] and 95% confidence intervals [ cis ] ) while the time ( days ) to achieve unassisted walking ( censored at 3 months ) was assessed using cox regression analyses ( adjusted hazard ratios with 95% cis ) . we analyzed mortality outcomes using binary logistic regression with death at 3 months ( mrs 6 ) as the dependent variable ( effect sizes : adjusted ors with 95% cis ) . we investigated dose effect on counts of saes using negative binomial regression ( effect sizes : adjusted incidence rate ratios with 95% cis ) . we used classification and regression tree ( cart ) advanced analysis ( salford predictive modeler software suite version 7 , salford systems , san diego , ca ) to further investigate the complex interactions between patient and dose characteristics and favorable outcome . cart is a binary partitioning statistical method that starts with the total sample and , in a stepwise manner , splits the sample into subsamples that are homogenous with respect to a defined outcome . the input variable that achieves the most effective split is dichotomized by automated analysis at an optimal threshold , maximizing the homogeneity within , and separation between , resulting subgroups . to maximize model performance ( assessed by area under the receiver operating characteristic [ roc ] curve ) , a 10-fold internal cross - validation , where data are randomly divided into 10 groups with 9 used to build the model ( training ) and in addition to the classification tree , cart numerically ranks each input used to build the tree by relative importance . our cart1 ( figure 1 ) analysis included all prespecified subgroup variables ( age , nihss , stroke type , recombinant tissue plasminogen activator [ rtpa ] treatment ) , group allocation , and the 3 dose characteristics ( ttfm , frequency , and daily amount ) . we explored the relative importance of each variable to achieving a favorable outcome ( mrs 02 ) . cart2 ( figure e-1 on the neurology web site at neurology.org ) was used to investigate multidimensional relationships between dose characteristics alone and favorable outcome . time to first mobilization , median daily number of out - of - bed sessions per day ( frequency ) , median daily out - of - bed activity session time ( amount ) , age ( in years ) , and stroke severity ( nih stroke scale [ nihss ] ) . between july 18 , 2006 , and october 16 , 2014 , we randomly assigned 2,104 patients to vem ( n = 1,054 ) and usual care ( n = 1,050 ) , with 2,083 ( 99% ) patients followed to the primary 3-month endpoint . 25% were over 80 years of age , few were disabled prior to stroke , more than 43% of patients experienced a moderate to severe stroke ( nihss > 7 ) , and 12% were diagnosed with intracerebral hemorrhage ( table 1 ) . a total of 1,584 patients ( 75% ) had no disability ( premorbid mrs 0 ) prior to stroke , a further 519 ( 24% ) had slight disability ( mrs 12 ) , and 1,833 ( 87% ) could walk without aids . the median ( interquartile range [ iqr ] ) time to first mobilization was 20.2 hours ( 14.723.8 ) , while 1,588 ( 75% ) participants commenced out - of - bed activity within 24 hours of stroke ( table 2 ) . baseline characteristics of all patients intervention summary , all patients greater ttfm was associated with a reduced odds of favorable outcome ( 0.99 , 0.981.00 , p = 0.036 ; table 3 ) . in model 1 ( favorable outcome ) , the effect of ttfm was adjusted for median daily number of sessions ( frequency ) , median daily minutes ( daily amount ) , age , and baseline nihss . in this example , the significant effect for ttfm on the odds of a favorable outcome should be interpreted as follows : for 2 patients of similar age and stroke severity , receiving a similar frequency and daily amount of out - of - bed activity , the patient who starts mobilization earlier has improved odds of a favorable outcome . effect of intervention characteristics on favorable outcome ( mrs 02 ) and unassisted walking for efficacy outcomes , favorable outcome ( mrs 02 ) and walking by 3 months , we found a similar pattern of association with each of the dose characteristics , and all associations were significant ( table 3 ) . in model 1 , ttfm , frequency , and daily amount all significantly influenced the odds of a favorable outcome . keeping ttfm and frequency constant , every extra 5 minutes of out - of - bed activity per day reduced the odds of a favorable outcome . increasing the frequency of sessions improved the odds of favorable outcome by 13% ( 95% ci 918 p < 0.001 ) and improved the odds of walking 50 meters unassisted by 66% ( 95% ci 5380 , p < 0.001 ) when ttfm and daily amount were kept constant . this pattern was similar in model 2 . when examining associations with intervention characteristics and death , increasing session frequency was the only characteristic that reduced the odds of death by approximately 20% ( table 4 ) . ttfm was not significant in any model , but frequency and amount were ( table 4 ) . given the relatively few immobility and neurologic saes , these results should be viewed with caution . effect of intervention characteristics on death and nonfatal serious adverse events in cart1 ( figure 1 ) , including ttfm , frequency and daily amount , age , nihss , stroke subtype , rtpa treatment , and randomization group , we found good to excellent performance , with a training roc of 0.78 and a testing roc of 0.77 . the relative contribution of variables to the model showed initial stroke severity ( nihss ) , a known predictor of recovery , was most important ( 100% ) , followed by session frequency ( 39.2% ) , age ( 32.4% ) , ttfm ( 10.5% ) , and daily amount ( 2.7% ) . younger patients ( 76.3 years ) and those with low nihss score ( 7.5 ) had high probability of a favorable outcome ( 78.2% ) . those with nihss score > 7.5 showed low ( 21.3% ) probability of achieving little to no disability ( mrs 02 ) . for example , at terminal node 4 ( figure 1 ) , patients between 76.3 and 86.1 years , with an nihss score > 4.5 ( but 7.5 ) , showed greater probability of a favorable outcome ( 63.6% ) if they received no more than 13.5 minutes per day distributed across frequent ( short ) sessions . frequency again split the tree for terminal nodes 5 and 6 , indicating that more frequent sessions to achieve a higher dose ( > 13.5 minutes per day ) was associated with greater odds of a good outcome . further exploration of cart1 large terminal nodes 1 ( younger age ) and 8 ( more severe stroke ) are shown in figures e-2 and e-3 . ttfm , frequency , amount , and group are all influential splitters in these models . we found a consistent pattern of association between the odds of experiencing little or no disability ( mrs 02 ) at 3 months and the intervention characteristics irrespective of treatment group . in particular , we saw 13% improvement in the odds of a favorable outcome with each additional session of out - of - bed activity per day ( keeping the time to first mobilization and daily amount constant ) . conversely , increasing the amount of time spent in out - of - bed activity , keeping the frequency and time to first mobilization constant , reduced the odds of a favorable outcome . the potentially beneficial effect of increasing the frequency of out - of - bed activity ( but not the amount ) was consistent across most of the efficacy and safety analyses . these findings begin to unpack the primary results , where we reported that vem ( very early , frequent , and higher - dose out - of - bed activity ) reduced the odds of a favorable outcome at 3 months . this dose - response analysis suggests that increased frequency of mobilization ( keeping other intervention characteristics constant ) helps reduce disability and increases the odds of walking by 3 months and reduces the odds of death . however , increasing the minutes of out - of - bed activity was more likely to result in worse outcomes . in other words , these findings indicate that short , frequent sessions may be preferable for many patients in the first weeks after stroke . addition of the cart analyses provided further support for the important influence of session frequency on outcome . even with the inclusion of patient characteristics considered strongly predictive of outcome after stroke ( such as stroke severity and age ) , intervention characteristics played an explicit and important role , defining homogenous groups of patients based on their chances of achieving the favorable outcome . indeed , in patients with more severe stroke ( nihss > 13.5 ) , a more favorable outcome was evident in those with more rather than less sessions ( figure e-3 ) . in humans and animals , there is limited discussion about the potential effect of frequency of intervention on stroke outcome . recently , bell et al . studied skilled reaching in stroke - affected mice and found that twice daily , higher - dose training accelerated recovery and improved final outcome compared to a once - a - day , lower - dose regimen . in humans , a number of systematic reviews and meta - analyses suggest a dose - response relationship , with more intensive therapy resulting in improved functional outcome after stroke . definitions for therapy intensity vary , but to date the focus has been on amount ( minutes ) rather than frequency ( repetitions of a task , or sessions per day ) . generally , inadequate reporting of therapy interventions , together with significant heterogeneity in the timing , amount , frequency , and intensity of training provided , complicates messaging of poststroke therapy , particularly in the first few weeks , where few studies exist . interestingly , a 3-arm early rehabilitation trial testing upper limb constraint regimens , although small ( n = 52 ) , also found that a higher amount of training was inferior to lower - dose usual care . a critical challenge in rehabilitation is determining who should be targeted and when and what is the optimal intervention . the influence of time to first mobilization was less clear , partly due to a compact distribution pattern with the median time ( iqr ) less than 24 hours ( 14.723.8 ) . the optimal time to commence out - of - bed activity remains unknown . while early animal studies showed that very - high - dose training within the first days poststroke increased brain lesion volume , our recent animal meta - analysis found that a shorter interval between stroke and exercise start reduced infarct volume ( effect size 0.24 , 95% ci 0.36 to 0.06 , p < 0.004 ) , without significantly influencing behavioral outcomes . a further animal systematic review showed that early initiated ( 2448 hours poststroke ) moderate exercise reduced lesion volume and protected perilesional tissue against oxidative damage and inflammation . given that animal research to date suggests that activity within 2448 hours of ischemic stroke onset may be helpful , an obvious translation gap exists . study strengths include that the dose - response analysis was prespecified to help understand our complex intervention , and we had a strong focus on the quality of the nurse and physiotherapy data collected in the trial . our main limitation is that this exploratory analysis is not an rct testing each of the intervention components separately ( time , amount , frequency ) . our results will need to be confirmed in further rcts . because we recorded physiotherapist - assisted out - of - bed therapy time only ( not nursing ) , these data underestimate the actual minutes each day that a patient spent undertaking out - of - bed activity . one could argue that the intervention protocol influenced our findings because intervention dose was greater when participants had a less severe stroke . however , given that we found the same relationship between time to first mobilization , frequency , and amount of time in the usual care group alone as found for the whole group , this seems unlikely . that is , more frequent sessions ( keeping mobilization time and median minutes of out - of - bed activity per day constant ) improved the odds of a good outcome by 12% ( or 1.12 , 95% ci 1.041.21 , p interestingly , less time to first mobilization was also associated with improved odds of a good outcome ( or 0.98 , 95% ci 0.970.99 , p = 0.002 ) , while more minutes of out - of - bed activity was not ( table e-1 ) . therefore , while the intervention protocol itself may have confounded some of this association , the results provide us with important clues on how the components of early and intensive rehabilitation affect outcome . these results provide insights into the drivers of outcome and provide clinicians with a guide to early rehabilitation practices . the first is that physiotherapist- and nurse - facilitated mobility interventions delivered in the acute phase of care can change a patient 's long - term outcomes , so it is critical that trialists carefully define and measure these interventions . second , these results suggest that the frequency of intervention may be a more important driver of outcome . the final message is that the currently accepted philosophy of more practice is always better needs to be reconsidered , particularly within the first days after stroke . the issue of timing , frequency , and amount of therapy is more complex that previously realized . j. bernhardt conceived and designed the study , wrote the first draft of the manuscript , and approved the final version . l. churilov was the study statistician who prepared the analyses , reviewed the manuscript , and provided input . the trial was supported by the national health and medical research council ( nhmrc ) of australia ( project grant nos . additional funding was received from chest heart and stroke scotland ( res08/a114 ) , northern ireland chest heart and stroke , singapore health ( shf / fg401p/2008 ) , the stroke association , uk ( tsa2009/09 ) , and the national institute of health research , uk ( grant no . the florey institute of neuroscience and mental health received support from the victorian government via the operational infrastructure support scheme . the content of the publication is solely the responsibility of the authors and does not reflect the views of the funders . j. bernhardt reports fellowship funding from the nhmrc ( 105863 ) , the australia research council ( 0991086 ) , and the national heart foundation ; nhmrc and stroke association uk project grants ; support to provide an overview of early mobilization research at a european stroke conference 2013 seminar sponsored by ever pharma neuro pharma gmbh ; and travel support to attend the european stroke conference and world stroke conference p. langhorne reports funding from chest heart and stroke scotland ( res08/a114 ) , the stroke association , uk ( tsa 2009/09 ) , and the national institute of health research , uk ( grant no : hta project 12/01/16 ) . r. lindley reports receiving compensation from boehringer ingelheim for chairing a scientific committee , lecturing honoraria from covidien and pfizer , is associate editor for the australasian journal on ageing , receives publishing royalties from 2 books , and has received various project grants from nhmrc . g. donnan reports membership on boehringer ingelheim , seriver , sanofi aventis , and bristol myer squibb scientific advisory boards and serves as editor - in - chief of international journal of stroke , as an advisory board member for stroke , journal of neuroimaging , and lancet neurology , and is an editorial board member of stroke research and treatment .

objective : our prespecified dose - response analyses of a very early rehabilitation trial ( avert ) aim to provide practical guidance for clinicians on the timing , frequency , and amount of mobilization following acute stroke.methods:eligible patients were aged 18 years , had confirmed first ( or recurrent ) stroke , and were admitted to a stroke unit within 24 hours of stroke onset . patients were randomized to receive very early and frequent mobilization , commencing within 24 hours , or usual care . we used regression analyses and classification and regression trees ( cart ) to investigate the effect of timing and dose of mobilization on efficacy and safety outcomes , irrespective of assigned treatment group.results:a total of 2,104 patients were enrolled , of whom 2,083 ( 99.0% ) were followed up at 3 months . we found a consistent pattern of improved odds of favorable outcome in efficacy and safety outcomes with increased daily frequency of out - of - bed sessions ( odds ratio [ or ] 1.13 , 95% confidence interval [ ci ] 1.09 to 1.18 , p < 0.001 ) , keeping time to first mobilization and mobilization amount constant . increased amount ( minutes per day ) of mobilization reduced the odds of a good outcome ( or 0.94 , 95% ci 0.91 to 0.97 , p < 0.001 ) . session frequency was the most important variable in the cart analysis , after prognostic variables age and baseline stroke severity.conclusion:these data suggest that shorter , more frequent mobilization early after acute stroke is associated with greater odds of favorable outcome at 3 months when controlling for age and stroke severity.classification of evidence : this study provides class iii evidence that shorter , more frequent early mobilization improves the chance of regaining independence after stroke .

Classification of evidence: METHODS Standard protocol approvals, registrations, and patient consents. Procedures. Outcome measures. Dose measures. Statistical analysis. RESULTS DISCUSSION Supplementary Material AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

this study provides class iii evidence that shorter , more frequent early mobilization improves the chance of regaining independence after stroke . patients with ischemic or hemorrhagic stroke , admitted within 24 hours of onset , were eligible . patients were randomized to receive either usual care or frequent out - of - bed activity ( mobilization ) in addition to usual care ( vem ) . vem patients commenced mobilization within 24 hours of stroke and trained physiotherapy and nursing staff helped them continue task - specific out - of - bed activity , targeting recovery of active sitting , standing , and walking activity , at a frequency and intensity ( amount ) guided by an intervention protocol . our primary outcome was a favorable outcome on the modified rankin scale ( mrs 02 ) at 3 months poststroke . secondary outcomes were time ( days ) to achieve unassisted walking over 50 meters , the proportion of patients achieving unassisted walking by 3 months , death , and the number of serious adverse events ( saes ) at 3 months . the dose - response analyses examine the 3 main characteristics of dose : ( 1 ) time from stroke onset to first mobilization out of bed ( ttfm , hours ) , ( 2 ) median number of out - of - bed sessions per patient per day ( frequency ) , and ( 3 ) median minutes of out - of - bed activity per patient per day ( daily amount ) . total minutes of out - of - bed activity over the intervention period ( total amount ) accounts for varying lengths of hospital stay . physiotherapists recorded activity type , time the activity began , and total out - of - bed activity time ( minutes ) , consistent with their routine practice . consequently , daily amount ( minutes ) and total amount ( minutes ) of out - of - bed activity reflect physiotherapy data alone , while ttfm and frequency of mobilizations is derived from both nurse and physiotherapist data . these dose - response analyses repeat our major primary and secondary analyses with dose characteristics ( ttfm , frequency , daily amount , total amount ) as independent variables , and were prespecified in our statistical analysis plan . to avoid excessive collinearity between daily amount and total amount , we tested 2 separate models , adjusted for age and baseline stroke severity ( nih stroke scale [ nihss ] ) , for all analyses , as follows : model 1 : ttfm , median daily number of out - of - bed sessions ( frequency ) , median daily out - of - bed session time ( in 5-minute increments).model 2 : ttfm , median daily number of out - of - bed sessions ( frequency ) , total minutes in out - of - bed activity over the intervention period ( in 5-minute increments ) . model 1 : ttfm , median daily number of out - of - bed sessions ( frequency ) , median daily out - of - bed session time ( in 5-minute increments ) . model 2 : ttfm , median daily number of out - of - bed sessions ( frequency ) , total minutes in out - of - bed activity over the intervention period ( in 5-minute increments ) . the primary analysis , with favorable outcome ( mrs 02 ) at 3 months as the dependent variable , was conducted using binary logistic regression models . the dose effect on the odds of achieving unassisted walking by 3 months was investigated using binary logistic regression analyses ( effect sizes : adjusted odds ratios [ ors ] and 95% confidence intervals [ cis ] ) while the time ( days ) to achieve unassisted walking ( censored at 3 months ) was assessed using cox regression analyses ( adjusted hazard ratios with 95% cis ) . we used classification and regression tree ( cart ) advanced analysis ( salford predictive modeler software suite version 7 , salford systems , san diego , ca ) to further investigate the complex interactions between patient and dose characteristics and favorable outcome . our cart1 ( figure 1 ) analysis included all prespecified subgroup variables ( age , nihss , stroke type , recombinant tissue plasminogen activator [ rtpa ] treatment ) , group allocation , and the 3 dose characteristics ( ttfm , frequency , and daily amount ) . cart2 ( figure e-1 on the neurology web site at neurology.org ) was used to investigate multidimensional relationships between dose characteristics alone and favorable outcome . time to first mobilization , median daily number of out - of - bed sessions per day ( frequency ) , median daily out - of - bed activity session time ( amount ) , age ( in years ) , and stroke severity ( nih stroke scale [ nihss ] ) . patients were randomized to receive either usual care or frequent out - of - bed activity ( mobilization ) in addition to usual care ( vem ) . vem patients commenced mobilization within 24 hours of stroke and trained physiotherapy and nursing staff helped them continue task - specific out - of - bed activity , targeting recovery of active sitting , standing , and walking activity , at a frequency and intensity ( amount ) guided by an intervention protocol . for example , in low arousal , dependent patients ( level 1 ) , active sitting with assistance was the mobilization target , with each session lasting a minimum of 10 and maximum of 30 minutes . our primary outcome was a favorable outcome on the modified rankin scale ( mrs 02 ) at 3 months poststroke . secondary outcomes were time ( days ) to achieve unassisted walking over 50 meters , the proportion of patients achieving unassisted walking by 3 months , death , and the number of serious adverse events ( saes ) at 3 months . the dose - response analyses examine the 3 main characteristics of dose : ( 1 ) time from stroke onset to first mobilization out of bed ( ttfm , hours ) , ( 2 ) median number of out - of - bed sessions per patient per day ( frequency ) , and ( 3 ) median minutes of out - of - bed activity per patient per day ( daily amount ) . total minutes of out - of - bed activity over the intervention period ( total amount ) accounts for varying lengths of hospital stay . physiotherapists recorded activity type , time the activity began , and total out - of - bed activity time ( minutes ) , consistent with their routine practice . consequently , daily amount ( minutes ) and total amount ( minutes ) of out - of - bed activity reflect physiotherapy data alone , while ttfm and frequency of mobilizations is derived from both nurse and physiotherapist data . these dose - response analyses repeat our major primary and secondary analyses with dose characteristics ( ttfm , frequency , daily amount , total amount ) as independent variables , and were prespecified in our statistical analysis plan . to avoid excessive collinearity between daily amount and total amount , we tested 2 separate models , adjusted for age and baseline stroke severity ( nih stroke scale [ nihss ] ) , for all analyses , as follows : model 1 : ttfm , median daily number of out - of - bed sessions ( frequency ) , median daily out - of - bed session time ( in 5-minute increments).model 2 : ttfm , median daily number of out - of - bed sessions ( frequency ) , total minutes in out - of - bed activity over the intervention period ( in 5-minute increments ) . model 1 : ttfm , median daily number of out - of - bed sessions ( frequency ) , median daily out - of - bed session time ( in 5-minute increments ) . model 2 : ttfm , median daily number of out - of - bed sessions ( frequency ) , total minutes in out - of - bed activity over the intervention period ( in 5-minute increments ) . the primary analysis , with favorable outcome ( mrs 02 ) at 3 months as the dependent variable , was conducted using binary logistic regression models . the dose effect on the odds of achieving unassisted walking by 3 months was investigated using binary logistic regression analyses ( effect sizes : adjusted odds ratios [ ors ] and 95% confidence intervals [ cis ] ) while the time ( days ) to achieve unassisted walking ( censored at 3 months ) was assessed using cox regression analyses ( adjusted hazard ratios with 95% cis ) . we analyzed mortality outcomes using binary logistic regression with death at 3 months ( mrs 6 ) as the dependent variable ( effect sizes : adjusted ors with 95% cis ) . we used classification and regression tree ( cart ) advanced analysis ( salford predictive modeler software suite version 7 , salford systems , san diego , ca ) to further investigate the complex interactions between patient and dose characteristics and favorable outcome . our cart1 ( figure 1 ) analysis included all prespecified subgroup variables ( age , nihss , stroke type , recombinant tissue plasminogen activator [ rtpa ] treatment ) , group allocation , and the 3 dose characteristics ( ttfm , frequency , and daily amount ) . cart2 ( figure e-1 on the neurology web site at neurology.org ) was used to investigate multidimensional relationships between dose characteristics alone and favorable outcome . time to first mobilization , median daily number of out - of - bed sessions per day ( frequency ) , median daily out - of - bed activity session time ( amount ) , age ( in years ) , and stroke severity ( nih stroke scale [ nihss ] ) . between july 18 , 2006 , and october 16 , 2014 , we randomly assigned 2,104 patients to vem ( n = 1,054 ) and usual care ( n = 1,050 ) , with 2,083 ( 99% ) patients followed to the primary 3-month endpoint . 25% were over 80 years of age , few were disabled prior to stroke , more than 43% of patients experienced a moderate to severe stroke ( nihss > 7 ) , and 12% were diagnosed with intracerebral hemorrhage ( table 1 ) . a total of 1,584 patients ( 75% ) had no disability ( premorbid mrs 0 ) prior to stroke , a further 519 ( 24% ) had slight disability ( mrs 12 ) , and 1,833 ( 87% ) could walk without aids . the median ( interquartile range [ iqr ] ) time to first mobilization was 20.2 hours ( 14.723.8 ) , while 1,588 ( 75% ) participants commenced out - of - bed activity within 24 hours of stroke ( table 2 ) . baseline characteristics of all patients intervention summary , all patients greater ttfm was associated with a reduced odds of favorable outcome ( 0.99 , 0.981.00 , p = 0.036 ; table 3 ) . in model 1 ( favorable outcome ) , the effect of ttfm was adjusted for median daily number of sessions ( frequency ) , median daily minutes ( daily amount ) , age , and baseline nihss . in this example , the significant effect for ttfm on the odds of a favorable outcome should be interpreted as follows : for 2 patients of similar age and stroke severity , receiving a similar frequency and daily amount of out - of - bed activity , the patient who starts mobilization earlier has improved odds of a favorable outcome . effect of intervention characteristics on favorable outcome ( mrs 02 ) and unassisted walking for efficacy outcomes , favorable outcome ( mrs 02 ) and walking by 3 months , we found a similar pattern of association with each of the dose characteristics , and all associations were significant ( table 3 ) . in model 1 , ttfm , frequency , and daily amount all significantly influenced the odds of a favorable outcome . keeping ttfm and frequency constant , every extra 5 minutes of out - of - bed activity per day reduced the odds of a favorable outcome . increasing the frequency of sessions improved the odds of favorable outcome by 13% ( 95% ci 918 p < 0.001 ) and improved the odds of walking 50 meters unassisted by 66% ( 95% ci 5380 , p < 0.001 ) when ttfm and daily amount were kept constant . when examining associations with intervention characteristics and death , increasing session frequency was the only characteristic that reduced the odds of death by approximately 20% ( table 4 ) . effect of intervention characteristics on death and nonfatal serious adverse events in cart1 ( figure 1 ) , including ttfm , frequency and daily amount , age , nihss , stroke subtype , rtpa treatment , and randomization group , we found good to excellent performance , with a training roc of 0.78 and a testing roc of 0.77 . the relative contribution of variables to the model showed initial stroke severity ( nihss ) , a known predictor of recovery , was most important ( 100% ) , followed by session frequency ( 39.2% ) , age ( 32.4% ) , ttfm ( 10.5% ) , and daily amount ( 2.7% ) . younger patients ( 76.3 years ) and those with low nihss score ( 7.5 ) had high probability of a favorable outcome ( 78.2% ) . for example , at terminal node 4 ( figure 1 ) , patients between 76.3 and 86.1 years , with an nihss score > 4.5 ( but 7.5 ) , showed greater probability of a favorable outcome ( 63.6% ) if they received no more than 13.5 minutes per day distributed across frequent ( short ) sessions . frequency again split the tree for terminal nodes 5 and 6 , indicating that more frequent sessions to achieve a higher dose ( > 13.5 minutes per day ) was associated with greater odds of a good outcome . ttfm , frequency , amount , and group are all influential splitters in these models . we found a consistent pattern of association between the odds of experiencing little or no disability ( mrs 02 ) at 3 months and the intervention characteristics irrespective of treatment group . in particular , we saw 13% improvement in the odds of a favorable outcome with each additional session of out - of - bed activity per day ( keeping the time to first mobilization and daily amount constant ) . conversely , increasing the amount of time spent in out - of - bed activity , keeping the frequency and time to first mobilization constant , reduced the odds of a favorable outcome . the potentially beneficial effect of increasing the frequency of out - of - bed activity ( but not the amount ) was consistent across most of the efficacy and safety analyses . these findings begin to unpack the primary results , where we reported that vem ( very early , frequent , and higher - dose out - of - bed activity ) reduced the odds of a favorable outcome at 3 months . this dose - response analysis suggests that increased frequency of mobilization ( keeping other intervention characteristics constant ) helps reduce disability and increases the odds of walking by 3 months and reduces the odds of death . however , increasing the minutes of out - of - bed activity was more likely to result in worse outcomes . in other words , these findings indicate that short , frequent sessions may be preferable for many patients in the first weeks after stroke . even with the inclusion of patient characteristics considered strongly predictive of outcome after stroke ( such as stroke severity and age ) , intervention characteristics played an explicit and important role , defining homogenous groups of patients based on their chances of achieving the favorable outcome . indeed , in patients with more severe stroke ( nihss > 13.5 ) , a more favorable outcome was evident in those with more rather than less sessions ( figure e-3 ) . in humans and animals , there is limited discussion about the potential effect of frequency of intervention on stroke outcome . in humans , a number of systematic reviews and meta - analyses suggest a dose - response relationship , with more intensive therapy resulting in improved functional outcome after stroke . definitions for therapy intensity vary , but to date the focus has been on amount ( minutes ) rather than frequency ( repetitions of a task , or sessions per day ) . generally , inadequate reporting of therapy interventions , together with significant heterogeneity in the timing , amount , frequency , and intensity of training provided , complicates messaging of poststroke therapy , particularly in the first few weeks , where few studies exist . interestingly , a 3-arm early rehabilitation trial testing upper limb constraint regimens , although small ( n = 52 ) , also found that a higher amount of training was inferior to lower - dose usual care . the influence of time to first mobilization was less clear , partly due to a compact distribution pattern with the median time ( iqr ) less than 24 hours ( 14.723.8 ) . the optimal time to commence out - of - bed activity remains unknown . while early animal studies showed that very - high - dose training within the first days poststroke increased brain lesion volume , our recent animal meta - analysis found that a shorter interval between stroke and exercise start reduced infarct volume ( effect size 0.24 , 95% ci 0.36 to 0.06 , p < 0.004 ) , without significantly influencing behavioral outcomes . study strengths include that the dose - response analysis was prespecified to help understand our complex intervention , and we had a strong focus on the quality of the nurse and physiotherapy data collected in the trial . because we recorded physiotherapist - assisted out - of - bed therapy time only ( not nursing ) , these data underestimate the actual minutes each day that a patient spent undertaking out - of - bed activity . however , given that we found the same relationship between time to first mobilization , frequency , and amount of time in the usual care group alone as found for the whole group , this seems unlikely . that is , more frequent sessions ( keeping mobilization time and median minutes of out - of - bed activity per day constant ) improved the odds of a good outcome by 12% ( or 1.12 , 95% ci 1.041.21 , p interestingly , less time to first mobilization was also associated with improved odds of a good outcome ( or 0.98 , 95% ci 0.970.99 , p = 0.002 ) , while more minutes of out - of - bed activity was not ( table e-1 ) . the first is that physiotherapist- and nurse - facilitated mobility interventions delivered in the acute phase of care can change a patient 's long - term outcomes , so it is critical that trialists carefully define and measure these interventions . second , these results suggest that the frequency of intervention may be a more important driver of outcome . the issue of timing , frequency , and amount of therapy is more complex that previously realized . additional funding was received from chest heart and stroke scotland ( res08/a114 ) , northern ireland chest heart and stroke , singapore health ( shf / fg401p/2008 ) , the stroke association , uk ( tsa2009/09 ) , and the national institute of health research , uk ( grant no . j. bernhardt reports fellowship funding from the nhmrc ( 105863 ) , the australia research council ( 0991086 ) , and the national heart foundation ; nhmrc and stroke association uk project grants ; support to provide an overview of early mobilization research at a european stroke conference 2013 seminar sponsored by ever pharma neuro pharma gmbh ; and travel support to attend the european stroke conference and world stroke conference p. langhorne reports funding from chest heart and stroke scotland ( res08/a114 ) , the stroke association , uk ( tsa 2009/09 ) , and the national institute of health research , uk ( grant no : hta project 12/01/16 ) . g. donnan reports membership on boehringer ingelheim , seriver , sanofi aventis , and bristol myer squibb scientific advisory boards and serves as editor - in - chief of international journal of stroke , as an advisory board member for stroke , journal of neuroimaging , and lancet neurology , and is an editorial board member of stroke research and treatment .

genome editing has recently emerged as a powerful technique that allows any chosen gene to be precisely modified in a predetermined way.1 , 2 the cre - loxp system is a popular and widely used site - specific genetic manipulation tool to conditionally knockout specific genes in cell lines and animal models , which modulates expression of selected genes at a certain developmental stage or in specific tissues , greatly facilitating our understanding of gene function and developmental mechanisms3 , 4 , 5 , 6 , 7 , 8 as well as flp / frt . cre recombinase originates from p1 phage and recognizes the 34-bp loxp site ( 5ataacttcgtataatgtatgctatacgaagttat-3 ) , whereas flp recombinase originates from the yeast 2-m plasmid and recognizes the distinct 34-bp frt site ( 5-gaagttcctattctctagaaagtataggaacttc-3 ) . both cre and flp recombinase can excise a region of dna surrounded by two loxp or frt sites , respectively . at the present time , the choice of tools for site - specific genetic manipulation is limited to just cre - loxp , flp - frt , phic31/attp , or attb and the dre recombinase / rox.8 , 9 , 10 additional genetic tools would be of benefit in the genetic modification of cell lines , especially to achieve genetic modification of both alleles . in vivo , double - allele knock out can be obtained by the mating of single - allele knockout animals . however , this strategy is not practical for cell lines . for this reason , new site - specific genetic manipulation tools are crispr / cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research . crispr / cas9 technology has been widely applied for functional genomic studies in a variety of organisms , including mouse and human cells.12 , 13 it can be programmed by single - guide rnas ( sgrnas ) to cleave specific genomic loci complementary to the sgrna with a downstream protospacer adjacent motif ( pam ) , where cas9 creates double - stranded dna breaks ( dsbs ) . these dsbs mediate error - prone non - homologous end - joining ( nhej ) or precise homologous recombination ( hr ) . the most widely used customized crispr / cas9 ( spcas9 ) is derived from streptococcus pyogenes , and the corresponding pam sequence is 5-ngg-3 , where n is any base . whether the traditional site - specific genetic manipulation tool and crispr technologies can be combined as a novel genetic tool for specific gene knockout studies is not clear ( figure s1 ) . to address this question , we took advantage of the double fluorescent reporter systems with crispr / cas9 because it is easily detectable by flow cytometry and microscopy . this approach was used to test the efficiency of a crispr / cas9-loxp system in human cells . to develop a simple reporter system for visualizing crispr / spcas9-loxp effects , we used the mt / mg system , with the structure cag(promoter)-loxp - mtomato(mt)-loxp - poly(a)- egfp ( mgreen , mg)-poly(a ) ( figure 1a ) . we hypothesized that if we introduced two crispr / cas9-mediated dna double strands break via targeting loxp flanking the mtomato cassette , the expression of the egfp gene will be directly driven by the cag promoter . because there are two identical loxp sequences flanking the mtomato cassette , one sgrna would be able to target them . however , loxp sites do not contain an ngg pam sequence , and the additional tagg sequence at the 3 end of the loxp ( 5-ataacttcgtataatgtatgctatacgaagttattagg-3 ) , named extended loxp ( eloxp ) , was created ( figure 1a ; table s1 ) . thus , the reformed loxp with the addition of an ngg pam sequence can be recognized by crispr /spcas9 . as we expected , co - transfection of two plasmids ( px330- eloxp expressing cas9 and sgrnas to target eloxp and pmtmg ) in human hek293 cells indeed led to egfp expression ( figure 1c ; the schematic of the protocol is shown in figure 1b ) , which implied that the crispr / spcas9-eloxp system can be used to excise the mtomato gene and allow the cag promoter to drive the expression of the egfp gene directly . specially , in the crispr / spcas9-eloxp group , 56.0%63.6% of cells were egfp - positive cells ( figures 1c , 1d , and s2 ) . pcr was then used to confirm the nhej events triggered appropriately by crispr / spcas9-eloxp ( figure 1e).figure 1performance of crispr / spcas9-eloxp system in transient reporter - gene expression assays(a ) schematic of the mt / mg cassette ( loxp - mt - pa - loxp - mg - pa ) before and after crispr / spcas9-eloxp - mediated recombination . mt / mg consists of a promoter driving a loxp - flanked coding sequence of membrane - targeted tandem dimer tomato ( mt ) , resulting in mtomato expression with membrane localization . after crispr / spcas9-eloxp - mediated recombination , the mtomato sequence is excised , allowing the promoter to drive expression of membrane - targeted egfp ( mg ) . ( b ) an illustration of the crispr / spcas9-eloxp system in transient reporter - gene expression assays . ( c ) hek293 cells were co - transfected with pmtmg and crispr / spcas9-eloxp ( 0.75 ug each ) , and images were obtained at 48 hr post transfection ( red indicates mt ; green indicates mg ) . ( d ) the level of fluorescence was quantified ( red indicates mt ; green indicates mg ) . the p value was calculated by student s t test ; * p < 0.05 . ( e ) performance of crispr / spcas9-eloxp system in transient reporter - gene expression assays ( a ) schematic of the mt / mg cassette ( loxp - mt - pa - loxp - mg - pa ) before and after crispr / spcas9-eloxp - mediated recombination . mt / mg consists of a promoter driving a loxp - flanked coding sequence of membrane - targeted tandem dimer tomato ( mt ) , resulting in mtomato expression with membrane localization . after crispr / spcas9-eloxp - mediated recombination , the mtomato sequence is excised , allowing the promoter to drive expression of membrane - targeted egfp ( mg ) . ( b ) an illustration of the crispr / spcas9-eloxp system in transient reporter - gene expression assays . ( c ) hek293 cells were co - transfected with pmtmg and crispr / spcas9-eloxp ( 0.75 ug each ) , and images were obtained at 48 hr post transfection ( red indicates mt ; green indicates mg ) . ( d ) the level of fluorescence was quantified ( red indicates mt ; green indicates mg ) . the p value was calculated by student s t test ; * p < 0.05 . to gain insights into the occurrence of the nhej events , we mapped the sequences of crispr / cas9-eloxp - mediated nhej products . comparing one loxp sequence left in the cre group , notably , there was one dominant sequence ( named 3 t ins , 14/25 , 56% ) in the crispr / cas9-eloxp group , which comprises the insertion of one additional t base 3 nt upstream of the pam ( figure 1e ) . other fusion sites included one loxp sequence , an additional gt at 3 nt upstream of the pam as well as 1- , 2- , 3- , 9- , 12- , and 14-bp multiple variable deletions at lower frequencies ( figure 1e ) . taken together , with plasmids ( transient reporter - gene expression ) , our data showed that crispr / cas9-eloxp system - based gene editing can achieve high excision efficiency of a specific gene between two loxp sites . because our system showed high excision efficiency on the specific gene between two loxp sites at the plasmid co - transfection level , we wondered whether it would also be functional at human chromosomal loci . to address this , we sought to generate a double - fluorescent reporter system at the human aavs1 locus . aavs1 ( also known as the ppp1r12c locus ) on human chromosome 19 is a well - validated safe harbor for hosting dna transgenes.17 , 18 , 19 it has an open chromatin structure and contains native insulators that prevent the integrated genes silencing . most importantly , there are no known adverse effects on cells as a result of dna fragment insertion . for the above reasons , we selected aavs1 as a targeting site for harboring the double - fluorescent reporter system . we designed one sgrna targeting the aavs1 locus to introduce dsbs and the pmtmg plasmid , which carries two arms homologous to the aavs1 locus , as the donor vector to knock in a double - fluorescent reporter gene at the human aavs1 locus ( figures 2a and 2b ) . to screen positive colonies , which maintain robust mtomato gene expression under the microscope ( figure 2c ) , we initially picked colonies that showed red fluorescence under the microscope . then , we re - picked the colonies among the red fluorescent candidates and assumed that most random integration events will lose the red fluorescent signal , whereas the knockin cell line would maintain red fluorescence . after six passages of the re - picked candidate colonies , we screened them with gene - specific pcr ( figure 2d ; table s2 ) . because one primer anneals to sequences located outside of the homologous arm , all the positive colonies should contain the homologous recombinant knockin gene . the four pcr - negative colonies should be due to random integration of the cassette , even if it has mtomato gene expression , which would not be suitable for further experiments.figure 2generation of a double - fluorescent reporter system at the human aavs1 locus(a ) human aavs1 locus knockin via crispr / cas9 . ( c ) images of the cells for picking up positive clones that specifically integrated mt / mg cassette into the aavs1 locus at chromosome 19 . from the top to the bottom , it shows the stable knockin colonies were generated . ( d ) genotyping of the mt / mg cassette knockin candidate colonies with f3/r3 primers . generation of a double - fluorescent reporter system at the human aavs1 locus ( a ) human aavs1 locus knockin via crispr / cas9 . ( c ) images of the cells for picking up positive clones that specifically integrated mt / mg cassette into the aavs1 locus at chromosome 19 . from the top to the bottom ( d ) genotyping of the mt / mg cassette knockin candidate colonies with f3/r3 primers . for further studies , one of the positive colonies was named 293-aavs - mtmg , which has red fluorescence and also has a clean homogeneous genetic background . 293-aavs - mtmg should only be a monoallelic , but not biallelic , knockin cell line because we could amplify the fragment of the wild - type aavs1 allele with the expected size , which indicates there is one intact wild - type aavs1 allele ( figure s3 ) . the plasmids for the crispr / spcas9 expression system targeting eloxp were transfected into 293-aavs - mtmg , and the level of effective genome editing was measured by identifying egfp - positive cells with flow cytometry ( figure 3a ) . specifically , transfection of 0.5 , 1.5 , and 2.5 g of plasmid per well of a six - well plate achieved approximately 15.1% , 20.4% , and 24.5% recombination efficiency ( figures 3b , 3c , and s4 ) , respectively . dna sequence chromatograms confirmed the occurrence of nhej in these cells ( figure 3d ) . these results showed that crispr / spcas9-eloxp - mediated site - specific genome editing has been achieved at the human aavs1 locus.figure 3crispr / spcas9-eloxp - mediated site - specific gene editing in 293-aavs - mtmg cells(a ) an illustration of the crispr / spcas9-eloxp system that mediated site - specific gene editing in 293-aavs - mtmg cells . ( b ) 293-aavs - mtmg cells were transfected with 2.0 ug plasmids ( plasmids coding for cre or crispr / spcas9-eloxp ) , and images were obtained at 48 hr post - transfection ( red indicates mt ; green indicates mg ) . the p value was calculated by student s t test ; * * p < 0.01 . ( d ) targeting dna sequence chromatograms of cells transfected with crispr / spcas9-eloxp are clustered together compared with one loxp remaining in the cre group . ( e ) crispr / spcas9-eloxp - mediated nhej pattern at the human aavs1 locus . crispr / spcas9-eloxp - mediated site - specific gene editing in 293-aavs - mtmg cells ( a ) an illustration of the crispr / spcas9-eloxp system that mediated site - specific gene editing in 293-aavs - mtmg cells . ( b ) 293-aavs - mtmg cells were transfected with 2.0 ug plasmids ( plasmids coding for cre or crispr / spcas9-eloxp ) , and images were obtained at 48 hr post - transfection ( red indicates mt ; green indicates mg ) . the p value was calculated by student s t test ; * * p < 0.01 . ( d ) targeting dna sequence chromatograms of cells transfected with crispr / spcas9-eloxp are clustered together compared with one loxp remaining in the cre group . ( e ) crispr / spcas9-eloxp - mediated nhej pattern at the human aavs1 locus . we then sought to ask whether there was the same nhej pattern between the plasmid - based nhejs and reporter genes at the human aavs1 locus . after transfection and isolation of the corresponding genomic dna from 293-aavs - mtmg cells , fragments harboring nhej sites were amplified and sequenced . surprisingly , at the human aavs1 locus , we did not identify the fusion sequence ( one intact loxp sequence ) , which was present at the group of plasmid - based nhejs ( figures 3e and s5 ) . the recurrent dominant sequence ( 3 t ins ) was with the insertion of an additional t base 3 nt upstream of the pam and was detected at high frequency ( 8/25 , 32% ) . also , 107- , 3- , and 2-bp deletions were the major nhejs in addition to 3 t ins ( figure 3e ) . notably , the majority ( 64% ) of the nhejs have deletion at the human aavs1 locus , whereas the majority ( 64% ) of plasmid ( transient gene expression)-based nhejs have insertion . these results implied that the target sequence under a different environment ( plasmid and at the chromosome ) may affect the formation of the nhej pattern of crispr / spcas9 . our previous study clearly demonstrated that nag may not be the universally predominant non - canonical pam for crispr / cas9-mediated dna cleavage in human cells .. we also tested nga or nag pam , and the results showed that crispr / cas9-loxp with nga non - canonical pam , but not nag , could achieve relative highly efficient ( 10% ) excision of a specific gene between two loxp sites ( figure s6 ) . recently , a novel type of cas9 ( sacas9 ) was derived from staphylococcus aureus , and it can edit the genome with efficiencies similar to those of spcas9 , despite being 1 kb shorter . its smaller size allows packaging into a single aav vector , with its sgrna expression cassette , for in vivo genome editing . nngrr(t ) , and the corresponding eloxp sequence is ataacttcgtataatgtatgctatacgaagttat attaagggt ( figure 4a).figure 4crispr / sacas9-eloxp - mediated site - specific gene editing in 293-aavs - mtmg cells(a ) targeting sequence and corresponding pams for crispr / spcas9 ( px330 ) and crispr / sacas9 ( px601 ) . the p value was calculated by student s t test ; * p < 0.05 ; * * p < 0.01 . ( c ) crispr / sacas9-eloxp - mediated nhej pattern at the human aavs1 locus . ( d ) crispr / sacas9-eloxp - mediated nhej pattern in transient reporter - gene expression assays . crispr / sacas9-eloxp - mediated site - specific gene editing in 293-aavs - mtmg cells ( a ) targeting sequence and corresponding pams for crispr / spcas9 ( px330 ) and crispr / sacas9 ( px601 ) . the p value was calculated by student s t test ; * p < 0.05 ; * * p < 0.01 . ( c ) crispr / sacas9-eloxp - mediated nhej pattern at the human aavs1 locus . ( d ) crispr / sacas9-eloxp - mediated nhej pattern in transient reporter - gene expression assays . because site - specific genome editing is very important in vivo , here , we sought to test the performance of the crispr / sacas9-eloxp - mediated novel site - specific genome editing in 293-aavs - mtmg cells . we observed a relatively high efficiency of crispr / sacas9-eloxp - mediated genome editing ( 27.6%31.4% versus 19.5%24.9% ; figures 4b and s7 ) . also , we found co - transfection of crispr / cas9- and pmtmg - based gene editing could achieve a higher efficiency comparing genome editing at the aavs1 locus ( figure 4b ) . like the crispr / spcas9-eloxp at aavs1 locus , the majority ( 64% ) of the fusion site ( nhejs ) has deletion , including 2- to 37-bp deletion ( figures 4c and s8 ) . the results of the nhej patterns of transient crispr / sacas9-eloxp expression showed that the minority ( 8% ) of the fusion site is deletion , which is different from that of the crispr / sacas9-eloxp at aavs1 locus ( 64% of the fusion sites have deletions ) . taken together , our study demonstrated that crispr / cas9-loxp ( sa cas9 or sp cas9 ) could be used as a tool to perform site - specific genome editing at the human aavs1 locus . meanwhile , crispr / cas9-mediated nhej patterns may be modulated by the environment of targeting sequence . off - target effects of crispr / cas9 remain a key issue for its application in genome editing , including pam - related off - target effects.20 , 22 in cell lines , it is not practical to re - correct the off - target effects , although these could be minimized through outcrossing in animals or plants . new strategies using double nickase ( dn ) and foki - dcas9 have been proposed,23 , 24 but the presence of off - target effects due to cas9/sgrna may still exist . in the present study , we sought to know the off - targets of crispr / cas9-eloxp . with online software ( http://www.rgenome.net/cas-offinder/ ) , we predicted the potential off - target sites in the human genome . 20 fragments harboring potential off - target sites for crispr / sacas9-eloxp and crispr / spcas9-eloxp have been amplified and sequenced ( tables s3 and s4 ) . compared with the parental cells , no additional multi - peaks in the chromatogram have been observed around the potential off - target sites ( table s4 ) . this indicated that there are non - detectable off - target effects of crispr / cas9-eloxp by sanger sequencing . further studies also need to characterize additional potential genome modifications inducing off - target crispr / cas9-eloxp with a genome analysis tool , i.e. , whole genome sequencing . meanwhile , it is meaningful to screen and identify specific cas9 mutants , which are optimized for genome engineering to minimize the off - target effects . recently , novel high - fidelity cas9s have been reported , which may strengthen the present novel crispr / cas9-eloxp tool.26 , 27 in the present study , we successfully generated a crispr / cas9-loxp system to perform site - specific genome editing in human cells , at the level of both plasmids ( episomal ) and chromosomes . it provides the proof of principle that these two technologies ( traditional site - specific genetic manipulation tool and crispr technologies ) can be used together . we also showed that distinct nhej patterns from crispr / cas9-mediated gene editing of the targeting sequence locates at the level of plasmids ( episomal ) and chromosomes . although crispr / cas9-eloxp - based knockout results are certainly encouraging , improvements still need be made . for example , if we place a specifically mutated loxp flanking the targeting gene , which cre can not recognize , with the specific sgrna - targeting mutated loxp , it could be used to perform conditional knockouts , and thus could be an attractive alternative genetic tool for the compensation of cre - loxp . the same strategy may be applied for the flp - frt or even two identical sequences at different locations in the genome . in the present study , we used eloxp for crispr / cas9 . there are also two loxp mutants , loxp66 and loxp71 , which both have ngg sequences or nngrr(t ) ( figure 5a ) . thus , these two additional loxp mutants may be more suitable for crispr / cas9-loxp - mediated genome editing . also , the wild - type and loxp mutants could be combined and the specific gene flanking them could be excised . we propose that additional crispr / sacas9-loxp genetic tools could be developed ( figure 5b ) . for example , as illustrated in figure 5b , genes a d were flanked by different loxp sites . with cre - mediated gene recombination , genes a and c could be excised . additional sgrnas targeting loxp or its mutants could allow different genes to be selectively excised or activated . meanwhile , tissue - specific or inducible promoter could be used to drive cas9 expression for conditional gene knockouts for animal models and in human embryonic stem cell or induced pluripotent stem cell studies.29 , 30 these tools will be useful for conditional knockout and studies of gene - gene interaction.figure 5crispr / cas9-loxp - mediated gene editing as a novel site - specific genetic manipulation tool(a ) schematics illustrating the wild loxp and mutant loxp pair lox66/lox71 sequence and the position of the guide rna . ( b ) schematic strategy of the switch gene expression employing the crispr / cas9-loxp system . ( c ) distinct nhej patterns from crispr / cas9-mediated gene editing of the targeting sequence located at the level of plasmids ( episomal ) and chromosomes . crispr / cas9-loxp - mediated gene editing as a novel site - specific genetic manipulation tool ( a ) schematics illustrating the wild loxp and mutant loxp pair lox66/lox71 sequence and the position of the guide rna . ( b ) schematic strategy of the switch gene expression employing the crispr / cas9-loxp system . ( c ) distinct nhej patterns from crispr / cas9-mediated gene editing of the targeting sequence located at the level of plasmids ( episomal ) and chromosomes . we also showed that there is a difference between the crispr / cas9-mediated nhej patterns of the targeting sequence located at the level of plasmids ( episomal ) and chromosomes . in particular , the crispr / cas9-mediated nhej pattern in the nuclear genome is in favor of deletions ( 64%68% at the human aavs1 locus versus 8%28% plasmid dna ) . we speculate that the difference between these two groups may be due to one of the following mechanisms ( figure 5c ) . first , the nuclear genome and plasmid dna exist in different genetic environments , with the nuclear genome heavily regulated by histones and other dna - interacting proteins , whereas the non - nuclear genome , including plasmids as episomes , are naked , with very few proteins binding . second , different nhej - related enzymes may be involved or they may be at different concentrations in the two scenarios . this study may provide insights for nhej - related dna repair and provides a platform for the comparison of chromosomal and extra - chromosomal dna repair . it is challenging but of interest to compare crispr / cas9 editing of the same dna sequence integrated into the nuclear genome ( at the chromosomal level ) and extra - chromosomal dna ( such as plasmids transiently existing in the cells ) . here , we demonstrated that with the same dna target sequence and crispr / cas9 , in the same cell type , extra - chromosomal genome crispr / cas9-based gene editing could achieve a high efficiency compared with gene editing at chromosomes ( 55.0%69.0% versus 19.5%31.4% at the aavs1 locus ) . we rationalize the results from these two groups are not readily comparable because there are still several factors that affect the comparison , especially target sequence copy number , which is higher in the case of plasmid transfection . this still has important implications for the future extensive use of this genome engineering to edit additional dna outside the nuclear genome , such as in virus - related disease and mitochondrial diseases . in summary , we demonstrated that crispr / cas9-loxp , a novel site - specific genetic manipulation tool , offers a genetic platform for the dissection of gene function and molecular insights into dna - repair pathways . the vector plasmid pmtmg contains the following : mtomato , egfp , two loxp sites , and two homologous arms ( figures 1a and s1 ) . the cre - expression vector was generated using the cre gene downstream of the elongation factor-1 alpha ( efi ) promoter . plasmids px330 and px601 were gifts from feng zhang ( addgene plasmid # 42230 and # 61591 ) . sgrna oligos were annealed and cloned into the px330 or px601 vectors using a standard protocol . hek293 cells were cultured as previously described . to generate aavs1 knockin cell lines containing mt / mg expression cassettes , hek293 cells were seeded on day 0 at 2.5 10 cells in six - well plates , and on day 1 , pmtmg and px330-aavs1 plasmids were transfected by the calcium - phosphate precipitation method . individual clonony with the expression of red fluorescent reporter genes pcr was used for the confirmation of the gene knockin at the aavs1 locus with specific primers , which is shown in figure 2 . the cell line with mt / mg expression cassette knockin at the aavs1 locus , 5 10 293-aavs - mtmg cells were seeded in six - well plates . on day 1 , the cells were transfected with px330-eloxp or efi-cre plasmids by the calcium - phosphate precipitation method . on day 2 , the transfected 293-aavs - mtmg cells were treated with trypsin and replated in a six - well plate . on day 3 , the expression of red / green fluorescent reporter genes was observed under the microscope . on day 3.5 , cells were harvested for flow cytometry and genomic dna isolation ( figure 2a ) . green / red percentage was determined by the formula 100 ( a/(1b ) ) , where a is the green fluorescent frequencies and b is the no fluorescent frequencies . the reporter gene sequence flanking the crispr target site was pcr amplified , and products were cloned into the vector pjet1.2 ( clonejet pcr cloning kit , thermo fisher scientific ) . the pcr products and vectors were purified and then sequenced on an abi prism 3730 dna sequencer ( sequencing primers are shown in table s1 ) . we examined the possibility that crispr / cas9-eloxp induced off - target mutations in 293-aavs - mtmg . the fragments harboring potential off - target sites have been amplified ( primer information in tables s3 and s4 ) and sequenced on an abi prism 3730 dna sequencer .

cre - loxp , as one of the site - specific genetic manipulation tools , offers a method to study the spatial and temporal regulation of gene expression / inactivation in order to decipher gene function . crispr / cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research . whether the traditional site - specific genetic manipulation tool and crispr / cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear . here , we successfully generated a crispr / cas9-loxp system to perform gene editing in human cells , providing the proof of principle that these two technologies can be used together for the first time . we also showed that distinct non - homologous end - joining ( nhej ) patterns from crispr / cas9-mediated gene editing of the targeting sequence locates at the level of plasmids ( episomal ) and chromosomes . specially , the crispr / cas9-mediated nhej pattern in the nuclear genome favors deletions ( 64%68% at the human aavs1 locus versus 4%28% plasmid dna ) . crispr / cas9-loxp , a novel site - specific genetic manipulation tool , offers a platform for the dissection of gene function and molecular insights into dna - repair pathways .

Introduction Results Discussion Materials and Methods Author Contributions Conflicts of Interest

genome editing has recently emerged as a powerful technique that allows any chosen gene to be precisely modified in a predetermined way.1 , 2 the cre - loxp system is a popular and widely used site - specific genetic manipulation tool to conditionally knockout specific genes in cell lines and animal models , which modulates expression of selected genes at a certain developmental stage or in specific tissues , greatly facilitating our understanding of gene function and developmental mechanisms3 , 4 , 5 , 6 , 7 , 8 as well as flp / frt . at the present time , the choice of tools for site - specific genetic manipulation is limited to just cre - loxp , flp - frt , phic31/attp , or attb and the dre recombinase / rox.8 , 9 , 10 additional genetic tools would be of benefit in the genetic modification of cell lines , especially to achieve genetic modification of both alleles . for this reason , new site - specific genetic manipulation tools are crispr / cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research . crispr / cas9 technology has been widely applied for functional genomic studies in a variety of organisms , including mouse and human cells.12 , 13 it can be programmed by single - guide rnas ( sgrnas ) to cleave specific genomic loci complementary to the sgrna with a downstream protospacer adjacent motif ( pam ) , where cas9 creates double - stranded dna breaks ( dsbs ) . these dsbs mediate error - prone non - homologous end - joining ( nhej ) or precise homologous recombination ( hr ) . the most widely used customized crispr / cas9 ( spcas9 ) is derived from streptococcus pyogenes , and the corresponding pam sequence is 5-ngg-3 , where n is any base . whether the traditional site - specific genetic manipulation tool and crispr technologies can be combined as a novel genetic tool for specific gene knockout studies is not clear ( figure s1 ) . to address this question , we took advantage of the double fluorescent reporter systems with crispr / cas9 because it is easily detectable by flow cytometry and microscopy . this approach was used to test the efficiency of a crispr / cas9-loxp system in human cells . we hypothesized that if we introduced two crispr / cas9-mediated dna double strands break via targeting loxp flanking the mtomato cassette , the expression of the egfp gene will be directly driven by the cag promoter . as we expected , co - transfection of two plasmids ( px330- eloxp expressing cas9 and sgrnas to target eloxp and pmtmg ) in human hek293 cells indeed led to egfp expression ( figure 1c ; the schematic of the protocol is shown in figure 1b ) , which implied that the crispr / spcas9-eloxp system can be used to excise the mtomato gene and allow the cag promoter to drive the expression of the egfp gene directly . specially , in the crispr / spcas9-eloxp group , 56.0%63.6% of cells were egfp - positive cells ( figures 1c , 1d , and s2 ) . pcr was then used to confirm the nhej events triggered appropriately by crispr / spcas9-eloxp ( figure 1e).figure 1performance of crispr / spcas9-eloxp system in transient reporter - gene expression assays(a ) schematic of the mt / mg cassette ( loxp - mt - pa - loxp - mg - pa ) before and after crispr / spcas9-eloxp - mediated recombination . after crispr / spcas9-eloxp - mediated recombination , the mtomato sequence is excised , allowing the promoter to drive expression of membrane - targeted egfp ( mg ) . ( b ) an illustration of the crispr / spcas9-eloxp system in transient reporter - gene expression assays . ( c ) hek293 cells were co - transfected with pmtmg and crispr / spcas9-eloxp ( 0.75 ug each ) , and images were obtained at 48 hr post transfection ( red indicates mt ; green indicates mg ) . ( e ) performance of crispr / spcas9-eloxp system in transient reporter - gene expression assays ( a ) schematic of the mt / mg cassette ( loxp - mt - pa - loxp - mg - pa ) before and after crispr / spcas9-eloxp - mediated recombination . ( b ) an illustration of the crispr / spcas9-eloxp system in transient reporter - gene expression assays . ( d ) the level of fluorescence was quantified ( red indicates mt ; green indicates mg ) . to gain insights into the occurrence of the nhej events , we mapped the sequences of crispr / cas9-eloxp - mediated nhej products . comparing one loxp sequence left in the cre group , notably , there was one dominant sequence ( named 3 t ins , 14/25 , 56% ) in the crispr / cas9-eloxp group , which comprises the insertion of one additional t base 3 nt upstream of the pam ( figure 1e ) . taken together , with plasmids ( transient reporter - gene expression ) , our data showed that crispr / cas9-eloxp system - based gene editing can achieve high excision efficiency of a specific gene between two loxp sites . because our system showed high excision efficiency on the specific gene between two loxp sites at the plasmid co - transfection level , we wondered whether it would also be functional at human chromosomal loci . to address this , we sought to generate a double - fluorescent reporter system at the human aavs1 locus . for the above reasons , we selected aavs1 as a targeting site for harboring the double - fluorescent reporter system . we designed one sgrna targeting the aavs1 locus to introduce dsbs and the pmtmg plasmid , which carries two arms homologous to the aavs1 locus , as the donor vector to knock in a double - fluorescent reporter gene at the human aavs1 locus ( figures 2a and 2b ) . to screen positive colonies , which maintain robust mtomato gene expression under the microscope ( figure 2c ) , we initially picked colonies that showed red fluorescence under the microscope . after six passages of the re - picked candidate colonies , we screened them with gene - specific pcr ( figure 2d ; table s2 ) . the four pcr - negative colonies should be due to random integration of the cassette , even if it has mtomato gene expression , which would not be suitable for further experiments.figure 2generation of a double - fluorescent reporter system at the human aavs1 locus(a ) human aavs1 locus knockin via crispr / cas9 . generation of a double - fluorescent reporter system at the human aavs1 locus ( a ) human aavs1 locus knockin via crispr / cas9 . ( c ) images of the cells for picking up positive clones that specifically integrated mt / mg cassette into the aavs1 locus at chromosome 19 . for further studies , one of the positive colonies was named 293-aavs - mtmg , which has red fluorescence and also has a clean homogeneous genetic background . the plasmids for the crispr / spcas9 expression system targeting eloxp were transfected into 293-aavs - mtmg , and the level of effective genome editing was measured by identifying egfp - positive cells with flow cytometry ( figure 3a ) . these results showed that crispr / spcas9-eloxp - mediated site - specific genome editing has been achieved at the human aavs1 locus.figure 3crispr / spcas9-eloxp - mediated site - specific gene editing in 293-aavs - mtmg cells(a ) an illustration of the crispr / spcas9-eloxp system that mediated site - specific gene editing in 293-aavs - mtmg cells . ( d ) targeting dna sequence chromatograms of cells transfected with crispr / spcas9-eloxp are clustered together compared with one loxp remaining in the cre group . ( e ) crispr / spcas9-eloxp - mediated nhej pattern at the human aavs1 locus . crispr / spcas9-eloxp - mediated site - specific gene editing in 293-aavs - mtmg cells ( a ) an illustration of the crispr / spcas9-eloxp system that mediated site - specific gene editing in 293-aavs - mtmg cells . ( e ) crispr / spcas9-eloxp - mediated nhej pattern at the human aavs1 locus . we then sought to ask whether there was the same nhej pattern between the plasmid - based nhejs and reporter genes at the human aavs1 locus . after transfection and isolation of the corresponding genomic dna from 293-aavs - mtmg cells , fragments harboring nhej sites were amplified and sequenced . surprisingly , at the human aavs1 locus , we did not identify the fusion sequence ( one intact loxp sequence ) , which was present at the group of plasmid - based nhejs ( figures 3e and s5 ) . notably , the majority ( 64% ) of the nhejs have deletion at the human aavs1 locus , whereas the majority ( 64% ) of plasmid ( transient gene expression)-based nhejs have insertion . these results implied that the target sequence under a different environment ( plasmid and at the chromosome ) may affect the formation of the nhej pattern of crispr / spcas9 . our previous study clearly demonstrated that nag may not be the universally predominant non - canonical pam for crispr / cas9-mediated dna cleavage in human cells .. we also tested nga or nag pam , and the results showed that crispr / cas9-loxp with nga non - canonical pam , but not nag , could achieve relative highly efficient ( 10% ) excision of a specific gene between two loxp sites ( figure s6 ) . nngrr(t ) , and the corresponding eloxp sequence is ataacttcgtataatgtatgctatacgaagttat attaagggt ( figure 4a).figure 4crispr / sacas9-eloxp - mediated site - specific gene editing in 293-aavs - mtmg cells(a ) targeting sequence and corresponding pams for crispr / spcas9 ( px330 ) and crispr / sacas9 ( px601 ) . ( c ) crispr / sacas9-eloxp - mediated nhej pattern at the human aavs1 locus . ( d ) crispr / sacas9-eloxp - mediated nhej pattern in transient reporter - gene expression assays . crispr / sacas9-eloxp - mediated site - specific gene editing in 293-aavs - mtmg cells ( a ) targeting sequence and corresponding pams for crispr / spcas9 ( px330 ) and crispr / sacas9 ( px601 ) . ( c ) crispr / sacas9-eloxp - mediated nhej pattern at the human aavs1 locus . ( d ) crispr / sacas9-eloxp - mediated nhej pattern in transient reporter - gene expression assays . because site - specific genome editing is very important in vivo , here , we sought to test the performance of the crispr / sacas9-eloxp - mediated novel site - specific genome editing in 293-aavs - mtmg cells . also , we found co - transfection of crispr / cas9- and pmtmg - based gene editing could achieve a higher efficiency comparing genome editing at the aavs1 locus ( figure 4b ) . like the crispr / spcas9-eloxp at aavs1 locus , the majority ( 64% ) of the fusion site ( nhejs ) has deletion , including 2- to 37-bp deletion ( figures 4c and s8 ) . the results of the nhej patterns of transient crispr / sacas9-eloxp expression showed that the minority ( 8% ) of the fusion site is deletion , which is different from that of the crispr / sacas9-eloxp at aavs1 locus ( 64% of the fusion sites have deletions ) . taken together , our study demonstrated that crispr / cas9-loxp ( sa cas9 or sp cas9 ) could be used as a tool to perform site - specific genome editing at the human aavs1 locus . meanwhile , crispr / cas9-mediated nhej patterns may be modulated by the environment of targeting sequence . off - target effects of crispr / cas9 remain a key issue for its application in genome editing , including pam - related off - target effects.20 , 22 in cell lines , it is not practical to re - correct the off - target effects , although these could be minimized through outcrossing in animals or plants . in the present study , we sought to know the off - targets of crispr / cas9-eloxp . with online software ( http://www.rgenome.net/cas-offinder/ ) , we predicted the potential off - target sites in the human genome . further studies also need to characterize additional potential genome modifications inducing off - target crispr / cas9-eloxp with a genome analysis tool , i.e. recently , novel high - fidelity cas9s have been reported , which may strengthen the present novel crispr / cas9-eloxp tool.26 , 27 in the present study , we successfully generated a crispr / cas9-loxp system to perform site - specific genome editing in human cells , at the level of both plasmids ( episomal ) and chromosomes . it provides the proof of principle that these two technologies ( traditional site - specific genetic manipulation tool and crispr technologies ) can be used together . we also showed that distinct nhej patterns from crispr / cas9-mediated gene editing of the targeting sequence locates at the level of plasmids ( episomal ) and chromosomes . for example , if we place a specifically mutated loxp flanking the targeting gene , which cre can not recognize , with the specific sgrna - targeting mutated loxp , it could be used to perform conditional knockouts , and thus could be an attractive alternative genetic tool for the compensation of cre - loxp . in the present study , we used eloxp for crispr / cas9 . thus , these two additional loxp mutants may be more suitable for crispr / cas9-loxp - mediated genome editing . also , the wild - type and loxp mutants could be combined and the specific gene flanking them could be excised . meanwhile , tissue - specific or inducible promoter could be used to drive cas9 expression for conditional gene knockouts for animal models and in human embryonic stem cell or induced pluripotent stem cell studies.29 , 30 these tools will be useful for conditional knockout and studies of gene - gene interaction.figure 5crispr / cas9-loxp - mediated gene editing as a novel site - specific genetic manipulation tool(a ) schematics illustrating the wild loxp and mutant loxp pair lox66/lox71 sequence and the position of the guide rna . ( b ) schematic strategy of the switch gene expression employing the crispr / cas9-loxp system . ( c ) distinct nhej patterns from crispr / cas9-mediated gene editing of the targeting sequence located at the level of plasmids ( episomal ) and chromosomes . crispr / cas9-loxp - mediated gene editing as a novel site - specific genetic manipulation tool ( a ) schematics illustrating the wild loxp and mutant loxp pair lox66/lox71 sequence and the position of the guide rna . ( b ) schematic strategy of the switch gene expression employing the crispr / cas9-loxp system . ( c ) distinct nhej patterns from crispr / cas9-mediated gene editing of the targeting sequence located at the level of plasmids ( episomal ) and chromosomes . we also showed that there is a difference between the crispr / cas9-mediated nhej patterns of the targeting sequence located at the level of plasmids ( episomal ) and chromosomes . in particular , the crispr / cas9-mediated nhej pattern in the nuclear genome is in favor of deletions ( 64%68% at the human aavs1 locus versus 8%28% plasmid dna ) . we speculate that the difference between these two groups may be due to one of the following mechanisms ( figure 5c ) . first , the nuclear genome and plasmid dna exist in different genetic environments , with the nuclear genome heavily regulated by histones and other dna - interacting proteins , whereas the non - nuclear genome , including plasmids as episomes , are naked , with very few proteins binding . this study may provide insights for nhej - related dna repair and provides a platform for the comparison of chromosomal and extra - chromosomal dna repair . it is challenging but of interest to compare crispr / cas9 editing of the same dna sequence integrated into the nuclear genome ( at the chromosomal level ) and extra - chromosomal dna ( such as plasmids transiently existing in the cells ) . here , we demonstrated that with the same dna target sequence and crispr / cas9 , in the same cell type , extra - chromosomal genome crispr / cas9-based gene editing could achieve a high efficiency compared with gene editing at chromosomes ( 55.0%69.0% versus 19.5%31.4% at the aavs1 locus ) . we rationalize the results from these two groups are not readily comparable because there are still several factors that affect the comparison , especially target sequence copy number , which is higher in the case of plasmid transfection . this still has important implications for the future extensive use of this genome engineering to edit additional dna outside the nuclear genome , such as in virus - related disease and mitochondrial diseases . in summary , we demonstrated that crispr / cas9-loxp , a novel site - specific genetic manipulation tool , offers a genetic platform for the dissection of gene function and molecular insights into dna - repair pathways . individual clonony with the expression of red fluorescent reporter genes pcr was used for the confirmation of the gene knockin at the aavs1 locus with specific primers , which is shown in figure 2 . the cell line with mt / mg expression cassette knockin at the aavs1 locus , 5 10 293-aavs - mtmg cells were seeded in six - well plates .

a detailed history and examination form the cornerstone of assessment in men with benign prostatic obstruction ( bpo ) . the international continence society has made large strides in standardizing and categorizing lower urinary tract symptoms ( luts ) . the three categories that have been devised relate to phases of voiding and are described as storage , voiding and post - void [ table 1 ] . lower urinary tract symptoms , common symptoms of bpo in red numerous investigations may help in the diagnosis and management of bpo . they are not all necessary and a clinician needs to weigh the benefits of improved evidence against the cost , time and invasiveness of each test . all tests should have a formulated question of what needs answering and how the result will affect the management decision . a serum creatinine test should be considered in men with suspected renal impairment , i.e. , with a palpable bladder , nocturnal enuresis or history of stone disease . prostate - specific antigen ( psa ) has a useful role in the assessment of men with bpo by acting as a surrogate marker of benign enlargement . a psa of 4 - 6 ng / ml is predictive of boo in 65% of the cases , whereas a psa of 6 - 10 ng / ml is 81% predictive . the evidence suggests that men with a psa > 1.4 ng / ml should be considered to be at an increased risk of symptom progression . the qmax in healthy males is 20.3 ml / s , which reduces with age . in elderly men , the uroflowmetry parameters have recently been studied for the first time in an indian population in over 1000 men . the qmax was shown to increase up to the age of 15 years , followed by a slow decline up to the age of 50 years . the mean qmax in 16 - 50 year olds was found to be 22.5 + 9.2 ml / s . the proportion of men with bladder outflow obstruction ( boo ) with a qmax < 10 ml / s has been reported at 90% , whereas 67% with a qmax between 11 and 14 ml / s and 30% with a qmax greater than 15 ml / s will have boo . this distinction is important as some men with low pressure low flow will not improve after prostatic surgery . cystometry is perhaps not essential in men with clear obstructive symptoms and a classical flow rate pattern . it is more likely to be useful in men with mixed lower urinary tract symptoms or chronic urinary retention where detrusor overactivity and underactivity are under question , respectively . the assessment of detrusor contractility includes the pdetqmax , which allows the calculation of the bladder outlet obstruction index ( booi ) . the booi = pdetqmax - ( 2 qmax ) . if the booi is greater than 40 cmh2o , then the patient is considered obstructed . a booi value of 20 - 40 cmh2o is equivocal and a value less than 20 cmh2o suggests detrusor underactivity rather than obstruction . similarly , the voiding trace may be plotted on an international continence society ics nomogram . these are pre - marked graphs with the qmax plotted on the x - axis and pdetqmax plotted on the y - axis . the patients void phase is plotted on the graph by the urodynamics machine , and the furthest point on the x - axis is equivalent to the booi . the graph contains a booi greater than 40 cmh2o cut - off for each qmax value to signify obstruction , a booi less than 20 cmh2o for unobstructed and a booi of 20 - 40 cmh2o range for equivocal results . the bladder contractility index ( bci ) is calculated as pdet qmax + 5 qmax . bci greater than 150 cmh2o reveals good bladder contractility , 100 - 150 cmh2o is normal contractility and less than 100 cmh2o is reduced contractility . the booi and bci will be elevated in men with bpo and will be low in men with detrusor underactivity . this investigation may reveal the level of obstruction that guides the clinician to what may be causing the obstruction . this may be followed by cystoscopy or magnetic resonance imaging depending on the level and nature of the obstruction . because of the invasiveness of cystometry , a number of non - invasive urodynamic investigations have been proposed . these provide either traditional cystometric measures in a non - invasive manner or they provide a surrogate marker of obstruction . if the length of the prostatic protrusion from its base is greater than 10 mm , then it has been shown that alpha blockers may be less effective at relieving symptoms ; conversely , surgery is likely to be more effective . a recent study has reported an area under the receiver - operating characteristic ( roc ) curve of 0.71 for predicting boo symptoms . another study reported an area under the roc curve of 0.84 compared with the booi . also , there is a higher probability of failing a trial without catheter in men with acute urinary retention if the ipp is > 10 mm . progression , defined as worsening of the international prostate symptom score ( ipps ) by 4 points , or development of urinary retention , has been shown to occur with a higher frequency in men with an ipp > 10 mm . from 259 men , followed for a mean of 32 months , 52 progressed ; of these men who progressed , 44% had an ipp > 10 mm compared with 6% with an ipp < 5 mm . the prostatic urethral angle occurs between the membranous and prostatic urethra . when correlated with the booi , the area under the roc curve was 0.63 in one study . an angle of > 35 was shown to correlate with boo , but an increasing angle did not correlate with degree of boo . these surrogate measures can not replace cystometry but may be useful in helping clinicians decide which treatments to give to patients . detrusor wall thickness ( dwt ) has been shown to correlate better with boo than bladder wall thickness . have prospectively compared anterior dwt > 2 mm with a 7.5-mhz ultrasound to standard urodynamics . the technique had a sensitivity of 83% , specificity of 95% and positive and negative predictive values of 94% and 86% respectively . the area under the roc curve for dwt was found to be 0.723 for successful transurethral resection of prostate turp in 239 men . the limitations of this technique are that minimal changes of less than 2 mm need to be interpreted , the filling volume affects the readings and the region of measurement has not been standardized . interobserver variability is reported in up to 12% and is affected by frequency of the ultrasound probe . however , using a higher cut - off of > 2.9 mm has been reported to have a 100% sensitivity for obstruction and , therefore , this technique will allow for exclusion of men who do not then need to progress to cystometry . more recently , ultrasound - estimated bladder weight ( uebw ) has been proposed to increase accuracy over dwt as it also takes account of the bladder filling volume . the area under the roc curve for this technique is reported at 0.72 for success with turp . men with an uebw > 35 g were 13-times more likely to go into acute urinary retention . these studies , however , have all been in japanese men and , when performed in south americans and europeans , a significant correlation between uebw and boo has not been shown . a limitation may be that uebw varies according to height and weight and therefore these factors need to be considered . near - infrared spectroscopy ( nirs ) measures the concentration of oxyhemoglobin and deoxyhemoglobin levels in the tissue . increased work by the detrusor muscle ( as occurs in boo ) leads to a reduction in the oxyhemoglobin levels ( downward slope ) , whereas in an unobstructed system the oxyhemoglobin levels increase ( upward slope ) . however algorithms vary between studies and may also include the qmax and post void residual pvr assessments in a combined score . problems with this technique are , however , that motion artefact and chronic health conditions such as peripheral vascular disease / diabetes may the affect readings . moreover , these patient groups have been excluded from these studies . this measures the detrusor pressure against a closed bladder outlet , the iso - volumetric pressure ( pves.iso ) . categorization of men into obstructed and non - obstructed categories was found in 42 of 46 patients with reference to urodynamics , and also has good repeatability . problems occur if the flow rates are less than 5.4 ml / s or if the bladder volumes are less than 250 ml . the penile cuff test assumes a continuous flow of fluid from the urethra to the bladder . a pneumatic cuff is placed around the penis and is inflated to interrupt the urine flow and is thereafter rapidly deflated , resulting in a surge of urine ( qsurg ) , followed by a steady state flow ( qss ) . the maximum value of cuff interruption pressure plotted on a non - invasive pressure flow nomogram has been proposed to provide the best diagnostic accuracy . a positive predictive value of 82% and a negative predictive value of 88% have been reported . more interestingly , men shown to be obstructed had an 87% chance of a good outcome after turp compared with only 56% who were shown to be unobstructed . the limiting factors are the high trace exclusion rate , technical failures and a high proportion of equivocal outcomes . doppler assessment of urine flow through the prostatic urethra allows the plotting of flow velocity curves . in obstructed men , the qmax / max velocity ratio is lower and has been suggested to correlate with obstruction . in a small study of 22 men , a ratio > 1.6 correctly classified men according to urodynamic criteria . limitations are the lack of large studies and the cost and expertise to perform this procedure . histological assessment of detrusor muscle has been shown to correlate with detrusor failure and obstruction . urodynamically , obstructed men have detrusor myo - hypertrophy with wide spaces between the muscle cells . contrarily , unobstructed men show muscular and axonal degeneration followed by fibrosis . as it is much more invasive than urodynamics and highly subjective in its interpretation , this methodology is unlikely to replace urodynamic assessment but may be useful in studying and identifying new methods for treating unobstructed men . clearly , at present , an important area for standardization of terminology and assessment of the underlying pathophysiology is the subject of underactive detrusor function . they are not all necessary and a clinician needs to weigh the benefits of improved evidence against the cost , time and invasiveness of each test . all tests should have a formulated question of what needs answering and how the result will affect the management decision . a serum creatinine test should be considered in men with suspected renal impairment , i.e. , with a palpable bladder , nocturnal enuresis or history of stone disease . prostate - specific antigen ( psa ) has a useful role in the assessment of men with bpo by acting as a surrogate marker of benign enlargement . a psa of 4 - 6 ng / ml is predictive of boo in 65% of the cases , whereas a psa of 6 - 10 ng / ml is 81% predictive . the evidence suggests that men with a psa > 1.4 ng / ml should be considered to be at an increased risk of symptom progression . the qmax in healthy males is 20.3 ml / s , which reduces with age . in elderly men , the uroflowmetry parameters have recently been studied for the first time in an indian population in over 1000 men . the qmax was shown to increase up to the age of 15 years , followed by a slow decline up to the age of 50 years . the mean qmax in 16 - 50 year olds was found to be 22.5 + 9.2 ml / s . the proportion of men with bladder outflow obstruction ( boo ) with a qmax < 10 ml / s has been reported at 90% , whereas 67% with a qmax between 11 and 14 ml / s and 30% with a qmax this distinction is important as some men with low pressure low flow will not improve after prostatic surgery . cystometry is perhaps not essential in men with clear obstructive symptoms and a classical flow rate pattern . it is more likely to be useful in men with mixed lower urinary tract symptoms or chronic urinary retention where detrusor overactivity and underactivity are under question , respectively . the assessment of detrusor contractility includes the pdetqmax , which allows the calculation of the bladder outlet obstruction index ( booi ) . the booi = pdetqmax - ( 2 qmax ) . if the booi is greater than 40 cmh2o , then the patient is considered obstructed . a booi value of 20 - 40 cmh2o is equivocal and a value less than 20 cmh2o suggests detrusor underactivity rather than obstruction . similarly , the voiding trace may be plotted on an international continence society ics nomogram . these are pre - marked graphs with the qmax plotted on the x - axis and pdetqmax plotted on the y - axis . the patients void phase is plotted on the graph by the urodynamics machine , and the furthest point on the x - axis is equivalent to the booi . the graph contains a booi greater than 40 cmh2o cut - off for each qmax value to signify obstruction , a booi less than 20 cmh2o for unobstructed and a booi of 20 - 40 cmh2o range for equivocal results . bladder contractility has also been defined by the ics . the bladder contractility index ( bci ) bci greater than 150 cmh2o reveals good bladder contractility , 100 - 150 cmh2o is normal contractility and less than 100 cmh2o is reduced contractility . the booi and bci will be elevated in men with bpo and will be low in men with detrusor underactivity . this investigation may reveal the level of obstruction that guides the clinician to what may be causing the obstruction . this may be followed by cystoscopy or magnetic resonance imaging depending on the level and nature of the obstruction . because of the invasiveness of cystometry , a number of non - invasive urodynamic investigations have been proposed . these provide either traditional cystometric measures in a non - invasive manner or they provide a surrogate marker of obstruction . if the length of the prostatic protrusion from its base is greater than 10 mm , then it has been shown that alpha blockers may be less effective at relieving symptoms ; conversely , surgery is likely to be more effective . a recent study has reported an area under the receiver - operating characteristic ( roc ) curve of 0.71 for predicting boo symptoms . another study reported an area under the roc curve of 0.84 compared with the booi . also , there is a higher probability of failing a trial without catheter in men with acute urinary retention if the ipp is > 10 mm . progression , defined as worsening of the international prostate symptom score ( ipps ) by 4 points , or development of urinary retention , has been shown to occur with a higher frequency in men with an ipp > 10 mm . from 259 men , followed for a mean of 32 months , 52 progressed ; of these men who progressed , 44% had an ipp > 10 mm compared with 6% with an ipp < 5 mm . the prostatic urethral angle occurs between the membranous and prostatic urethra . when correlated with the booi , the area under the roc curve was 0.63 in one study . an angle of > 35 was shown to correlate with boo , but an increasing angle did not correlate with degree of boo . these surrogate measures can not replace cystometry but may be useful in helping clinicians decide which treatments to give to patients . detrusor wall thickness ( dwt ) has been shown to correlate better with boo than bladder wall thickness . have prospectively compared anterior dwt > 2 mm with a 7.5-mhz ultrasound to standard urodynamics . the technique had a sensitivity of 83% , specificity of 95% and positive and negative predictive values of 94% and 86% respectively . the area under the roc curve for dwt was found to be 0.723 for successful transurethral resection of prostate turp in 239 men . the limitations of this technique are that minimal changes of less than 2 mm need to be interpreted , the filling volume affects the readings and the region of measurement has not been standardized . interobserver variability is reported in up to 12% and is affected by frequency of the ultrasound probe . however , using a higher cut - off of > 2.9 mm has been reported to have a 100% sensitivity for obstruction and , therefore , this technique will allow for exclusion of men who do not then need to progress to cystometry . more recently , ultrasound - estimated bladder weight ( uebw ) has been proposed to increase accuracy over dwt as it also takes account of the bladder filling volume . the area under the roc curve for this technique is reported at 0.72 for success with turp . men with an uebw > 35 g were 13-times more likely to go into acute urinary retention . these studies , however , have all been in japanese men and , when performed in south americans and europeans , a significant correlation between uebw and boo has not been shown . a limitation may be that uebw varies according to height and weight and therefore these factors need to be considered . near - infrared spectroscopy ( nirs ) measures the concentration of oxyhemoglobin and deoxyhemoglobin levels in the tissue . increased work by the detrusor muscle ( as occurs in boo ) leads to a reduction in the oxyhemoglobin levels ( downward slope ) , whereas in an unobstructed system the oxyhemoglobin levels increase ( upward slope ) . however algorithms vary between studies and may also include the qmax and post void residual pvr assessments in a combined score . problems with this technique are , however , that motion artefact and chronic health conditions such as peripheral vascular disease / diabetes may the affect readings . moreover , these patient groups have been excluded from these studies . this measures the detrusor pressure against a closed bladder outlet , the iso - volumetric pressure ( pves.iso ) . with the patient voiding , correct categorization of men into obstructed and non - obstructed categories was found in 42 of 46 patients with reference to urodynamics , and also has good repeatability . problems occur if the flow rates are less than 5.4 ml / s or if the bladder volumes are less than 250 ml . the penile cuff test assumes a continuous flow of fluid from the urethra to the bladder . a pneumatic cuff is placed around the penis and is inflated to interrupt the urine flow and is thereafter rapidly deflated , resulting in a surge of urine ( qsurg ) , followed by a steady state flow ( qss ) . the maximum value of cuff interruption pressure plotted on a non - invasive pressure flow nomogram has been proposed to provide the best diagnostic accuracy . a positive predictive value of 82% and a negative predictive value of 88% have been reported . more interestingly , men shown to be obstructed had an 87% chance of a good outcome after turp compared with only 56% who were shown to be unobstructed . the limiting factors are the high trace exclusion rate , technical failures and a high proportion of equivocal outcomes . doppler assessment of urine flow through the prostatic urethra allows the plotting of flow velocity curves . in obstructed men , the qmax / max velocity ratio is lower and has been suggested to correlate with obstruction . in a small study of 22 men , a ratio > 1.6 correctly classified men according to urodynamic criteria . limitations are the lack of large studies and the cost and expertise to perform this procedure . histological assessment of detrusor muscle has been shown to correlate with detrusor failure and obstruction . urodynamically , obstructed men have detrusor myo - hypertrophy with wide spaces between the muscle cells . as it is much more invasive than urodynamics and highly subjective in its interpretation , this methodology is unlikely to replace urodynamic assessment but may be useful in studying and identifying new methods for treating unobstructed men . clearly , at present , an important area for standardization of terminology and assessment of the underlying pathophysiology is the subject of underactive detrusor function . they are not all necessary and a clinician needs to weigh the benefits of improved evidence against the cost , time and invasiveness of each test . all tests should have a formulated question of what needs answering and how the result will affect the management decision . a serum creatinine test should be considered in men with suspected renal impairment , i.e. , with a palpable bladder , nocturnal enuresis or history of stone disease . prostate - specific antigen ( psa ) has a useful role in the assessment of men with bpo by acting as a surrogate marker of benign enlargement . a psa of 4 - 6 ng / ml is predictive of boo in 65% of the cases , whereas a psa of 6 - 10 ng / ml is 81% predictive . the evidence suggests that men with a psa > 1.4 ng / ml should be considered to be at an increased risk of symptom progression . the qmax in healthy males is 20.3 ml / s , which reduces with age . in elderly men , the uroflowmetry parameters have recently been studied for the first time in an indian population in over 1000 men . the qmax was shown to increase up to the age of 15 years , followed by a slow decline up to the age of 50 years . the mean qmax in 16 - 50 year olds was found to be 22.5 + 9.2 ml / s . the proportion of men with bladder outflow obstruction ( boo ) with a qmax < 10 ml / s has been reported at 90% , whereas 67% with a qmax between 11 and 14 ml / s and 30% with a qmax greater than 15 ml / s will have boo . this distinction is important as some men with low pressure low flow will not improve after prostatic surgery . cystometry is perhaps not essential in men with clear obstructive symptoms and a classical flow rate pattern . it is more likely to be useful in men with mixed lower urinary tract symptoms or chronic urinary retention where detrusor overactivity and underactivity are under question , respectively the assessment of detrusor contractility includes the pdetqmax , which allows the calculation of the bladder outlet obstruction index ( booi ) . the booi = pdetqmax - ( 2 qmax ) . if the booi is greater than 40 cmh2o , then the patient is considered obstructed . a booi value of 20 - 40 cmh2o is equivocal and a value less than 20 cmh2o suggests detrusor underactivity rather than obstruction . similarly , the voiding trace may be plotted on an international continence society ics nomogram . these are pre - marked graphs with the qmax plotted on the x - axis and pdetqmax plotted on the y - axis . the patients void phase is plotted on the graph by the urodynamics machine , and the furthest point on the x - axis is equivalent to the booi . the graph contains a booi greater than 40 cmh2o cut - off for each qmax value to signify obstruction , a booi less than 20 cmh2o for unobstructed and a booi of 20 - 40 cmh2o range for equivocal results . bladder contractility has also been defined by the ics . the bladder contractility index ( bci ) bci greater than 150 cmh2o reveals good bladder contractility , 100 - 150 cmh2o is normal contractility and less than 100 cmh2o is reduced contractility . the booi and bci will be elevated in men with bpo and will be low in men with detrusor underactivity . this investigation may reveal the level of obstruction that guides the clinician to what may be causing the obstruction . this may be followed by cystoscopy or magnetic resonance imaging depending on the level and nature of the obstruction . because of the invasiveness of cystometry , a number of non - invasive urodynamic investigations have been proposed . these provide either traditional cystometric measures in a non - invasive manner or they provide a surrogate marker of obstruction . if the length of the prostatic protrusion from its base is greater than 10 mm , then it has been shown that alpha blockers may be less effective at relieving symptoms ; conversely , surgery is likely to be more effective . a recent study has reported an area under the receiver - operating characteristic ( roc ) curve of 0.71 for predicting boo symptoms . another study reported an area under the roc curve of 0.84 compared with the booi . also , there is a higher probability of failing a trial without catheter in men with acute urinary retention if the ipp is > 10 mm . progression , defined as worsening of the international prostate symptom score ( ipps ) by 4 points , or development of urinary retention , has been shown to occur with a higher frequency in men with an ipp > 10 mm . from 259 men , followed for a mean of 32 months , 52 progressed ; of these men who progressed , 44% had an ipp > 10 mm compared with 6% with an ipp < 5 mm . the prostatic urethral angle occurs between the membranous and prostatic urethra . when correlated with the booi , the area under the roc curve was 0.63 in one study . an angle of > 35 was shown to correlate with boo , but an increasing angle did not correlate with degree of boo . these surrogate measures can not replace cystometry but may be useful in helping clinicians decide which treatments to give to patients . detrusor wall thickness ( dwt ) has been shown to correlate better with boo than bladder wall thickness . have prospectively compared anterior dwt > 2 mm with a 7.5-mhz ultrasound to standard urodynamics . the technique had a sensitivity of 83% , specificity of 95% and positive and negative predictive values of 94% and 86% respectively . the area under the roc curve for dwt was found to be 0.723 for successful transurethral resection of prostate turp in 239 men . the limitations of this technique are that minimal changes of less than 2 mm need to be interpreted , the filling volume affects the readings and the region of measurement has not been standardized . interobserver variability is reported in up to 12% and is affected by frequency of the ultrasound probe . however , using a higher cut - off of > 2.9 mm has been reported to have a 100% sensitivity for obstruction and , therefore , this technique will allow for exclusion of men who do not then need to progress to cystometry . more recently , ultrasound - estimated bladder weight ( uebw ) has been proposed to increase accuracy over dwt as it also takes account of the bladder filling volume . the area under the roc curve for this technique is reported at 0.72 for success with turp . men with an uebw > 35 g were 13-times more likely to go into acute urinary retention . these studies , however , have all been in japanese men and , when performed in south americans and europeans , a significant correlation between uebw and boo has not been shown . a limitation may be that uebw varies according to height and weight and therefore these factors need to be considered . near - infrared spectroscopy ( nirs ) measures the concentration of oxyhemoglobin and deoxyhemoglobin levels in the tissue . increased work by the detrusor muscle ( as occurs in boo ) leads to a reduction in the oxyhemoglobin levels ( downward slope ) , whereas in an unobstructed system the oxyhemoglobin levels increase ( upward slope ) . however algorithms vary between studies and may also include the qmax and post void residual pvr assessments in a combined score . problems with this technique are , however , that motion artefact and chronic health conditions such as peripheral vascular disease / diabetes may the affect readings . moreover , these patient groups have been excluded from these studies . this measures the detrusor pressure against a closed bladder outlet , the iso - volumetric pressure ( pves.iso ) . with the patient voiding , correct categorization of men into obstructed and non - obstructed categories was found in 42 of 46 patients with reference to urodynamics , and also has good repeatability . problems occur if the flow rates are less than 5.4 ml / s or if the bladder volumes are less than 250 ml . the penile cuff test assumes a continuous flow of fluid from the urethra to the bladder . a pneumatic cuff is placed around the penis and is inflated to interrupt the urine flow and is thereafter rapidly deflated , resulting in a surge of urine ( qsurg ) , followed by a steady state flow ( qss ) . the maximum value of cuff interruption pressure plotted on a non - invasive pressure flow nomogram has been proposed to provide the best diagnostic accuracy . a positive predictive value of 82% and a negative predictive value of 88% have been reported . more interestingly , men shown to be obstructed had an 87% chance of a good outcome after turp compared with only 56% who were shown to be unobstructed . the limiting factors are the high trace exclusion rate , technical failures and a high proportion of equivocal outcomes . doppler assessment of urine flow through the prostatic urethra allows the plotting of flow velocity curves . in obstructed men , the qmax / max velocity ratio is lower and has been suggested to correlate with obstruction . in a small study of 22 men , a ratio > 1.6 correctly classified men according to urodynamic criteria . limitations are the lack of large studies and the cost and expertise to perform this procedure . histological assessment of detrusor muscle has been shown to correlate with detrusor failure and obstruction . urodynamically , obstructed men have detrusor myo - hypertrophy with wide spaces between the muscle cells . as it is much more invasive than urodynamics and highly subjective in its interpretation , this methodology is unlikely to replace urodynamic assessment but may be useful in studying and identifying new methods for treating unobstructed men . clearly , at present , an important area for standardization of terminology and assessment of the underlying pathophysiology is the subject of underactive detrusor function . the majority of men with the correct history and examination findings probably do not need to undergo invasive and expensive urodynamic assessment . however , emerging evidence does suggest a role for psa assessment and ipp measurement in deciding which men are likely to progress . the use of prostatic measurements such as ipp and prostatic - urethral angle will not inform about bladder contractility . the use of dwt does reflect bladder contractility better , but is still a limited technique that requires further standardization . similarly , the penile cuff test and condom catheter method require more evidence in larger populations but do carry the benefit of being less invasive , potentially providing more information than a flow rate , but being more labor intensive and costly to perform and not providing the same degree of accuracy as a pressure flow study .

the assessment of men with bladder outflow obstruction relies on an adequate history and examination . uroflowmetry and post - void residue estimation are very revealing and may be sufficient in the majority of men . the prostate - specific antigen test may be used to select men who are at a high risk of progression . in specific situations , cystometry may be required . we discuss the use of cystometry and the newer less - invasive methods of assessment that have emerged over the last few years , including ultrasound estimation of intravesical prostatic protrusion , prostatic urethra angle , detrusor wall thickness , ultrasound - estimated bladder weight , near - infrared spectroscopy and the condom catheter and penile cuff tests . although these techniques show promise , they still require further modifications , standardization and testing in larger populations . in addition , they should be used in men where only specific questions need to be answered .

HISTORY AND EXAMINATION None Investigations Blood tests Uroflowmetry Cystometry Non-invasive cystometry CONCLUSIONS

a detailed history and examination form the cornerstone of assessment in men with benign prostatic obstruction ( bpo ) . the three categories that have been devised relate to phases of voiding and are described as storage , voiding and post - void [ table 1 ] . lower urinary tract symptoms , common symptoms of bpo in red numerous investigations may help in the diagnosis and management of bpo . a serum creatinine test should be considered in men with suspected renal impairment , i.e. prostate - specific antigen ( psa ) has a useful role in the assessment of men with bpo by acting as a surrogate marker of benign enlargement . the evidence suggests that men with a psa > 1.4 ng / ml should be considered to be at an increased risk of symptom progression . the qmax was shown to increase up to the age of 15 years , followed by a slow decline up to the age of 50 years . the mean qmax in 16 - 50 year olds was found to be 22.5 + 9.2 ml / s . the proportion of men with bladder outflow obstruction ( boo ) with a qmax < 10 ml / s has been reported at 90% , whereas 67% with a qmax between 11 and 14 ml / s and 30% with a qmax greater than 15 ml / s will have boo . this distinction is important as some men with low pressure low flow will not improve after prostatic surgery . cystometry is perhaps not essential in men with clear obstructive symptoms and a classical flow rate pattern . it is more likely to be useful in men with mixed lower urinary tract symptoms or chronic urinary retention where detrusor overactivity and underactivity are under question , respectively . the assessment of detrusor contractility includes the pdetqmax , which allows the calculation of the bladder outlet obstruction index ( booi ) . similarly , the voiding trace may be plotted on an international continence society ics nomogram . the patients void phase is plotted on the graph by the urodynamics machine , and the furthest point on the x - axis is equivalent to the booi . the booi and bci will be elevated in men with bpo and will be low in men with detrusor underactivity . this investigation may reveal the level of obstruction that guides the clinician to what may be causing the obstruction . this may be followed by cystoscopy or magnetic resonance imaging depending on the level and nature of the obstruction . because of the invasiveness of cystometry , a number of non - invasive urodynamic investigations have been proposed . these provide either traditional cystometric measures in a non - invasive manner or they provide a surrogate marker of obstruction . if the length of the prostatic protrusion from its base is greater than 10 mm , then it has been shown that alpha blockers may be less effective at relieving symptoms ; conversely , surgery is likely to be more effective . also , there is a higher probability of failing a trial without catheter in men with acute urinary retention if the ipp is > 10 mm . progression , defined as worsening of the international prostate symptom score ( ipps ) by 4 points , or development of urinary retention , has been shown to occur with a higher frequency in men with an ipp > 10 mm . from 259 men , followed for a mean of 32 months , 52 progressed ; of these men who progressed , 44% had an ipp > 10 mm compared with 6% with an ipp < 5 mm . the prostatic urethral angle occurs between the membranous and prostatic urethra . these surrogate measures can not replace cystometry but may be useful in helping clinicians decide which treatments to give to patients . detrusor wall thickness ( dwt ) has been shown to correlate better with boo than bladder wall thickness . the area under the roc curve for dwt was found to be 0.723 for successful transurethral resection of prostate turp in 239 men . the limitations of this technique are that minimal changes of less than 2 mm need to be interpreted , the filling volume affects the readings and the region of measurement has not been standardized . however , using a higher cut - off of > 2.9 mm has been reported to have a 100% sensitivity for obstruction and , therefore , this technique will allow for exclusion of men who do not then need to progress to cystometry . more recently , ultrasound - estimated bladder weight ( uebw ) has been proposed to increase accuracy over dwt as it also takes account of the bladder filling volume . men with an uebw > 35 g were 13-times more likely to go into acute urinary retention . a limitation may be that uebw varies according to height and weight and therefore these factors need to be considered . near - infrared spectroscopy ( nirs ) measures the concentration of oxyhemoglobin and deoxyhemoglobin levels in the tissue . increased work by the detrusor muscle ( as occurs in boo ) leads to a reduction in the oxyhemoglobin levels ( downward slope ) , whereas in an unobstructed system the oxyhemoglobin levels increase ( upward slope ) . however algorithms vary between studies and may also include the qmax and post void residual pvr assessments in a combined score . categorization of men into obstructed and non - obstructed categories was found in 42 of 46 patients with reference to urodynamics , and also has good repeatability . the penile cuff test assumes a continuous flow of fluid from the urethra to the bladder . the maximum value of cuff interruption pressure plotted on a non - invasive pressure flow nomogram has been proposed to provide the best diagnostic accuracy . more interestingly , men shown to be obstructed had an 87% chance of a good outcome after turp compared with only 56% who were shown to be unobstructed . the limiting factors are the high trace exclusion rate , technical failures and a high proportion of equivocal outcomes . doppler assessment of urine flow through the prostatic urethra allows the plotting of flow velocity curves . limitations are the lack of large studies and the cost and expertise to perform this procedure . histological assessment of detrusor muscle has been shown to correlate with detrusor failure and obstruction . as it is much more invasive than urodynamics and highly subjective in its interpretation , this methodology is unlikely to replace urodynamic assessment but may be useful in studying and identifying new methods for treating unobstructed men . clearly , at present , an important area for standardization of terminology and assessment of the underlying pathophysiology is the subject of underactive detrusor function . a serum creatinine test should be considered in men with suspected renal impairment , i.e. , with a palpable bladder , nocturnal enuresis or history of stone disease . prostate - specific antigen ( psa ) has a useful role in the assessment of men with bpo by acting as a surrogate marker of benign enlargement . the evidence suggests that men with a psa > 1.4 ng / ml should be considered to be at an increased risk of symptom progression . the qmax was shown to increase up to the age of 15 years , followed by a slow decline up to the age of 50 years . the mean qmax in 16 - 50 year olds was found to be 22.5 + 9.2 ml / s . the proportion of men with bladder outflow obstruction ( boo ) with a qmax < 10 ml / s has been reported at 90% , whereas 67% with a qmax between 11 and 14 ml / s and 30% with a qmax this distinction is important as some men with low pressure low flow will not improve after prostatic surgery . cystometry is perhaps not essential in men with clear obstructive symptoms and a classical flow rate pattern . it is more likely to be useful in men with mixed lower urinary tract symptoms or chronic urinary retention where detrusor overactivity and underactivity are under question , respectively . the assessment of detrusor contractility includes the pdetqmax , which allows the calculation of the bladder outlet obstruction index ( booi ) . a booi value of 20 - 40 cmh2o is equivocal and a value less than 20 cmh2o suggests detrusor underactivity rather than obstruction . similarly , the voiding trace may be plotted on an international continence society ics nomogram . the patients void phase is plotted on the graph by the urodynamics machine , and the furthest point on the x - axis is equivalent to the booi . the booi and bci will be elevated in men with bpo and will be low in men with detrusor underactivity . this investigation may reveal the level of obstruction that guides the clinician to what may be causing the obstruction . this may be followed by cystoscopy or magnetic resonance imaging depending on the level and nature of the obstruction . because of the invasiveness of cystometry , a number of non - invasive urodynamic investigations have been proposed . these provide either traditional cystometric measures in a non - invasive manner or they provide a surrogate marker of obstruction . if the length of the prostatic protrusion from its base is greater than 10 mm , then it has been shown that alpha blockers may be less effective at relieving symptoms ; conversely , surgery is likely to be more effective . also , there is a higher probability of failing a trial without catheter in men with acute urinary retention if the ipp is > 10 mm . progression , defined as worsening of the international prostate symptom score ( ipps ) by 4 points , or development of urinary retention , has been shown to occur with a higher frequency in men with an ipp > 10 mm . from 259 men , followed for a mean of 32 months , 52 progressed ; of these men who progressed , 44% had an ipp > 10 mm compared with 6% with an ipp < 5 mm . the prostatic urethral angle occurs between the membranous and prostatic urethra . these surrogate measures can not replace cystometry but may be useful in helping clinicians decide which treatments to give to patients . detrusor wall thickness ( dwt ) has been shown to correlate better with boo than bladder wall thickness . the area under the roc curve for dwt was found to be 0.723 for successful transurethral resection of prostate turp in 239 men . the limitations of this technique are that minimal changes of less than 2 mm need to be interpreted , the filling volume affects the readings and the region of measurement has not been standardized . however , using a higher cut - off of > 2.9 mm has been reported to have a 100% sensitivity for obstruction and , therefore , this technique will allow for exclusion of men who do not then need to progress to cystometry . more recently , ultrasound - estimated bladder weight ( uebw ) has been proposed to increase accuracy over dwt as it also takes account of the bladder filling volume . men with an uebw > 35 g were 13-times more likely to go into acute urinary retention . a limitation may be that uebw varies according to height and weight and therefore these factors need to be considered . near - infrared spectroscopy ( nirs ) measures the concentration of oxyhemoglobin and deoxyhemoglobin levels in the tissue . increased work by the detrusor muscle ( as occurs in boo ) leads to a reduction in the oxyhemoglobin levels ( downward slope ) , whereas in an unobstructed system the oxyhemoglobin levels increase ( upward slope ) . however algorithms vary between studies and may also include the qmax and post void residual pvr assessments in a combined score . with the patient voiding , correct categorization of men into obstructed and non - obstructed categories was found in 42 of 46 patients with reference to urodynamics , and also has good repeatability . the penile cuff test assumes a continuous flow of fluid from the urethra to the bladder . the maximum value of cuff interruption pressure plotted on a non - invasive pressure flow nomogram has been proposed to provide the best diagnostic accuracy . more interestingly , men shown to be obstructed had an 87% chance of a good outcome after turp compared with only 56% who were shown to be unobstructed . the limiting factors are the high trace exclusion rate , technical failures and a high proportion of equivocal outcomes . doppler assessment of urine flow through the prostatic urethra allows the plotting of flow velocity curves . limitations are the lack of large studies and the cost and expertise to perform this procedure . histological assessment of detrusor muscle has been shown to correlate with detrusor failure and obstruction . as it is much more invasive than urodynamics and highly subjective in its interpretation , this methodology is unlikely to replace urodynamic assessment but may be useful in studying and identifying new methods for treating unobstructed men . clearly , at present , an important area for standardization of terminology and assessment of the underlying pathophysiology is the subject of underactive detrusor function . a serum creatinine test should be considered in men with suspected renal impairment , i.e. prostate - specific antigen ( psa ) has a useful role in the assessment of men with bpo by acting as a surrogate marker of benign enlargement . the evidence suggests that men with a psa > 1.4 ng / ml should be considered to be at an increased risk of symptom progression . the qmax was shown to increase up to the age of 15 years , followed by a slow decline up to the age of 50 years . the mean qmax in 16 - 50 year olds was found to be 22.5 + 9.2 ml / s . the proportion of men with bladder outflow obstruction ( boo ) with a qmax < 10 ml / s has been reported at 90% , whereas 67% with a qmax between 11 and 14 ml / s and 30% with a qmax greater than 15 ml / s will have boo . this distinction is important as some men with low pressure low flow will not improve after prostatic surgery . cystometry is perhaps not essential in men with clear obstructive symptoms and a classical flow rate pattern . it is more likely to be useful in men with mixed lower urinary tract symptoms or chronic urinary retention where detrusor overactivity and underactivity are under question , respectively the assessment of detrusor contractility includes the pdetqmax , which allows the calculation of the bladder outlet obstruction index ( booi ) . similarly , the voiding trace may be plotted on an international continence society ics nomogram . the patients void phase is plotted on the graph by the urodynamics machine , and the furthest point on the x - axis is equivalent to the booi . the booi and bci will be elevated in men with bpo and will be low in men with detrusor underactivity . this investigation may reveal the level of obstruction that guides the clinician to what may be causing the obstruction . this may be followed by cystoscopy or magnetic resonance imaging depending on the level and nature of the obstruction . because of the invasiveness of cystometry , a number of non - invasive urodynamic investigations have been proposed . these provide either traditional cystometric measures in a non - invasive manner or they provide a surrogate marker of obstruction . if the length of the prostatic protrusion from its base is greater than 10 mm , then it has been shown that alpha blockers may be less effective at relieving symptoms ; conversely , surgery is likely to be more effective . also , there is a higher probability of failing a trial without catheter in men with acute urinary retention if the ipp is > 10 mm . progression , defined as worsening of the international prostate symptom score ( ipps ) by 4 points , or development of urinary retention , has been shown to occur with a higher frequency in men with an ipp > 10 mm . from 259 men , followed for a mean of 32 months , 52 progressed ; of these men who progressed , 44% had an ipp > 10 mm compared with 6% with an ipp < 5 mm . the prostatic urethral angle occurs between the membranous and prostatic urethra . these surrogate measures can not replace cystometry but may be useful in helping clinicians decide which treatments to give to patients . detrusor wall thickness ( dwt ) has been shown to correlate better with boo than bladder wall thickness . the area under the roc curve for dwt was found to be 0.723 for successful transurethral resection of prostate turp in 239 men . the limitations of this technique are that minimal changes of less than 2 mm need to be interpreted , the filling volume affects the readings and the region of measurement has not been standardized . interobserver variability is reported in up to 12% and is affected by frequency of the ultrasound probe . however , using a higher cut - off of > 2.9 mm has been reported to have a 100% sensitivity for obstruction and , therefore , this technique will allow for exclusion of men who do not then need to progress to cystometry . more recently , ultrasound - estimated bladder weight ( uebw ) has been proposed to increase accuracy over dwt as it also takes account of the bladder filling volume . men with an uebw > 35 g were 13-times more likely to go into acute urinary retention . a limitation may be that uebw varies according to height and weight and therefore these factors need to be considered . near - infrared spectroscopy ( nirs ) measures the concentration of oxyhemoglobin and deoxyhemoglobin levels in the tissue . increased work by the detrusor muscle ( as occurs in boo ) leads to a reduction in the oxyhemoglobin levels ( downward slope ) , whereas in an unobstructed system the oxyhemoglobin levels increase ( upward slope ) . however algorithms vary between studies and may also include the qmax and post void residual pvr assessments in a combined score . with the patient voiding , correct categorization of men into obstructed and non - obstructed categories was found in 42 of 46 patients with reference to urodynamics , and also has good repeatability . the penile cuff test assumes a continuous flow of fluid from the urethra to the bladder . the maximum value of cuff interruption pressure plotted on a non - invasive pressure flow nomogram has been proposed to provide the best diagnostic accuracy . more interestingly , men shown to be obstructed had an 87% chance of a good outcome after turp compared with only 56% who were shown to be unobstructed . the limiting factors are the high trace exclusion rate , technical failures and a high proportion of equivocal outcomes . doppler assessment of urine flow through the prostatic urethra allows the plotting of flow velocity curves . limitations are the lack of large studies and the cost and expertise to perform this procedure . histological assessment of detrusor muscle has been shown to correlate with detrusor failure and obstruction . as it is much more invasive than urodynamics and highly subjective in its interpretation , this methodology is unlikely to replace urodynamic assessment but may be useful in studying and identifying new methods for treating unobstructed men . clearly , at present , an important area for standardization of terminology and assessment of the underlying pathophysiology is the subject of underactive detrusor function . the majority of men with the correct history and examination findings probably do not need to undergo invasive and expensive urodynamic assessment . the use of prostatic measurements such as ipp and prostatic - urethral angle will not inform about bladder contractility . the use of dwt does reflect bladder contractility better , but is still a limited technique that requires further standardization . similarly , the penile cuff test and condom catheter method require more evidence in larger populations but do carry the benefit of being less invasive , potentially providing more information than a flow rate , but being more labor intensive and costly to perform and not providing the same degree of accuracy as a pressure flow study .

strains and media bacteria and plasmids used in this study are listed in supplemental table s1 . l. pneumophila was grown on cye agar plates or in aye broth ; escherichia coli was cultured in lb medium . antibiotics were added at the following concentrations : 5 g / ml chloramphenicol or 50 g / ml kanamycin for l. pneumophila and 30 g / ml chloramphenicol or 100 g / ml ampicillin for e. coli . the d. discoideum wild - type ax3 strain was grown axenically in hl-5 medium at 23 c as described , adding 20 g / ml g418 ( 32 ) , if required ( psu01 ) . drosophila kc167 phagocytes were grown at 25 c in schneider 's medium with 10% heat - inactivated fetal bovine serum ( invitrogen ) . translational n - terminal gst or m45 fusions of ralf , lida , sidm , and fragments of sidm were constructed by pcr amplification from chromosomal l. pneumophila jr32 dna using the primers listed in supplemental table s2 . the pcr fragments were cloned into the vectors pgex-4t-1 , pgex-6p-1 , and pcr33 , respectively , yielding the plasmids listed in supplemental table s1 . sidc-(1586)-dl - m9/m13 was produced by cloning pcr fragments generated with ombglii444fw or ombglii544fw and ocr117 into the bglii site of sidc and sali site of pgex , resulting in the insertion of two additional amino acids ( asp and leu ) between the sidc and sidm fragments . expression of m45 and sidc fusion proteins was verified by western blot analysis using a monoclonal mouse anti - m45 hybridoma supernatant or an affinity - purified polyclonal rabbit anti - sidc antibody ( 32 ) , followed by a goat anti - mouse or -rabbit secondary peroxidase - labeled antibody ( sigma ) . the chromosomal deletions of ralf and sidm were performed following a protocol described previously ( 38 , 39 ) , and gst fusion proteins were produced as described ( 32 , 33 ) . pneumophila jr32 was grown in aye medium to an od600 of 3 , harvested at 4 c , washed once in cold w - buffer ( 10 mm hepes ( ph 7.4 ) , 150 mm nacl ) , and lysed with a french press . after addition of 1 mm phenylmethylsulfonyl fluoride , cell debris was removed by centrifugation ( 10 min , 3,300 g ) , followed by ultracentrifugation ( 1 h , 155,000 g ) . the amount of soluble protein was estimated using the bradford assay ( bio - rad ) . for pulldown assays 12 ml of lysate containing 1030 mg of total protein was incubated overnight at 4 c with 50100 l of pi - coated agarose beads ( 10 pm ptdins/l slurry ; echelon ) . bound proteins were eluted by adding 20 l of laemmli buffer ( 5 min , 95 c ) and analyzed by sds - page / coomassie brilliant blue or silver staining . the proteins were digested with trypsin and identified by matrix - assisted laser desorption ionization - tandem mass spectrometry or , alternatively , by liquid chromatography - electrospray ionization - tandem mass spectrometry at the functional genomics center zurich . using the same protocol , pulldown assays were also performed with 100 pmol of purified gst fusion protein samples , which were incubated with 50 l of ptdins(4)p - coated agarose beads suspended in w - buffer supplemented with 0.25% nonidet p-40 . binding of the different proteins to phosphoinositides in vitro the binding specificity of different proteins to pis was tested in a protein - lipid overlay assay ( 32 , 33 , 40 ) using 200 nm gst fusion proteins expressed from pgex-4t-1 plasmids as detailed in the supplemental material . discoideum was infected with l. pneumophila ( m.o.i . of 50 ) and analyzed by fluorescence microscopy as described ( 4 , 32 ) . the bacteria were stained with a monoclonal rhodamine - conjugated rabbit anti - l . pneumophila philadelphia-1 serogroup 1 antibody ( m - tech ) , and m45-tagged proteins were labeled with a monoclonal mouse anti m45 hybridoma , followed by a cy5-conjugated goat anti - mouse antibody ( the jackson laboratories ) . pneumophila philadelphia-1 serogroup 1 antibody ( santa cruz biotechnology ) , and sidc was stained using an affinity - purified polyclonal rabbit anti sidc antibody ( 32 ) , followed by a cy3-conjugated goat anti - mouse and a fluorescein isothiocyanate - labeled anti - rabbit antibody ( the jackson laboratories ) . in other experiments , dsred - labeled l. pneumophila ( 41 ) were used to infect calnexin - gfp - producing d. discoideum , and sidc on lcvs was visualized by an affinity - purified polyclonal rabbit anti - sidc antibody ( 32 ) and a secondary goat anti - rabbit cy5-labeled antibody ( the jackson laboratories ) . the amount of sidc was quantified only on calnexin - positive lcvs by determining the fluorescence intensity of the area covering individual lcvs after local background correction using the quantityone software ( bio - rad ) . rna interference rna silencing was performed with drosophila kc167 phagocytes as described ( 29 , 30 ) . briefly , 1 10 cells / ml were plated in schneider 's medium without serum and incubated for 4 h with dsrna ( 20 g / ml ) . the transfection process was terminated by adding schneider 's medium with fetal calf serum to a final concentration of 10% , and the cells were incubated for 45 days at 25 c prior to the infections . drosophila genomic dna and the oligonucleotides listed in supplemental table s2 were used to amplify by pcr the template dna containing a t7 promoter for in vitro transcription . the pcr products were transcribed in vitro into dsrna using the megascript rna kit ( ambion ) , and the quality of the dsrna was assessed by gel electrophoresis . gene silencing by specific dsrna was confirmed by rt - pcr . to determine effects of gene silencing on the recruitment of sidc to lcvs , 2.5 10 kc167 cells were plated in 24-well dishes containing a coverslip and transfected with dsrna followed by incubation for 5 days . the cells were infected with l. pneumophila ( m.o.i . of 50 ) and incubated at 25 c for a further 15 min , and sidc - positive lcvs were quantified by immunofluorescence as described ( 32 ) . analytical ultracentrifugation to produce full - length sidm for analysis by analytical ultracentrifugation ( auc ) , the gst - sidm fusion protein was produced , and the gst fragment was cleaved off as described ( 33 ) . a beckman xl - i analytical ultracentrifuge using an 8-cell 50ti rotor was used for the auc studies . samples of sidm were prepared in 20 mm tris - hcl ( ph 7.4 ) containing 100 mm nacl and 1 mm dithiothreitol and were centrifuged at 20,000 , 22,000 , and 24,000 rpm for 20 h at 4 c . the absorbance of the sample was measured at a wavelength of 280 nm throughout the cell . a total of three measurements were taken at 1-h intervals at the end of each run . parameters for the partial specific volume of the protein were calculated using sednterp ( 43 ) . far - uv circular dichroism spectroscopy the cd spectra were measured on a jasco j-810 spectropolarimeter using a 0.02-cm path length cuvette . protein solutions were prepared in 20 mm tris - hcl buffer ( ph 7.4 ) including 100 mm nacl at a concentration of 0.4 mg / ml ( 5.4 , 8.0 , 16.8 , and 32.5 m for full - length sidm , m7 , m9 , and m13 , respectively ) . the scanned wavelength range was 185300 nm , and the spectra were collected at 20 c . the secondary structure content was estimated from the cd spectra using the cdsstr algorithm ( 44 ) with reference data set 7 ( which contains spectra from 48 proteins , including 5 , which are denatured ) at the dichroweb server ( 45 , 46 ) . the back - calculated spectra and experimental spectra were compared to estimate the normalized root mean square deviation values , which were below 0.1 . thermofluor assay thermofluor experiments were carried out with a real time pcr machine mx3005p ( stratagene ) . the protein was mixed with the fluorescent dye sypro orange ( molecular probes ) in a thermo - fast 96-well pcr plate ( abgene ) , resulting in final protein concentrations of 5 m . the plate was heated at a rate of 1 c / min from 25 to 93 c , and fluorescence was measured in 1 c increments . fluorescence was filtered through custom interference excitation ( 492 nm ) and emission ( 568 nm ) filters . the primary data ( relative fluorescence intensity versus temperature ) were fit to standard equations describing protein thermal stability , as described previously ( 47 ) . bioinformatics and statistical analysis homology searches were performed using the following software packages : blast ( www.ncbi.nlm.nih ) , scansite , phyre ( protein homology / analogy recognition engine ) , and elm ( eukaryotic linear motif resource ) . prediction of coiled coils was carried out by the coils server , and prediction of secondary structure by dompred and psipred ( protein structure prediction server ) ( 48 ) . for statistical analysis , the one - tailed student 's t test was used , considering p < 0.05 as significant . identification of sidm as a pi - binding protein the signaling lipid ptdins(4)p was recently discovered to be specifically recognized by the icm / dot substrate sidc on lcvs ( 32 , 33 ) . to identify additional pi - binding proteins of l. pneumophila , we performed pulldown assays using agarose beads coated with individual pis . staining of the proteins eluting from washed beads by coomassie brilliant blue revealed large amounts of a protein with an apparent molecular mass of 75 kda , which predominantly bound to ptdins(4)p ( fig . the major ptdins(4)p interactor was identified by mass spectrometry as the 73-kda protein sidm , a known icm / dot substrate previously characterized as a rab1 gef ( 19 , 20 ) . upon visualizing proteins eluting from pi - coated beads by silver staining , the 75-kda protein was also present in eluates from agarose beads coupled to ptdins(4,5)p2 or ptdins(3,4,5)p3 ( fig . the ptdins(4)p - binding icm / dot substrate sidc , which is not similar to sidm in sequence , was expected to also be identified in this screen for pi - binding l. pneumophila proteins . although we did not identify full - length sidc under the above conditions , small amounts of a 50-kda c - terminal sidc fragment , including the ptdins(4)p - binding domain p4c ( 33 ) , were retained by ptdins(4)p - coated agarose beads and identified by mass spectrometry ( data not shown ) . to determine whether sidc or other l. pneumophila proteins bind to ptdins(4)p - coated agarose beads more efficiently in the absence of sidm , we repeated the pulldown experiments using lysates of an l. pneumophila sidm strain . whereas in the absence of sidm no protein bound in high amounts to ptdins(4)p - coated beads , in the absence of sidc only sidm was detected ( fig . these results suggest that sidm is a major ptdins(4)p - binding protein of l. pneumophila . we also tested whether sidc was not recovered from the enrichment because of proteolytic degradation in l. pneumophila lysates ( supplemental fig . sidc was found to be stable in the absence and in presence of ptdins(4)p - coated agarose beads for at least 20 h , and thus , proteolysis does not account for the failure to recover significant amounts of sidc under the conditions used . figure 1.identification of sidm in a screen for pi - binding l. pneumophila proteins . pulldown of lysate from l. pneumophila wild - type ( a and b ) , and sidc or sidm mutant strains ( c ) using agarose beads coated with different pis or ptdins . bacterial proteins retained by washed beads were separated by sds - page and visualized by staining with coomassie brilliant blue ( a and c ) or silver ( b ) . the dominant protein with an apparent molecular mass of 75 kda eluting from beads coated with ptdins(4)p or ptdins(3,4)p2 and to a smaller extent from beads coated with ptdins(4,5)p2 or ptdins(3,4,5)p3 was identified by mass spectrometry as the rab1 gef sidm / drra . pulldown of lysate from l. pneumophila wild - type ( a and b ) , and sidc or sidm mutant strains ( c ) using agarose beads coated with different pis or ptdins . bacterial proteins retained by washed beads were separated by sds - page and visualized by staining with coomassie brilliant blue ( a and c ) or silver ( b ) . the dominant protein with an apparent molecular mass of 75 kda eluting from beads coated with ptdins(4)p or ptdins(3,4)p2 and to a smaller extent from beads coated with ptdins(4,5)p2 or ptdins(3,4,5)p3 was identified by mass spectrometry as the rab1 gef sidm / drra . sidm specifically binds to ptdins(4)p in vitro to assess the pi - binding specificity of heterologously produced , purified sidm , we tested binding of an n - terminal gst - sidm fusion protein to agarose beads coated with different pis . under the conditions used , the 99-kda gst - sidm fusion protein bound only to ptdins(4)p - coated beads , but not to beads coated with any other pis , ptdins , or to agarose beads alone ( fig . these results indicate that sidm specifically and directly binds to ptdins(4)p in vitro without requiring any co - factors . sds gels stained with coomassie brilliant blue of pulldown of affinity - purified gst - sidm with agarose beads coated with different pis or ptdins ( a ) , or gst - sidm , gst - sidc , gst - ralf , and gst - lida ( 3 g ) ( left panel ) and eluate from ptdins(4)p - coated agarose beads incubated with the gst fusion proteins ( right panel ) ( b ) . protein - lipid overlay assay of 100 pmol ( c ) or serial 2-fold dilutions of the lipids indicated ( d ) . the binding of affinity - purified gst fusion proteins to lipids immobilized on nitrocellulose membranes was analyzed using an anti - gst antibody . lpa , lysophosphatidic acid ; lpc , lysophosphocholine ; pe , phosphatidylethanolamine ; pc , phosphatidylcholine ; s1p , sphingosine 1-phosphate ; pa , phosphatidic acid ; ps , phosphatidylserine ; ptdins , phosphatidylinositol . sds gels stained with coomassie brilliant blue of pulldown of affinity - purified gst - sidm with agarose beads coated with different pis or ptdins ( a ) , or gst - sidm , gst - sidc , gst - ralf , and gst - lida ( 3 g ) ( left panel ) and eluate from ptdins(4)p - coated agarose beads incubated with the gst fusion proteins ( right panel ) ( b ) . protein - lipid overlay assay of 100 pmol ( c ) or serial 2-fold dilutions of the lipids indicated ( d ) . the binding of affinity - purified gst fusion proteins to lipids immobilized on nitrocellulose membranes was analyzed using an anti - gst antibody . lpa , lysophosphatidic acid ; lpc , lysophosphocholine ; pe , phosphatidylethanolamine ; pc , phosphatidylcholine ; s1p , sphingosine 1-phosphate ; pa , phosphatidic acid ; ps , phosphatidylserine ; ptdins , phosphatidylinositol . 1 ) , even though sidc is also produced by l. pneumophila under the conditions used for the screen ( 33 ) ( supplemental fig . possibly , sidm binds to ptdins(4)p more strongly than sidc . to compare the ptdins(4)p affinities of icm / dot substrates localizing to lcvs , we directly compared binding of the corresponding gst fusion proteins in pulldown assays . agarose beads coated with ptdins(4)p were incubated with equal amounts of the purified gst fusion proteins of sidc , sidm , ralf , and lida . proteins retained by the beads were separated by sds - page and visualized by coomassie brilliant blue staining ( fig . similar amounts of purified gst - sidc and gst - sidm eluted from the beads , indicating that the affinities of the two different effectors to ptdins(4)p are comparable . compared with the amount of protein applied , 20% of gst - sidc or gst - sidm eluted from the beads . in contrast , gst - ralf was not retained by ptdins(4)p - coated agarose beads , and only a very faint band was observed for gst - lida eluting from the beads . a , sidm fragments fused to gst were affinity - purified and used in protein - lipid overlay assay to test binding to 100 pmol ( b ) or serial 2-fold dilutions of ptdins(4)p and ptdins(4,5)p2 spotted onto nitrocellulose membranes ( c ) . a , sidm fragments fused to gst were affinity - purified and used in protein - lipid overlay assay to test binding to 100 pmol ( b ) or serial 2-fold dilutions of ptdins(4)p and ptdins(4,5)p2 spotted onto nitrocellulose membranes ( c ) . next , we tested binding of purified gst fusion proteins of sidm , ralf , lida , and sidc to pis and other lipids immobilized on nitrocellulose membranes ( fig . 2c ) . the rab1 gef sidm preferentially bound to ptdins(4)p and weakly also to ptdins(3)p . in contrast , the arf1 gef ralf did not bind to any pis or other lipids on the membrane , suggesting that l. pneumophila produces distinct classes of gefs that localize to lcvs either pi - dependently or pi - independently . interestingly , the sidm auxiliary protein lida preferentially bound to ptdins(3)p but also significantly to ptdins(4)p . these results were confirmed using pi array nitrocellulose membranes , onto which the pis are spotted in 2-fold dilution series ( fig . sidm and sidc specifically bound to ptdins(4)p , whereas lida showed a more relaxed pi - binding specificity and preferentially bound to ptdins(3)p but also weakly to ptdins(4)p . identification of the ptdins(4)p - binding domain of sidm to map the ptdins(4)p - binding domain of sidm , we constructed n - terminal fusions of gst with fragments of sidm of different lengths and visualized binding of the fusion proteins to ptdins(4)p by protein - lipid overlay assays ( fig . full - length sidm ( 73 kda ) and the c - terminal fragments m7 ( 49 kda , sidm-(214647 ) ) , m9 ( 23 kda , sidm-(444647 ) ) , and m13 ( 12 kda , sidm-(544647 ) ) bound to ptdins(4)p but not to ptdins(4,5)p2 , which was used as a negative control ( fig . m13 was the smallest ptdins(4)p - binding fragment identified , and upon further cleavage into the n- and c - terminal fragments m17 and m19 , ptdins(4)p binding activity was completely lost . the m13 ptdins(4)p - binding domain does not show any homology to the ptdins(4)p - binding pleckstrin homology ( ph ) domain of the eukaryotic adaptor protein fapp1 ( 40 ) , the p4c domain of l. pneumophila sidc ( 33 ) , or any other prokaryotic or eukaryotic pi - binding protein . the n - terminal fragments of sidm , m1 , and m3 , or the internal fragments m5 , m11 , and m15 , did not bind to ptdins(4)p ( fig . 3 ) . notably , only the 49-kda fragment m7 , comprising amino acid residues 214647 of sidm , bound ptdins(4)p as efficiently as full - length sidm . the affinity of the smaller fragments m9 ( 23 kda , sidm-(444647 ) ) and m13 ( 12 kda , sidm-(544647 ) ) appeared to be 50-fold reduced , as estimated by a 2-fold dilution series of ptdins(4)p ( fig . we suggest that this may be attributed to the lack of predicted coiled coil regions or other structurally stabilizing elements in these fragments . structural analysis of sidm and fragments auc of purified full - length sidm revealed a single species of 71,282 586 da , indicating a homogeneous monomeric state ( fig . further structural analysis of full - length sidm and the fragments m7 , m9 , and m13 by cd revealed that the -helical content of the full - length protein and m7 fragment was similar and 67 or 71% , respectively ( fig . in contrast , the m9 and m13 fragments were found by cd spectroscopy to adopt only 48 and 59% -helical structure , compared with predictions of 69 or 73% , respectively . these results suggest that the m9 and m13 fragments are structurally less stable , a finding that is reinforced by the poorly resolved nmr spectra of n - labeled m13 protein ( data not shown ) . as a corollary , we suggest that the entire ptdins(4)p binding structural domain of sidm includes residues n - terminal to residue 444 , which are present in the m7 construct . in agreement with this notion , the m9 and m13 fragments were found by thermofluor assays to lack a thermal unfolding transition typical of a globular fold , whereas the longer constructs revealed an unfolding transition between 65 and 72 c ( fig . 4c ) . this instability of the m9 and m13 sidm fragments likely accounts for their apparent 50-fold reduced affinity for ptdins(4)p , compared with full - length sidm and the m7 fragment ( fig . table 1secondary structure contents of sidm and fragments estimated by cdsstrfragmentaaturndisordered % % % % sidm 67 ( 67.0 ) 6 ( 3.4 ) 12 15 m7 71 ( 71.7 ) 9 ( 3.0 ) 10 10 m9 48 ( 68.8 ) 24 ( 4.4 ) 10 18 m13 59 ( 73.1 ) 8 ( 5.8 ) 12 20 avalues in parentheses indicate predicted -helix or -strand contents from secondary structure prediction using dompred . secondary structure contents of sidm and fragments estimated by cdsstr values in parentheses indicate predicted -helix or -strand contents from secondary structure prediction using dompred . a , sedimentation equilibrium analysis of full - length sidm revealed an 71-kda species corresponding to a monomeric state . b , far - uv cd spectra of the full - length protein sidm ( red ) and the fragments m7 ( blue ) , m9 ( green ) , and m13 ( black ) . the helical structure is evidenced by strong negative ellipticities at around 220 and 208 nm . c , thermofluor assay for the full - length sidm protein and the fragments m7 , m9 , and m13 . the estimated unfolding transition temperatures of full - length sidm and the fragment m7 were 62.3 and 71.4 c , respectively , whereas the m9 and m13 fragments did not display cooperative unfolding transitions . a , sedimentation equilibrium analysis of full - length sidm revealed an 71-kda species corresponding to a monomeric state . b , far - uv cd spectra of the full - length protein sidm ( red ) and the fragments m7 ( blue ) , m9 ( green ) , and m13 ( black ) . the signal unit is converted into mean residue ellipticity ( mre ) . the helical structure is evidenced by strong negative ellipticities at around 220 and 208 nm . c , thermofluor assay for the full - length sidm protein and the fragments m7 , m9 , and m13 . the estimated unfolding transition temperatures of full - length sidm and the fragment m7 were 62.3 and 71.4 c , respectively , whereas the m9 and m13 fragments did not display cooperative unfolding transitions . c - terminal fragments of sidm localize to lcvs in vivo in vitro , 2 ) , a compound that has been identified as a lipid component of lcvs ( 32 ) . we used d. discoideum to address the question of whether sidm not only localizes to the lcv membrane in infected macrophages ( 19 , 20 ) but also in amoebae , and whether n - terminal ptdins(4)p - binding fragments of sidm are still translocated by the icm / dot t4ss and bind to the lcv membrane . to this end , we produced m45-tagged sidm and fragments thereof in the wild - type l. pneumophila strain jr32 , infected the amoebae , and analyzed the localization by immunofluorescence microscopy ( fig . full - length m45-sidm as well as m45-sidc , used as a positive control ( 33 ) , were translocated and bound to lcv membranes in d. discoideum amoebae , as expected . the sidm fragment m45-m7 , including the p4 m domain but lacking a 24-kda n - terminal fragment , was also translocated into d. discoideum and bound to lcv membranes , indicating that sidm possesses a c - terminal translocation signal . the translocation of m45-m7 required a functional icm / dot t4ss and did not occur in a icmt mutant strain . in a similar way however , using an anti - m45 antibody we neither detected translocation of m45-m9 or m45-m13 , nor production of these fragments in lysates of l. pneumophila ( data not shown ) . presumably , these small fragments are not sufficiently stable when produced in the bacteria , as indicated by the thermofluor experiments described above ( fig . 4c ) . to possibly stabilize the small sidm fragments , we constructed fusion proteins with a 67-kda n - terminal fragment of sidc ( sidc-(1586 ) ) that does not bind to ptdins(4)p in vitro and is not translocated to lcv membranes in vivo ( 33 ) . in addition , this strategy allowed the use of a polyclonal anti - sidc antibody , which is more sensitive than the monoclonal anti m45 antibody ( data not shown ) . whereas sidc-(1586)-m13 was still not detectable by western blot , sidc-(1586)-m9 was produced by l. pneumophila , although at a much reduced level ( 4% ) compared with full - length sidc ( data not shown ) . upon infection of d. discoideum with an l. pneumophila sidc - sdca mutant strain producing sidc-(1586)-m9 , the fusion protein was detected on lcvs by immunofluorescence using an anti - sidc antibody ( fig . this result indicates that the 23-kda sidm fragment m9 is translocated into d. discoideum and binds to lcvs , in agreement with the notion that the 12-kda n - terminal p4 m domain anchors sidm to the lcv membrane . as observed previously , full - length sidc but not sidc-(1608 ) was translocated and bound to lcvs ( 33 ) . sidm was the predominant protein bound by ptdins(4)p - coated agarose beads in l. pneumophila lysates ( fig . 1 ) , suggesting that this effector is a major ptdins(4)p - binding protein . as a corollary , higher amounts of other l. pneumophila ptdins(4)p - binding proteins are predicted to bind to lcvs in the absence of sidm . to test this hypothesis , we quantified on lcvs harboring different l. pneumophila strains the icm / dot substrate sidc , which binds to ptdins(4)p in vitro with an affinity comparable with sidm ( fig . the amount of sidc on lcvs was determined by immunofluorescence after infecting calnexin - gfp producing d. discoideum with l. pneumophila wild - type , sidm , sidc - sdca , or ralf mutant strains ( fig . the median fluorescence intensity of sidc bound to lcvs significantly increased 1.5 times in the absence of sidm ( p < 10 ) , whereas the absence of the pi - independent gef ralf did not affect binding of sidc to lcvs ( fig . the assay was specific for sidc , because upon infection of d. discoideum with an l. pneumophila strain lacking sidc and its paralogue sdca , only background fluorescence was measured on lcvs . moreover , whereas the sidc - sdca mutant strain did not produce any sidc as expected , the levels of sidc produced by wild - type , sidm , and ralf l. pneumophila were the same ( supplemental fig . s1 ) , ruling out that the different amounts of sidc on lcvs were because of different production levels of sidc in the bacterial strains . figure 5.c - terminal fragments of sidm localize to lcvs . a , confocal laser scanning micrographs of calnexin - gfp - labeled d. discoideum ax3 ( green ) , infected at an m.o.i . of 50 for 1 h with l. pneumophila labeled with a serogroup 1-specific antibody ( red ) and immunostained for m45-sidm , m45-m7 , and m45-sidc with an anti - m45 antibody ( blue ) . h with l. pneumophila sidc - sdca harboring plasmid pcr34 ( m45-sidc ) , pcr52 ( m45-sidc-(1608 ) ) , or peb216 ( sidc-(1586)-m9 ) and immunostained using antibodies against l. pneumophila serogroup 1 ( red ) and sidc ( green ) . the experiments were reproduced three ( a ) or two ( b ) independent times with similar results . c - terminal fragments of sidm localize to lcvs . a , confocal laser scanning micrographs of calnexin - gfp - labeled d. discoideum ax3 ( green ) , infected at an m.o.i . of 50 for 1 h with l. pneumophila labeled with a serogroup 1-specific antibody ( red ) and immunostained for m45-sidm , m45-m7 , and m45-sidc with an anti - m45 antibody ( blue ) . h with l. pneumophila sidc - sdca harboring plasmid pcr34 ( m45-sidc ) , pcr52 ( m45-sidc-(1608 ) ) , or peb216 ( sidc-(1586)-m9 ) and immunostained using antibodies against l. pneumophila serogroup 1 ( red ) and sidc ( green ) . the experiments were reproduced three ( a ) or two ( b ) independent times with similar results . in addition , we characterized lcvs harboring l. pneumophila wild - type , sidm , sidc - sdca , or ralf mutant strains with regard to the acquisition of the er marker calnexin . whereas lcvs harboring the sidc - sdca strain were defective for calnexin acquisition as described previously ( 33 ) , lcvs containing the sidm or ralf strains accumulated calnexin to the same extent as wild - type lcvs , indicating that trafficking and composition of these lcvs are similar ( data not shown ) . to confirm the findings obtained with sidc translocated by l. pneumophila , we ectopically produced the ptdins(4)p - binding probe gfp - sidcp4c in d. discoideum and quantified free ptdins(4)p on lcvs ( fig . using this probe , we observed that the gfp fluorescence intensity on lcvs containing either an l. pneumophila sidm or sidc - sdca mutant strain significantly increased 1.5 times ( p < 3 10 ) , compared with lcvs harboring wild - type l. pneumophila or a strain lacking the pi - independent gef ralf ( fig . hence , the results obtained with sidc endogenously produced by l. pneumophila and gfp - sidcp4c ectopically produced by d. discoideum are consistent . in summary , our findings indicate that sidm as well as sidc and sdca are major ptdins(4)p - binding effector proteins that compete for ptdins(4)p - binding sites on lcv membranes . production of ptdins(4)p on lcvs involves pi4k iiiptdins(4)p is synthesized from ptdins , which in metazoan cells is catalyzed by several pi4ks . these enzymes preferentially localize to different subcellular compartments : pi4k ii/ to the tgn , endosomes , and plasma membrane ; pi4k iii to the er , plasma membrane , and nucleus ; and pi4k iii to the golgi , respectively ( 34 , 49 ) . in the tgn , ptdins(4)p is formed by pi4k iii upon recruitment by arf1 ( 36 ) . to resolve whether a specific pi4k controls the levels of ptdins(4)p on lcvs , we knocked down the respective kinases by rna interference in drosophila kc167 phagocytes , which are permissive for intracellular replication of l. pneumophila ( 29 ) . although mrna of pi4k iii , pi4k iii , and pi4k ii was readily amplified by rt - pcr in control cells , dsrna oligonucleotides specific against individual pi4ks reduced gene expression to a level not detectable by rt - pcr ( supplemental fig . phagocytes with l. pneumophila , we quantified the amount of sidc on lcvs ( fig . 7a ) . using this assay , we found that the accumulation of sidc on lcvs was impaired upon depletion of pi4k iii , but not pi4k iii or pi4k ii ( fig . 7b ) . depletion of pi4k iii decreased the number of sidc - positive lcvs by 4.5-fold , indicating that this pi4k controls the level of ptdins(4)p on lcvs , which in turn is bound by sidc ( and other ptdins(4)p - binding effectors ) . because the depletion of pi4k iii did not impair intracellular replication of l. pneumophila ( data not shown ) , ptdins(4)p - dependent recruitment of sidc ( and other ptdins(4)p - binding effectors ) is not rate - limiting for intracellular replication . this result corresponds to the finding that l. pneumophila sidc - sdca ( 31 , 33 ) or sidm ( 19 , 20 ) mutant strains grow at wild - type rate . pneumophila forms a replicative vacuole within phagocytes by means of the icm / dot t4ss and more than 100 effector proteins , most of which have not been functionally characterized to date . we recently discovered that the icm / dot substrate sidc specifically binds to ptdins(4)p in vitro ( 32 ) . sidc is a bi - functional effector , which anchors to lcvs by binding to ptdins(4)p via its c - terminal p4c domain and promotes the interaction with er via its n - terminal domain ( 33 ) . based on these findings , we performed an unbiased screen using agarose beads coupled to different pis to discover other pi - binding l. pneumophila proteins . thus , we identified the rab1 gdf / gef sidm as a major ptdins(4)p - binding effector ( fig . 1 ) . this finding represents a novel link between the exploitation of pis and the modulation of host gtp metabolism by pathogenic bacteria . a , confocal laser scanning micrographs ; b , dot plot of sidc fluorescence on lcvs in calnexin - gfp - producing d. discoideum ax3 ( green ) , infected with dsred - labeled l. pneumophila ( red ) wild - type jr32 , sidm , ralf , or sidc - sdca and immunostained for sidc ( blue ) . the data and the median ( * , p < 10 ) are derived from three independent experiments ( n > 200 ) , which were normalized to the median of sidc fluorescence of wild - type jr32 . c , confocal laser scanning micrographs ; d , dot plot of gfp - sidcp4c fluorescence ( green ) on lcvs in d. discoideum ax3 harboring the plasmid psu01 , infected with dsred - labeled l. pneumophila ( red ) wild - type jr32 , sidm , ralf , or sidc - sdca . the data are combined from three independent experiments ( n > 143 ) , each normalized to the median fluorescence obtained with jr32 ( * , p < 3 10 ; * * , p < 5 10 ) . competition of sidm and sidc for ptdins(4)p on lcvs . a , confocal laser scanning micrographs ; b , dot plot of sidc fluorescence on lcvs in calnexin - gfp - producing d. discoideum ax3 ( green ) , infected with dsred - labeled l. pneumophila ( red ) wild - type jr32 , sidm , ralf , or sidc - sdca and immunostained for sidc ( blue ) . the data and the median ( * , p < 10 ) are derived from three independent experiments ( n > 200 ) , which were normalized to the median of sidc fluorescence of wild - type jr32 . c , confocal laser scanning micrographs ; d , dot plot of gfp - sidcp4c fluorescence ( green ) on lcvs in d. discoideum ax3 harboring the plasmid psu01 , infected with dsred - labeled l. pneumophila ( red ) wild - type jr32 , sidm , ralf , or sidc - sdca . the data are combined from three independent experiments ( n > 143 ) , each normalized to the median fluorescence obtained with jr32 ( * , p < 3 10 ; * * , p < 5 10 ) . sidm eluted as the predominant protein from ptdins(4)p - coated agarose beads , and no other proteins seemed to be strongly retained by any pi - coated beads . notably , a c - terminal fragment of sidc was identified in the eluate of ptdins(4)p - coated beads , but the effector was apparently not retained in high amounts by the beads . therefore , sidc might be either produced at lower levels compared with sidm , be less stable , or bind less strongly to ptdins(4)p - coated agarose beads . in lysates of l. pneumophila prepared like the samples used for the screen , sidc was readily detected by western blot ( 33 ) and stable for at least 20 h ( supplemental fig . s1 ) , indicating that sidc is indeed produced and not proteolytically degraded under these conditions . moreover , the binding affinity to ptdins(4)p of recombinant gst - sidc was comparable with that of gst - sidm ( fig . 2b ) , suggesting that the intrinsic affinity of the two purified effector proteins for ptdins(4)p is similar . to explain the paradox posed by the ptdins(4)p activities of sidc , we propose that the p4c ptdins(4)p - binding domain of sidc is masked , either in cis by one of its own domains or in trans by another protein . supporting the first notion , we found that in the absence of a 70-kda n - terminal fragment the 20-kda p4c fragment or a 36-kda c - terminal fragment seem to bind ptdins(4)p with higher affinity ( 33 ) . alternatively or additionally , sidc might be complexed by other l. pneumophila proteins in the bacterial cytoplasm ( lysate ) , thus preventing binding to ptdins(4)p . obvious candidates for such proteins are icms and icmw , which constitute a putative chaperone complex within the bacterial cell , necessary for icm / dot - mediated translocation of a subset of effectors ( 50 , 51 ) . translocation of sidc is significantly decreased in either l. pneumophila icms or icmw single mutant strains and occurs as much as 10-fold less efficiently in the icms - icmw double mutant ( 52 ) . in addition to sidc , the icms - icmw complex might bind other l. pneumophila effector proteins in the cytoplasm , thus preventing their interaction with ptdins(4)p in bacterial lysates . interestingly , l. pneumophila produces at least two families of pi - binding effector proteins , which display distinct preferences for pis . whereas sidm ( fig . 2 ) and sidc ( 32 , 33 ) almost exclusively bind ptdins(4)p , the icm / dot substrate lida preferentially binds ptdins(3)p but also ptdins(4)p ( fig . 2 ) , and the effector lpne ( 53 , 54 ) selectively binds ptdins(3)p ( 55 ) . accordingly , the specificity of l. pneumophila pi - binding effectors seems to be strongly biased toward mono - phosphorylated pis , in particular ptdins(4)p ( sidc and sidm ) and ptdins(3)p ( lida and lpne ) . because the cellular concentration of ptdins(4)p is much higher than ptdins(3)p , ptdins(4)p might actually be the dominant ligand for lida in vivo . this notion is in agreement with the function of lida as an auxiliary protein for the ptdins(4)p - binding effector sidm . ptdins(4)p - binding domain of sidm the minimal ptdins(4)p - binding domain of sidm was mapped to the 12-kda c - terminal m13 fragment and termed the p4 m domain ( fig . this domain includes amino acids 544647 and thus does not overlap with functional domains of sidm described previously . a number of functions of sidm have been assigned to amino acids 317545 , such as binding of the rab1 gtpase , as well as the gef and gdf activities ( 20 ) . the p4 m domain does not share homology with the ptdins(4)p - binding domain p4c of sidc ( 33 ) or with eukaryotic ptdins(4)p recognition folds , including the ph domain of fapp1 ( 40 ) , the px domain of bem1p ( 56 ) , and the vhs domain of gga2p ( 57 ) . however , the sidm topology may resemble the bar / imd domains , which are helical bundles that also bind pis and induce membrane curvature ( 58 ) . some bar domains form oligomers on membranes , and therefore we speculate that a ptdins(4)p- and membrane - dependent oligomerization of sidm might contribute to the higher affinity toward ptdins(4)p of the full - length protein compared with the m9 and m13 ( p4 m ) fragments ( fig . 3 ) . figure 7.production of ptdins(4)p on lcvs involves pi4k iii. a , confocal laser scanning micrographs of drosophila kc167 phagocytes treated with the dsrna indicated and infected at an m.o.i . of 50 for 15 min with dsred - labeled wild - type l. pneumophila ( red ) . recruitment of the ptdins(4)p - binding icm / dot substrate sidc was analyzed by immunofluorescence microscopy using an affinity - purified antibody against sidc ( green ) . means and standard deviations of three independent experiments are shown ( n = 303762 , * , p < 2 10 ) . production of ptdins(4)p on lcvs involves pi4k iii. a , confocal laser scanning micrographs of drosophila kc167 phagocytes treated with the dsrna indicated and infected at an m.o.i . of 50 for 15 min with dsred - labeled wild - type l. pneumophila ( red ) . recruitment of the ptdins(4)p - binding icm / dot substrate sidc was analyzed by immunofluorescence microscopy using an affinity - purified antibody against sidc ( green ) . means and standard deviations of three independent experiments are shown ( n = 303762 , * , p < 2 10 ) . the apparent ptdins(4)p affinity of the full - length sidm or the 49-kda m7 fragment is 50-fold higher than that of the 12-kda p4 m domain ( or the 23-kda m9 fragment ) ( fig . this stronger interaction is possibly caused by the greater structural stability of the longer forms , as evidenced by the cd and thermofluor experiments ( fig . , several coiled coils are predicted in the n - terminal 400 residues of sidm and are missing in the shorter constructs . however , this region does not appear to mediate obligatory homo - oligomerization of sidm , as the full - length protein is monomeric ( fig . the m3 , m5 , and m15 fragments are not directly involved in binding of ptdins(4)p ; however , these portions of sidm might contribute to stabilizing the p4 m domain , thereby increasing its affinity for ptdins(4)p . in contrast to sidm , the p4c domain of sidc , as well as its 36-kda c - terminal fragment , bound ptdins(4)p more tightly than the full - length effector protein ( 33 ) . thus , whereas the sidc p4c domain is a suitable probe for the analysis of ptdins(4)p in cell biological and biochemical experiments , only full - length sidm or the m7 fragment are recommended as stable ptdins(4)p - binding tools . both the p4 m and p4c domains are located in the c termini of the corresponding effector proteins . however , although the 12-kda p4 m domain constitutes the very c terminus of sidm , the 20-kda p4c domain lies 16-kda upstream of the c terminus of sidc . the c - terminal sidm fragment m7 and the fusion protein sidc-(1586)-m9 were translocated by icm / dot - proficient l. pneumophila into d. discoideum and bound to lcv membranes ( fig . 5 ) . this result suggests that sidm contains a c - terminal translocation signal , similar to the icm / dot substrates sidc ( 33 ) , ralf ( 59 ) , sdha , and its paralogue sidh ( 60 ) as well as sidg ( 52 ) . moreover , sidm features an isoleucine at position 4 in relation to the c terminus , which is in agreement with the finding that a hydrophobic amino acid at position 3 or 4 is critical for icm / dot - dependent secretion ( 59 ) . different classes of l. pneumophila gefs two different kinds of gefs can be classified in l. pneumophila based on the nature of their pi interactions . whereas the rab1 gef sidm localizes to lcvs ( 19 , 20 ) by binding to ptdins(4)p , the arf1 gef ralf localizes to lcvs ( 21 ) very likely independently of pis , because it does not bind to pis in vitro ( fig . sidm recruits and activates the small gtpase rab1 ( 19 , 20 ) , which is present in the host cytoplasm in its inactive state bound to a gdi . rab - gdi complexes are recognized by a specific gdf , and after gdi dissociation rab - gdp becomes membrane - associated before being activated by a membrane - bound gef ( 61 ) . in contrast , the small gtpase arf1 itself is able to associate with ptdins(4,5)p2 , and this in turn appears to promote a conformational change required for association with its gef ( 62 ) . these distinct features of the gtpases may account for the different characteristics of the two l. pneumophila gefs sidm and ralf with regard to pi binding . ptdins(4)p is present on lcvs ( 32 ) , and therefore , sidm as well as sidc likely anchor to the vacuole via this pi . even though these l. pneumophila effectors directly and selectively bound to ptdins(4)p in vitro , we can not rule out that binding on lcvs involves a co - receptor . the mammalian four - phosphate - adaptor proteins fapp1 and fapp2 interact with ptdins(4)p on the golgi through their ph domains , and additionally bind the gtp - bound form of the small gtpase arf1 ( 63 ) . l. pneumophila recruits and activates arf1 at the lcv membrane by means of the icm / dot substrate ralf ( 21 ) , and depletion of arf1 by rna interference abolishes binding of sidc to lcvs ( 30 ) . therefore , analogously to the fapps on the golgi , sidm and sidc might bind to ptdins(4)p in the context of activated arf1 on lcvs . on the other hand , depletion or inhibition of the pleiotropic small gtpase arf1 prevents the formation of replication - permissive lcvs altogether ( 7 ) , and thus , a drastically altered vacuole membrane composition might nonspecifically reduce the amounts of bound sidc . using rna interference in drosophila phagocytes , we showed that pi4k iii but not pi4k iii or pi4k ii promote the binding of sidc to lcvs ( fig . pi4k iii is recruited to the tgn by arf1 ( 36 ) , and therefore , the pi4k might localize to lcvs by direct fusion with the tgn or with other cellular compartments enriched in pi4k iii. alternatively , recruitment of cytoplasmic arf1 to lcvs by l. pneumophila ralf ( 21 ) might lead to an accumulation of pi4k iii. however , in the absence of ralf , the amount of sidc or ectopically expressed gfp - sidcp4c on lcvs in d. discoideum was not affected ( fig . 6 ) , indicating that the pathway involving arf1 and pi4k iii is probably not relevant on lcvs . in contrast to the deletion of ralf , deletion of sidm significantly increased the amount of sidc on lcvs ( fig . sidm recruits rab1 to lcvs ( 19 , 20 ) , yet knockdown of rab1 in drosophila cells did not affect the levels of sidc on lcvs ( 30 ) . therefore , the increased amounts of sidc on lcvs harboring l. pneumophila sidm mutant bacteria are likely caused by increased levels of free ptdins(4)p , which on lcvs harboring wild - type l. pneumophila is bound by sidm . in agreement with this notion , the amount of the ectopically produced ptdins(4)p probe sidcp4c , significantly increased on lcvs harboring either l. pneumophila sidm or sidc - sdca ( fig . together , these results support the general concept that l. pneumophila exploits specific host pis to anchor effector proteins to the lcv membrane , and furthermore , our findings suggest that sidm and sidc are ( the ) major ptdins(4)p - binding effectors , which compete for free ptdins(4)p - binding sites on lcvs .

the causative agent of legionnaires disease , legionella pneumophila , forms a replicative vacuole in phagocytes by means of the intracellular multiplication / defective organelle trafficking ( icm / dot ) type iv secretion system and translocated effector proteins , some of which subvert host gtp and phosphoinositide ( pi ) metabolism . the icm / dot substrate sidc anchors to the membrane of legionella - containing vacuoles ( lcvs ) by specifically binding to phosphatidylinositol 4-phosphate ( ptdins(4)p ) . using a nonbiased screen for novel l. pneumophila pi - binding proteins , we identified the rab1 guanine nucleotide exchange factor ( gef ) sidm / drra as the predominant ptdins(4)p - binding protein . purified sidm specifically and directly bound to ptdins(4)p , whereas the sidm - interacting icm / dot substrate lida preferentially bound ptdins(3)p but also ptdins(4)p , and the l. pneumophila arf1 gef ralf did not bind to any pis . the ptdins(4)p - binding domain of sidm was mapped to the 12-kda c - terminal sequence , termed p4 m ( ptdins4p binding of sidm / drra ) . the isolated p4 m domain is largely helical and displayed higher ptdins(4)p binding activity in the context of the -helical , monomeric full - length protein . sidm constructs containing p4 m were translocated by icm / dot - proficient l. pneumophila and localized to the lcv membrane , indicating that sidm anchors to ptdins(4)p on lcvs via its p4 m domain . an l. pneumophila sidm mutant strain displayed significantly higher amounts of sidc on lcvs , suggesting that sidm and sidc compete for limiting amounts of ptdins(4)p on the vacuole . finally , rna interference revealed that ptdins(4)p on lcvs is specifically formed by host ptdins 4-kinase iii. thus , l. pneumophila exploits ptdins(4)p produced by ptdins 4-kinase iii to anchor the effectors sidc and sidm to lcvs .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

identification of sidm as a pi - binding protein the signaling lipid ptdins(4)p was recently discovered to be specifically recognized by the icm / dot substrate sidc on lcvs ( 32 , 33 ) . to identify additional pi - binding proteins of l. pneumophila , we performed pulldown assays using agarose beads coated with individual pis . the ptdins(4)p - binding icm / dot substrate sidc , which is not similar to sidm in sequence , was expected to also be identified in this screen for pi - binding l. pneumophila proteins . although we did not identify full - length sidc under the above conditions , small amounts of a 50-kda c - terminal sidc fragment , including the ptdins(4)p - binding domain p4c ( 33 ) , were retained by ptdins(4)p - coated agarose beads and identified by mass spectrometry ( data not shown ) . to determine whether sidc or other l. pneumophila proteins bind to ptdins(4)p - coated agarose beads more efficiently in the absence of sidm , we repeated the pulldown experiments using lysates of an l. pneumophila sidm strain . whereas in the absence of sidm no protein bound in high amounts to ptdins(4)p - coated beads , in the absence of sidc only sidm was detected ( fig . sidc was found to be stable in the absence and in presence of ptdins(4)p - coated agarose beads for at least 20 h , and thus , proteolysis does not account for the failure to recover significant amounts of sidc under the conditions used . sidm specifically binds to ptdins(4)p in vitro to assess the pi - binding specificity of heterologously produced , purified sidm , we tested binding of an n - terminal gst - sidm fusion protein to agarose beads coated with different pis . to compare the ptdins(4)p affinities of icm / dot substrates localizing to lcvs , we directly compared binding of the corresponding gst fusion proteins in pulldown assays . similar amounts of purified gst - sidc and gst - sidm eluted from the beads , indicating that the affinities of the two different effectors to ptdins(4)p are comparable . in contrast , the arf1 gef ralf did not bind to any pis or other lipids on the membrane , suggesting that l. pneumophila produces distinct classes of gefs that localize to lcvs either pi - dependently or pi - independently . interestingly , the sidm auxiliary protein lida preferentially bound to ptdins(3)p but also significantly to ptdins(4)p . sidm and sidc specifically bound to ptdins(4)p , whereas lida showed a more relaxed pi - binding specificity and preferentially bound to ptdins(3)p but also weakly to ptdins(4)p . identification of the ptdins(4)p - binding domain of sidm to map the ptdins(4)p - binding domain of sidm , we constructed n - terminal fusions of gst with fragments of sidm of different lengths and visualized binding of the fusion proteins to ptdins(4)p by protein - lipid overlay assays ( fig . full - length sidm ( 73 kda ) and the c - terminal fragments m7 ( 49 kda , sidm-(214647 ) ) , m9 ( 23 kda , sidm-(444647 ) ) , and m13 ( 12 kda , sidm-(544647 ) ) bound to ptdins(4)p but not to ptdins(4,5)p2 , which was used as a negative control ( fig . m13 was the smallest ptdins(4)p - binding fragment identified , and upon further cleavage into the n- and c - terminal fragments m17 and m19 , ptdins(4)p binding activity was completely lost . the m13 ptdins(4)p - binding domain does not show any homology to the ptdins(4)p - binding pleckstrin homology ( ph ) domain of the eukaryotic adaptor protein fapp1 ( 40 ) , the p4c domain of l. pneumophila sidc ( 33 ) , or any other prokaryotic or eukaryotic pi - binding protein . the n - terminal fragments of sidm , m1 , and m3 , or the internal fragments m5 , m11 , and m15 , did not bind to ptdins(4)p ( fig . further structural analysis of full - length sidm and the fragments m7 , m9 , and m13 by cd revealed that the -helical content of the full - length protein and m7 fragment was similar and 67 or 71% , respectively ( fig . as a corollary , we suggest that the entire ptdins(4)p binding structural domain of sidm includes residues n - terminal to residue 444 , which are present in the m7 construct . this instability of the m9 and m13 sidm fragments likely accounts for their apparent 50-fold reduced affinity for ptdins(4)p , compared with full - length sidm and the m7 fragment ( fig . b , far - uv cd spectra of the full - length protein sidm ( red ) and the fragments m7 ( blue ) , m9 ( green ) , and m13 ( black ) . the estimated unfolding transition temperatures of full - length sidm and the fragment m7 were 62.3 and 71.4 c , respectively , whereas the m9 and m13 fragments did not display cooperative unfolding transitions . b , far - uv cd spectra of the full - length protein sidm ( red ) and the fragments m7 ( blue ) , m9 ( green ) , and m13 ( black ) . the estimated unfolding transition temperatures of full - length sidm and the fragment m7 were 62.3 and 71.4 c , respectively , whereas the m9 and m13 fragments did not display cooperative unfolding transitions . we used d. discoideum to address the question of whether sidm not only localizes to the lcv membrane in infected macrophages ( 19 , 20 ) but also in amoebae , and whether n - terminal ptdins(4)p - binding fragments of sidm are still translocated by the icm / dot t4ss and bind to the lcv membrane . the sidm fragment m45-m7 , including the p4 m domain but lacking a 24-kda n - terminal fragment , was also translocated into d. discoideum and bound to lcv membranes , indicating that sidm possesses a c - terminal translocation signal . to possibly stabilize the small sidm fragments , we constructed fusion proteins with a 67-kda n - terminal fragment of sidc ( sidc-(1586 ) ) that does not bind to ptdins(4)p in vitro and is not translocated to lcv membranes in vivo ( 33 ) . this result indicates that the 23-kda sidm fragment m9 is translocated into d. discoideum and binds to lcvs , in agreement with the notion that the 12-kda n - terminal p4 m domain anchors sidm to the lcv membrane . as a corollary , higher amounts of other l. pneumophila ptdins(4)p - binding proteins are predicted to bind to lcvs in the absence of sidm . to test this hypothesis , we quantified on lcvs harboring different l. pneumophila strains the icm / dot substrate sidc , which binds to ptdins(4)p in vitro with an affinity comparable with sidm ( fig . the median fluorescence intensity of sidc bound to lcvs significantly increased 1.5 times in the absence of sidm ( p < 10 ) , whereas the absence of the pi - independent gef ralf did not affect binding of sidc to lcvs ( fig . moreover , whereas the sidc - sdca mutant strain did not produce any sidc as expected , the levels of sidc produced by wild - type , sidm , and ralf l. pneumophila were the same ( supplemental fig . to confirm the findings obtained with sidc translocated by l. pneumophila , we ectopically produced the ptdins(4)p - binding probe gfp - sidcp4c in d. discoideum and quantified free ptdins(4)p on lcvs ( fig . using this probe , we observed that the gfp fluorescence intensity on lcvs containing either an l. pneumophila sidm or sidc - sdca mutant strain significantly increased 1.5 times ( p < 3 10 ) , compared with lcvs harboring wild - type l. pneumophila or a strain lacking the pi - independent gef ralf ( fig . in summary , our findings indicate that sidm as well as sidc and sdca are major ptdins(4)p - binding effector proteins that compete for ptdins(4)p - binding sites on lcv membranes . to resolve whether a specific pi4k controls the levels of ptdins(4)p on lcvs , we knocked down the respective kinases by rna interference in drosophila kc167 phagocytes , which are permissive for intracellular replication of l. pneumophila ( 29 ) . phagocytes with l. pneumophila , we quantified the amount of sidc on lcvs ( fig . depletion of pi4k iii decreased the number of sidc - positive lcvs by 4.5-fold , indicating that this pi4k controls the level of ptdins(4)p on lcvs , which in turn is bound by sidc ( and other ptdins(4)p - binding effectors ) . pneumophila forms a replicative vacuole within phagocytes by means of the icm / dot t4ss and more than 100 effector proteins , most of which have not been functionally characterized to date . we recently discovered that the icm / dot substrate sidc specifically binds to ptdins(4)p in vitro ( 32 ) . sidc is a bi - functional effector , which anchors to lcvs by binding to ptdins(4)p via its c - terminal p4c domain and promotes the interaction with er via its n - terminal domain ( 33 ) . thus , we identified the rab1 gdf / gef sidm as a major ptdins(4)p - binding effector ( fig . notably , a c - terminal fragment of sidc was identified in the eluate of ptdins(4)p - coated beads , but the effector was apparently not retained in high amounts by the beads . to explain the paradox posed by the ptdins(4)p activities of sidc , we propose that the p4c ptdins(4)p - binding domain of sidc is masked , either in cis by one of its own domains or in trans by another protein . interestingly , l. pneumophila produces at least two families of pi - binding effector proteins , which display distinct preferences for pis . 2 ) and sidc ( 32 , 33 ) almost exclusively bind ptdins(4)p , the icm / dot substrate lida preferentially binds ptdins(3)p but also ptdins(4)p ( fig . accordingly , the specificity of l. pneumophila pi - binding effectors seems to be strongly biased toward mono - phosphorylated pis , in particular ptdins(4)p ( sidc and sidm ) and ptdins(3)p ( lida and lpne ) . ptdins(4)p - binding domain of sidm the minimal ptdins(4)p - binding domain of sidm was mapped to the 12-kda c - terminal m13 fragment and termed the p4 m domain ( fig . the p4 m domain does not share homology with the ptdins(4)p - binding domain p4c of sidc ( 33 ) or with eukaryotic ptdins(4)p recognition folds , including the ph domain of fapp1 ( 40 ) , the px domain of bem1p ( 56 ) , and the vhs domain of gga2p ( 57 ) . some bar domains form oligomers on membranes , and therefore we speculate that a ptdins(4)p- and membrane - dependent oligomerization of sidm might contribute to the higher affinity toward ptdins(4)p of the full - length protein compared with the m9 and m13 ( p4 m ) fragments ( fig . recruitment of the ptdins(4)p - binding icm / dot substrate sidc was analyzed by immunofluorescence microscopy using an affinity - purified antibody against sidc ( green ) . recruitment of the ptdins(4)p - binding icm / dot substrate sidc was analyzed by immunofluorescence microscopy using an affinity - purified antibody against sidc ( green ) . the apparent ptdins(4)p affinity of the full - length sidm or the 49-kda m7 fragment is 50-fold higher than that of the 12-kda p4 m domain ( or the 23-kda m9 fragment ) ( fig . the m3 , m5 , and m15 fragments are not directly involved in binding of ptdins(4)p ; however , these portions of sidm might contribute to stabilizing the p4 m domain , thereby increasing its affinity for ptdins(4)p . in contrast to sidm , the p4c domain of sidc , as well as its 36-kda c - terminal fragment , bound ptdins(4)p more tightly than the full - length effector protein ( 33 ) . thus , whereas the sidc p4c domain is a suitable probe for the analysis of ptdins(4)p in cell biological and biochemical experiments , only full - length sidm or the m7 fragment are recommended as stable ptdins(4)p - binding tools . however , although the 12-kda p4 m domain constitutes the very c terminus of sidm , the 20-kda p4c domain lies 16-kda upstream of the c terminus of sidc . the c - terminal sidm fragment m7 and the fusion protein sidc-(1586)-m9 were translocated by icm / dot - proficient l. pneumophila into d. discoideum and bound to lcv membranes ( fig . this result suggests that sidm contains a c - terminal translocation signal , similar to the icm / dot substrates sidc ( 33 ) , ralf ( 59 ) , sdha , and its paralogue sidh ( 60 ) as well as sidg ( 52 ) . whereas the rab1 gef sidm localizes to lcvs ( 19 , 20 ) by binding to ptdins(4)p , the arf1 gef ralf localizes to lcvs ( 21 ) very likely independently of pis , because it does not bind to pis in vitro ( fig . l. pneumophila recruits and activates arf1 at the lcv membrane by means of the icm / dot substrate ralf ( 21 ) , and depletion of arf1 by rna interference abolishes binding of sidc to lcvs ( 30 ) . therefore , analogously to the fapps on the golgi , sidm and sidc might bind to ptdins(4)p in the context of activated arf1 on lcvs . therefore , the increased amounts of sidc on lcvs harboring l. pneumophila sidm mutant bacteria are likely caused by increased levels of free ptdins(4)p , which on lcvs harboring wild - type l. pneumophila is bound by sidm . together , these results support the general concept that l. pneumophila exploits specific host pis to anchor effector proteins to the lcv membrane , and furthermore , our findings suggest that sidm and sidc are ( the ) major ptdins(4)p - binding effectors , which compete for free ptdins(4)p - binding sites on lcvs .

microglia and astrocytes are well known to play an important role in homeostasis within the cns and to support neuronal cell function . however , these cell types are now recognized to be key players in the development of protective immune responses or the progression of damaging inflammation during cns disease states ( bauer et al . , 1995 ; stoll and jander , 1999 ; dong and benveniste , 2001 ; fischer and reichmann , 2001 ) . microglia are resident myeloid immune cells of the cns and , similar to other myeloid cells such as macrophages and dendritic cells , these cells are facultative phagocytes and express antigen presenting mhc class ii molecules ( hickey and kimura , 1988 ) . importantly , microglia produce key pro - inflammatory mediators such as il-1 ( martin et al . , 1993 ) , tnf- ( streit et al . , 1998 ) , il-6 ( kiefer et al . , 1993 ) , and bioactive il-12 p70 ( aloisi et al . , 1997 ; suzumura et al . , 1998 ) astrocytes are the major glial cell type in the brain and may also function as immune effector cells ( dong and benveniste , 2001 ) . stimulated astrocytes can express an array of inflammatory cytokines and chemokines that can initiate leukocyte migration across the blood brain barrier and promote effector functions in these infiltrating cells ( dong and benveniste , 2001 ) . as such , these cells are ideally situated to detect and respond to invading pathogens including neurotropic viruses . viral infections in the cns are known to broadly trigger glial activation and the release of pro - inflammatory molecules . for example , it is known that pro - inflammatory cytokines , ifn- and tnf- , are markedly increased in cns tissues during hsv-1 infection in the brain and microglia have been shown to respond to hsv-1 by secreting pro - inflammatory and chemotactic molecules such as tnf- , il-1 , il-6 , il-12 , ccl7 , ccl8 , ccl9 , cxcl1 , cxcl2 , cxcl4 , and cxcl5 ( lokensgard et al . , 2001 ; aravalli et al . , 2006 ) . human microglia have also been shown to respond to rna viruses including wnv by producing cytokines and chemokines ( cheeran et al . , 2005 ) . elevated levels of il-1 are readily detectable in neural tissue from wnv encephalitis patients and cultured human glia produce this potent inflammatory cytokine in response to wnv challenge ( van marle et al . , 2007 ) . we have also documented the ability of primary cultured microglia and astrocytes to respond to several rna and dna viruses , including vesicular stomatitis virus ( vsv ) , sendai virus , murine gammaherpesvirus ( mhv)-68 , and hsv-1 , by producing inflammatory mediators such as il-6 , tnf- , and il-1 ( rasley et al . , 2004 ; chauhan et al . , 2010 ; furr et al . , 2010 , 2011 ) . importantly , we confirmed that in vivo vsv administration results in viral infection of both glial cell types in situ , while earlier studies demonstrated that microglia and astrocytes respond to vsv by proliferating , producing inducible nitric oxide synthase , and increasing the expression of cell surface mhc class ii molecules ( bi et al . , 1995 ) , responses that are likely to set the stage for subsequent inflammatory damage . an immune response to a virus in the cns requires the recognition of this pathogen and the mechanisms by which innate immune cells achieve this have only recently become apparent with the discovery of a variety of cell surface and cytosolic molecules that serve as sensors for viral components . such pattern recognition receptors ( prrs ) are present on and within immune sentinel cells ( furr et al . , 2008 , 2011 ; kielian , 2009 ) and appear to bind conserved viral structures known as pathogen - associated molecular patterns ( pamps ) . first , pamps are unique to microbes and viruses allowing for self versus non - self recognition by the innate immune system . second , pamps are often conserved among similar pathogens allowing a relatively small number of prrs to detect a broad array of challenges . in addition , pamps tend to be essential for microbial / viral survival making pathogen evasion by genetic mutation or deletion unlikely ( medzhitov and janeway , 2002 ) . pattern recognition receptor engagement by pamps can precipitate the production of anti - viral type i interferons ( ifn ) and/or the release of chemotactic and inflammatory molecules that can then direct a subsequent adaptive immune response . three families of prrs have been defined : toll - like receptors ( tlrs ) , nucleotide oligomerization domain ( nod)-like receptors ( nlrs ) , and retinoic acid inducible gene ( rig)-i - like receptors ( rlrs ) . as such , these molecules represent important mechanisms by which a protective host response or potentially damaging inflammation is initiated ( kawai and akira , 2007 ; yoneyama and fujita , 2010 ) . toll was first identified as a gene critical for fruit fly embryogenesis ( hashimoto et al . , 1988 ) but its products were subsequently shown to play an important role in drosophila s immunity to fungal infections ( lemaitre et al . , 1996 ) . interestingly , similar gene products were found in mammalian cells and these tlrs have since been shown to play an important role in the initiation of innate immune responses to infection ( medzhitov et al . , 1997 ) . to date , at least 13 tlrs have been discovered in mammals and members of this family of cell surface receptors can elicit inflammatory and/or anti - viral mediator production and can serve to initiate or modify adaptive immune responses ( as reviewed in kawai and akira , 2008 ) . acute virally induced cns inflammatory responses occur within the first few days following infection and this argues against a central role for activated t cell clones in such responses . indeed , previous studies have shown that acute viral encephalitis can occur in athymic mice ( bi et al . , 1995 ) . that being said , t cell subpopulations , such as th1 and th17 cells , are almost certain to play a key role in sustained virally induced neuroinflammation ( kalinke and prinz , 2012 ) . as such , glial activation via tlrs and other prrs is likely to facilitate antigen presentation to , and costimulation of , such cells and the role of these receptors in the antigen - specific activation of t cells following viral challenge has been extensively reviewed elsewhere ( kawai and akira , 2008 ) . several tlrs have been demonstrated to specifically recognize viral motifs , including tlrs 2 , 3 , 7 , 8 , and 9 . while tlr2 is best known to bind a variety of microbial cell wall component including lipoproteins , peptidoglycans , and lipoteichoic acid present in bacterial cell walls and the yeast cell wall component zymosan , this sensor can also recognize as yet unidentified viral motifs . in contrast , endosomal tlrs such as tlr3 recognize viral double - stranded rna ( dsrna ) and its synthetic analog , polyinosine - deoxycytidylic acid ( poly(i : c ) ) , while tlr7 and tlr8 mediate responses to gu - rich single - stranded rna ( ssrna ) produced in virus - infected cells . studies from our group and others have demonstrated that microglia and astrocytes constitutively express low levels of tlr3 ( jack et al . 2006 ) , tlr7 ( jack et al . , 2005 ) , and tlr9 ( bowman et al . , 2003 ; jack et al . , 2005 ) , but such expression shows marked upregulation following viral infection and/or stimulation with viral components ( bowman et al . , 2003 ; carpentier et al . , 2005 ; jack et al . , 2005 ; mckimmie and fazakerley , 2005 ; mckimmie et al . , 2005 ; bsibsi et al . , while these findings suggest that glial cells could become sensitized to the presence of viral pathogens , it is presently unclear whether sustained stimulation via these sensors can alter the pattern of immune mediator production by glial cells to one that favors the resolution of inflammation . upon ligand binding , tlrs dimerize and undergo conformational changes precipitating a complex cascade of intracellular signaling events that ultimately result in activation of transcription factors including nuclear factor-b ( nf-b ) , and interferon - regulatory factors ( irf ) 3 and 7 ( as reviewed in kawai and akira , 2008 ; wilkins and gale , 2010 ) . nf-b is a critical transcription factor in the regulation of inflammatory cytokine production , while irf-3 and irf-7 activation stimulates expression of anti - viral genes including ifn-. hence , activation of cells via these receptors can initiate the repertoire of defense mechanisms used by the innate immune system against viral pathogens . tlr3 is perhaps the most widely studied prr in the perception of viral pathogens by glial cells . isolated murine wild - type microglia are known to respond to the tlr3 ligand poly(i : c ) by secreting tnf- and il-6 ( melton et al . , 2003 ) . in line with these findings , intracerebral administration of poly(i : c ) has been shown to elicit microglial ( melton et al . , 2003 ; town et al . , 2006 ) and astrocyte ( melton et al . , 2003 ) activation associated with neuronal loss , a response that is nearly absent in tlr3 knockout animals ( town et al . , circumstantial evidence for the involvement of tlr3 in viral encephalitis comes from the finding that the expression of this prr is upregulated following rabv infection prior to the onset of lethal neuroinflammation ( mckimmie and fazakerley , 2005 ; mckimmie et al . , dsrna , a ligand for tlr3 , is produced by wnv during infection and has been shown to trigger the release of cytokines and chemokines from human microglia ( cheeran et al . , 2005 ) . seemingly in keeping with these observations , tlr3 deficient mice were found to be resistant to lethal wnv - associated encephalitis . the fulminate cns inflammatory response seen in wild - type mice , characterized in part by activated microglia , was markedly reduced or absent in knockout animals following intraperitoneal wnv infection ( wang and fikrig , 2004 ) . however , this reduced cns disease occurs despite increased peripheral viral loads and reduced systemic production of anti - viral and pro - inflammatory cytokines ( wang et al . , these results suggest that decreased systemic inflammation in the absence of tlr3 expression may maintain blood brain barrier integrity and prevent wnv access into the cns ( wang et al . , 2004 ) . such a hypothesis is supported by the demonstration that direct intracerebral administration of wnv leads to identical outcomes in wild - type and tlr3 deficient mice ( wang et al . , 2004 ) . however , it must be noted that at least one study appears to contradict this finding in that neuronal wnv replication was increased in tlr3 knockout animals following subcutaneous infection and the reason for this apparent disparity is presently unclear ( daffis et al . , 2008 ) . another endosome - associated prr , tlr7 , also appears to play an important role in inflammatory and/or protective glial responses to viral pathogens . intracerebral administration of the tlr7 agonist imiquimod leads to pronounced neuroinflammation , with upregulated expression of tnf- , il-1 , il-1 , il-6 , and il-12 as well as the chemokines ccl2 , ccl3 , cxcl1 , cxcl9 , and cxcl10 ( butchi et al . , interestingly however , this effect appears to be predominantly due to the activation of astrocytes rather than microglia ( butchi et al . , 2008 ) . upregulation of tlr7 has been demonstrated in the brains of infected mice prior to lethal rabv - induced inflammation ( mckimmie et al . , 2005 ) . furthermore , mice lacking the expression of myd88 , a critical downstream effector molecule of tlrs including tlr7 , have increased susceptibility to an attenuated rabv strain and tlr7 deficient mice exhibited a phenotype with deficits in both the development of peripheral immunity and rabv clearance from the cns ( li et al . , 2011 ) . as such , these data indicate a possible role for tlr7 in viral clearance from infected brain tissue . in contrast , another report has shown that macrophages isolated from tlr7 deficient animals released less il-12 and il-23 in response to in vitro wnv infection and tlr7 knockout mice demonstrated an increased susceptibility to wnv encephalitis ( town et al . , 2009 ) . it has recently been shown that tlr2 contributes to detrimental inflammatory cytokine production in mice following intracranial administration with hsv-1 , with the absence of tlr2 expression leading to decreased peripheral ( kurt - jones et al . , 2004 ) and cns ( aravalli et al . , 2006 ) in addition , multiple reports have shown that maximal in vitro microglial and astrocyte responses to hsv-1 are dependent on the presence of tlr2 ( lokensgard et al . 2012 ) and hsv-1-infected microglia derived from tlr2 deficient mice have been demonstrated to elicit less neuronal oxidative damage and death in mixed neural cell cultures in comparison to infected wild - type microglia ( schachtele et al . , 2010 ) . however , it should be noted that these studies differ in their assessments of the relative importance of this prr in such responses , and marked elevations in serum il-6 concentrations have been found in tlr2 deficient mice following hsv-1 infection ( kurt - jones et al . , 2004 ) . together , these data suggest while tlr2 may play a major role in hsv - induced pathology and mortality , additional mechanism(s ) are likely to be involved . in addition to the detection of hsv virion motifs by tlr2 , genomic hsv dna can be recognized by tlr9 and at least one study suggests that this sensor might play a more significant role in lethal hsv-1 encephalitis than tlr2 ( lima et al . , 2010 ) . in contrast , others have shown that the absence of tlr9 alone does not impact survival , type i ifn levels , or viral replication in the brain following hsv infection ( wang et al . , since tlr2/9 double knockout mice show significantly greater susceptibility to hsv-1 infection than mice deficient in either tlr2 or tlr9 alone ( lima et al . , 2010 ) , it appears likely that these sensors act in a cooperative or synergistic manner . taken together , this body of work suggests that tlr activation is a significant contributor to sustained glial activation , inflammatory mediator production , and neuronal loss during viral cns infections . however , it is important to note that tlrs can also mediate beneficial effects . for example , the activation of glial cells via tlr2 , 3 , or 9 has been shown to initiate the production of type i ifns ( zhou et al . , 2009 ; conrady et al . , 2010 ) which limit viral replication through the induction of proteins such as rnase l and ifn - induced dsrna - activated protein kinase , resulting in mrna degradation and cessation of translation , respectively ( samuel , 2001 ) . furthermore , two recent studies ( zhang et al . , 2007 ; guo et al . , 2011 ) indicate that a genetic deficiency in tlr3 expression can predispose children to hsv-1 encephalitis and implicate a key role for this prr in protective cns immune responses against this , but not other , pathogens . however , the mechanisms that keep the balance between damaging and protective glial responses have not been identified and are likely to include a multitude of variables including the number of infectious organisms , the kinetics of glial activation and immune mediator production , the specific identity of the responsive glial cell , and the direct influence of the pathogen itself on the cellular response . moreover , evidence is accumulating that tlr expression is not limited to glial cells and that neurons can also express such prrs under certain conditions ( as reviewed in carty and bowie , 2011 ) , although , the functional relevance of tlr expression in neurons has not been resolved . while cell - surface receptors like tlrs appear to play an important role in glial responses to extracellular pathogens or those present in intracellular compartments , the ability of such cells to respond to pathogens in the absence of tlr expression suggests that additional mechanisms are present . furthermore , our recent studies employing the model neurotropic rna virus , vsv , demonstrate that viral replication is required to elicit robust immune responses by infected microglia and astrocytes , and indicate that mechanisms exist to sense the presence of replicative viral products within infected cells . we have shown that heat inactivated wild - type vsv or a host - range limited vsv mutant ( grdzelishvili et al . , 2005 ) elicit glial immune responses that are an order of magnitude smaller than those induced by wild - type viral particles ( chauhan et al . , 2010 ) . these results suggest that vsv - induced glial responses are not predominantly mediated by cell surface and/or endosomal prrs and indicate that active viral replication is a critical requirement for such responses . the recent demonstration that microglia and astrocytes express members of the newly described rlr family of prrs may provide a mechanism underlying the replication - dependent nature of glial responses ( furr et al . , 2008 ) . rlrs such as rig - i and melanoma differentiation - associated gene 5 ( mda5 ) have been shown to serve as intracellular prrs for viral nucleic acid motifs ( kato et al . , 2006 ; takeuchi and akira , 2008 ) , and their involvement in inflammatory and anti - viral responses in other cell types raises the intriguing possibility that these cytosolic sensors could play an important role in glial responses to viral infection . in contrast to cell - surface tlrs , rlrs detect viral rna motifs or processed self - rna in the cytoplasm to trigger innate immunity and inflammation ( takeuchi and akira , 2010 ) . these intracellular sensors are responsible for the production of type i ifns in most cell types in response to rna virus infection ( sumpter et al . , 2005 ; kato et al . , 2006 ; in addition to such anti - viral responses , rlr engagement also elicits robust inflammatory cytokine production ( takeuchi and akira , 2010 ) . as shown in figure 1 , helicase interaction with viral rna induces the recruitment of downstream effector molecules including interferon promoter stimulator ( ips)-1 ( also known as visa , cardif , or mavs ) , tnf receptor associated factor ( traf ) 3 ( takeuchi and akira , 2010 ) , and perhaps stimulator of interferon genes ( sting ; ishikawa and barber , 2008 ) , leading to the activation of mitogen activated protein ( map ) kinases , and the ib kinase ( ikk)-related kinases , traf family member - associated nf-b activator ( tank ) binding kinase 1 and ikk - i . these kinases activate the transcription factors nf-b and irf-3 , which translocate into the nucleus and activate transcription of ifn stimulated genes and inflammatory cytokines . rlrs are comprised of three domains : n - terminal caspase activation and recruitment domains ( cards ) , a helicase domain , and a c - terminal domain ( ctd ) . although rig - i and mda5 show structural homology ( yoneyama et al . , 2004 ) and share signaling features ( yoneyama et al . , 2005 ) , it is now known that the two helicases discriminate between differing ligands to trigger innate immune responses to rna viruses . retinoic acid inducible gene ( rig)-i - like receptors serve as cytosolic sensors for viral rna motifs and can initiate anti - viral and inflammatory cell responses . melanoma differentiation - associated gene 5 ( mda5 ) recognizes double - stranded rna moieties ( dsrna ) , while rig - i perceives short single or double - stranded rnas with 5 triphosphate ends ( ppp - ssrna ) . helicase interaction with these viral rnas induces the recruitment of downstream effector molecules including mitochondrial- or peroxisomal - associated interferon promoter stimulator ( ips)-1 , tnf receptor associated factor ( traf ) 3 , and/or stimulator of interferon genes ( sting ) , leading to the activation of ib kinase ( ikk)-related kinases , traf family member - associated nf-b activator binding kinase 1 ( tbk1 ) , and ikk - i . these kinases activate the transcription factors nf-b and interferon - regulatory factors ( irf ) 3 , which translocate into the nucleus and activate transcription of anti - viral and inflammatory cytokines . signaling by rig - i is triggered during infection by a number of rna viruses and by the presence of synthetic rna transcribed in vitro ( sumpter et al . , 2005 ; kato et al . , 2006 ; saito et al . , 2007 ; it appears that rig - i recognizes ssrna or dsrna moieties with 5 triphosphate ends ( 5ppp - ssrna and 5ppp - dsrna , respectively ; hornung et al . 2006 ) or rnas that assume complex secondary structures ( sumpter et al . , 2005 ; saito et al . , 2007 ) . inactive rig - i has an open conformation in which the card domains are sequestered by a helical domain inserted between the helicase moieties . when activated , atp and rna binding induce a major rearrangement in rig - i to a closed conformation in which the helicase and the ctd bind to the blunt end 5ppp - dsrna with perfect complementarity ( kowalinski et al . , 2011 ) . in contrast , mda5 has been reported to be more selective and is triggered during picornavirus infections or in the presence of poly(i : c ) ( gitlin et al . , 2006 ; kato et al . , 2006 ; loo et al . , 2008 ) . the ctd domains of mda5 and rig - i involved in rna binding have been suggested to be responsible for the differences in ligand affinity seen between these rlrs . each have a similar fold and basic surface but distinct rna binding loops exist within the ctd for rig - i and mda5 , and the conformation of this rna binding loop seems to be responsible for sensitivity to dsrna or 5ppp - ssrna . such a conclusion is supported by rlr structural analysis and studies featuring mutation of the basic surface and the rna binding loop ( li et al . , 2009 ; takahasi et al . , we have recently demonstrated that expression of both rig - i and mda5 is discernable in uninfected mouse brain tissue ( furr et al . importantly , we have shown that isolated murine microglia and astrocytes constitutively express rig - i and mda5 transcripts and protein , as well as the critical downstream effector molecule , ips-1 ( furr et al . , 2008 ) . furthermore , the expression of these novel viral sensors has been confirmed in human glial cells ( yoshida et al . circumstantial evidence of a role for these rlrs in rna virus infections of the cns comes from our observation that rig - i and mda5 expression is elevated in the brains of mice following intranasal administration of vsv ( furr et al . , vsv is a neurotropic negative - sense ssrna virus that closely resembles rabv . in mice , intranasal vsv infection results in a severe encephalitis with rapid activation and proliferation of microglia and astrocytes ( huneycutt et al . , 1993 ; bi et al . , 1995 ) and our in vitro studies show that this virus elicits marked increases in rig - i expression by murine and human glial cells ( furr et al . , 2008 , 2010 ) . more direct evidence for the functional nature of rig - i expression in glia comes from our observation that this molecule associates with ips-1 following vsv infection and from the finding that the specific rig - i ligand , 5ppp - ssrna , elicits human astrocyte immune responses ( furr et al . , importantly , we also showed that rig - i knockdown significantly reduces inflammatory cytokine production by vsv - infected human astrocytes and inhibits the production of soluble neurotoxic mediators by virally challenged cells ( furr et al . , 2010 ) . these findings directly implicate rig - i in the initiation of glial inflammatory immune responses and suggest a potential mechanism underlying the neuronal cell death associated with acute viral cns infections . in contrast to the proposed detrimental role for rlr activation following challenge with vsv , other work suggests that ips-1 is essential for triggering protective innate and adaptive immunity against wnv in the cns . in these studies , mice deficient in the rlr adaptor molecule ips-1 exhibited increased susceptibility to wnv infection , characterized by enhanced inflammation , viral replication , and rapid dissemination into the cns ( suthar et al . , 2010 ) . however , it is important to note that glial cells were not the focus of this study and ips-1 dependent ifn responses and limiting of viral replication was attributed to effects on systemically generated immune cells and cortical neurons , perhaps via changes in the numbers and/or function of regulatory t cells ( suthar et al . , 2010 ) . as such , these data suggest an innate / adaptive immune interface mediated through rlr signaling that regulates the balance of the immune response to wnv infection . together , these findings raise the exciting possibility that rlr molecules play important roles in the detection of viral cns pathogens and , depending upon the host cell type and viral pathogen , the initiation of protective immune responses or damaging inflammation within the brain . recently , novel cytosolic dna sensors have been discovered that include dna - dependent activator of interferon - regulatory factors ( dai ; also known as z - dna binding protein 1 ; takaoka et al . , 2007 ) and a dna - sensing inflammasome consisting of the hin200 protein , absent in melanoma 2 ( aim2 ; schroder and tschopp , 2010 ) . acting in concert with tlrs , these receptors may provide a diverse repertoire of mechanisms to alert the cell to viral and microbial dna , leading to the activation of the innate immune system in a similar manner to that seen with rlr - mediated responses to viral rna . dai reportedly senses cytosolic dna using two n - terminal z - dna binding domains ( zbds ) and a third putative dna binding domain located next to the second zbd . analysis of the dai / z - dna complex structure reveals that dai adopts an unusual binding mode for z - dna recognition ( ha et al . , 2008 ) . the dai zbds bind dna and both must be bound for full b - to - z conversion , and so it is conceivable that the binding of two dai proteins to each cytoplasmic dsdna elicits dai dimerization and subsequent innate immune activation ( ha et al . , 2008 ; wang et al . , 2008 ) . importantly , dai has been shown to recognize double - stranded dna in its canonical b helical form ( b - dna ; takaoka et al . , 2007 ) and elicit type i ifn and inflammatory cytokine production in a tlr9-independent manner ( ishii et al . as shown in figure 2 , dai elicits such immune functions via activation of the irf and nf-b transcription factors . dai - induced irf activation is dependent upon tank - binding kinase 1 ( tbk1 ) . in contrast , dai - induced nf-b activation is mediated through interactions between two receptor - interacting protein ( rip ) homotypic interaction motifs ( rhims ) in the dai protein and rhim - containing kinases , rip1 and rip3 ( kaiser et al . , 2008 ; rebsamen et al . , 2009 ) , as shown by the demonstration that knockdown of either rip1 or rip3 inhibits dai - induced nf-b activation . both of these activation pathways appear to be induced via the recruitment of the adaptor molecules ips-1 , traf3 , and sting . the importance of these adaptor molecules in host immune responses to dna viruses is exemplified by the demonstration that the loss of sting renders mice susceptible to lethal hsv-1 infection ( ishikawa and barber , 2008 ) . dna - dependent activator of ifn regulatory factors ( dai ) can serve as a cytosolic sensor for viral or microbial double - stranded dna ( dsdna ) motifs and initiate anti - viral and inflammatory cell responses . dai elicits such immune functions via possible interactions with mitochondrial- or peroxisomal - associated interferon promoter stimulator ( ips)-1 , and/or stimulator of interferon genes ( sting ) , facilitating the activation of the interferon - regulatory factors ( irf ) and nf-b transcription factors . dai - induced irf activation is dependent on tank - binding kinase 1 ( tbk1 ) . in contrast , dai - induced nf-b activation is mediated through interactions involving receptor - interacting protein ( rip ) 1 and rip3 kinases . overexpression of dai in mouse fibroblasts enhances dna - mediated type i ifn production while dai knockdown attenuates responses to viral dna ( takaoka et al . , 2007 ) . in an additional study , dna - mediated activation of nf-b and the induction of nf-b - dependent genes and their products were also significantly inhibited in cells following dai knockdown ( rebsamen et al . , 2009 ) . as such , these data are suggestive of a significant role for dai in dna - mediated activation of the nf-b pathway and viral recognition . however , it is interesting to note that dai - mediated nf-b activation appears to facilitate hiv-1 replication although the mechanisms underlying this effect remain unclear ( hayashi et al . dai is reported to be expressed in the lymph nodes and spleen constitutively , but detectable levels are also found in circulating leukocytes and other tissues including bone marrow and small intestines ( rothenburg et al . , 2002 ) . in the cns , we have shown that microglia and astrocytes constitutively express dai and its effector molecules rip3 and sting , and show that such expression is upregulated following dna virus challenge ( furr et al . in vivo hsv-1 infection elicited an upregulation in microglial dai expression by up to 23.8-fold over that seen in resting cells , and despite strong constitutive expression , hsv-1 exposure was also able to further increase dai expression in astrocytes with a maximal increase of 2.2-fold over that seen in unstimulated cells . interestingly , the ability of viral challenge to augment dai expression by primary glial cells was not limited to this neurotropic alpha herpesvirus , as the lymphotropic gammaherpesvirus , mhv-68 , was also capable of eliciting robust increases in microglial dai expression and caused modest increases in the expression of this molecule in astrocytes . importantly , we showed that transfection with the dai specific ligand b - dna elicits inflammatory cytokine production by isolated glial cells , with induced production of the inflammatory cytokines tnf- and il-6 as rapidly as 6 h post - transfection at levels that matched or exceeded those elicited following a 24-h hsv-1 challenge ( furr et al . , 2011 ) . to confirm the functional status of dai in glial cells and to begin to determine the relative importance of this innate immune sensor in their responses to neurotropic dna viruses , we also assessed the effect of dai knockdown on inflammatory cytokine production by hsv-1 challenged microglia and astrocytes ( furr et al . , 2011 ) . we found that sirna directed against dai significantly attenuated hsv-1 induced tnf- and il-6 production by murine microglia . such an approach also markedly reduced il-6 release by hsv-1 infected astrocytes but was not as effective in reducing tnf- expression by these cells , where a statistically significant reduction was only observed at the highest viral moi used ( furr et al . , 2011 ) . finally , we demonstrated that hsv-1 challenged microglia and astrocytes release neurotoxic mediators and showed that such production is significantly attenuated following dai knockdown ( furr et al . , the functional expression of dai by microglia and astrocytes may represent an important innate immune mechanism underlying the rapid and potentially lethal inflammation associated with neurotropic dna virus infection . however , the involvement of dna sensor(s ) other than dai can not be excluded . for example , a new dna sensor , aim2 , has been identified which binds to dna ( as reviewed in ranjan et al . , 2009 ) . upon dna ligation , aim2 associates with the apoptosis - associated speck - like protein containing a card ( asc ) leading to nf-b and caspase-1 activation and inflammatory cytokine production . aim2 knockdown has been shown to reduce such inflammasome formation and caspase-1 activation in response to dsdna administration and infection with vaccinia virus ( hornung et al . , 2009 ) and stable transfection of cell - lines with aim2 has been demonstrated to confer responsiveness to cytoplasmic dna ( fernandes - alnemri et al . , 2009 ) . to date , the role of aim2 in innate immune responses to dna viruses in the cns has not been explored . while the preceding sections have focused on the roles of specific individual prrs in the detection of viral pathogens by cns cells , it is highly likely that glial responses represent the sum of the inputs from disparate cell surface , endosomal , and cytosolic viral sensors ( as shown in figure 3 ) . we , and others , have demonstrated that the level of expression of a particular tlr , nlr , or rlr by microglia and astrocytes can be regulated by ligands for dissimilar prrs ( bowman et al . , 2003 ; jack et al . , 2005 ; carpentier et al . , furthermore , mechanisms of positive and negative regulation of rlr signaling have been identified that include signaling cross - talk between rlr , nlr and dna - sensing pathways , and caspase networks . for example , it has been shown that mice deficient in the expression of the dai downstream effector molecule sting are highly susceptible to infection by negative - stranded viruses including vsv suggesting a role for this signaling molecule in both dai and rig - i mediated cell activation ( ishikawa and barber , 2008 ) . immunoprecipitation studies indicate sting associates with rig - i complexes in close proximity to endoplasmic reticulum associated mitochondria ( ishikawa et al . , 2009 ; interestingly , a number of rna viruses including yellow fever virus , dengue virus , and hepatitis c virus , encode proteins with significant structural homology to sting and at least one of these proteins produced by the dengue virus has been shown to inhibit sting function ( ishikawa et al . , 2009 ) . as such , these observations suggest a role for sting in anti - viral host responses to both dna and rna viruses . possible interactions between toll - like receptors ( tlrs ; e.g. , tlr3 and tlr7/8 ) , nucleotide oligomerization domain ( nod)-like receptors ( nlrs ; e.g. , nod2 ) , retinoic acid inducible gene ( rig)-i - like receptors ( rlrs ; e.g. , rig - i and mda5 ) and dna - dependent activator of ifn regulatory factors ( dai ) in the detection of viruses and the initiation of glial immune responses . these disparate cell surface , endosomal , and cytosolic sensors share common signaling components including interferon promoter stimulator ( ips)-1 and stimulator of interferon genes ( sting ) , leading to the activation of the transcription factors nf-b and interferon - regulatory factor ( irf ) 3 , which translocate into the nucleus and initiate anti - viral and inflammatory cytokine production . in addition , the activity of rna polymerase iii ( pol iii ) could provide an additional mechanism for the perception of dna viruses via rlrs . another example of cross - talk between viral sensing pathways is the recent demonstration that rig - i may be important in the production of type i ifns by cells in response to dna viral challenge in a dna - dependent rna polymerase iii manner ( ablasser et al . , 2009 ; chiu et al . , 2009 ) . in the proposed model , at - rich double - stranded dna serves as a template for rna polymerase iii and is transcribed into rna containing a 5-triphosphate , the ligand for rig - i . subsequent activation of rig - i by this ligand has been shown to induce type i ifn production and to activate nf-b , and studies employing rna polymerase iii knockdown or pharmacological inhibitors of this enzyme have shown that this pathway is important in cell responses to the gammaherpesvirus epstein - barr virus ( ablasser et al . , 2009 ; chiu et al . , 2009 ) . together , these studies suggest that the transcription of cytosolic viral dna by rna polymerase iii and subsequent rna recognition by rig - i could provide an additional mechanism by which glial cells perceive dna viruses . interestingly , there has been a recent report of a role for the well - characterized bacterial sensor , nod2 , in the recognition of viral infections and the activation of irf-3 via ips-1 ( sabbah et al . , 2009 ) . while nod2 is known to recognize the bacterial cell wall component muramyl dipeptide and trigger nf-b activation via the adaptor molecule rip2 , sabbah et al . ( 2009 ) have provided evidence that this sensor is also important in the detection of ssrna derived from respiratory syncytial virus . however , it is currently unknown if nod2 is directly activated by this viral ligand , or is activated secondarily following association with other prrs or rna binding factors . furthermore , it remains to be determined whether such interactions between nlrs and rig - i - associated signaling components occur in glial cells or whether they play a significant role during viral cns infections . microglia and astrocytes express a diverse array of cell surface , endosomal , and cytosolic molecules that can serve as sensors for rna and dna viruses , either constitutively or following activation . it is also clear that resident cns cells can respond to a diverse array of ligands and that exquisite control of the associated signaling pathways exists leading to the observed complexity in the interactions between the innate and adaptive arms of the host immune response . however , it is obvious that we still do not fully understand the precise nature and structure of the ligands for many of these prrs or their mode of generation during infection . furthermore , the available data suggest that models that ascribe discrete prrs to each particular pathogen are overly simplistic . rather , it appears that tlrs , rlrs , nlrs , and dai function as prrs and/or adaptor molecules that act in a cooperative / synergistic manner to promote glial responses to viral pathogens . in addition to these caveats , it is almost certain that additional prrs await discovery in glial cells and the nature of the interactions between these viral sensors and cellular activation pathways have yet to be determined . finally , it is unclear whether and to what degree viral cns pathogens evade detection or manipulate immune responses to their advantage . answering these questions will lead to greater understanding of the fundamental mechanisms underlying glial activation by neurotropic viruses and will contribute significantly to our understanding of the events that underlie the development of either protective host responses or life threatening neuroinflammation . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

viruses are the major causative agents of central nervous system ( cns ) infection worldwide . rna and dna viruses trigger broad activation of glial cells including microglia and astrocytes , eliciting the release of an array of mediators that can promote innate and adaptive immune responses . such responses can limit viral replication and dissemination leading to infection resolution . however , a defining feature of viral cns infection is the rapid onset of severe neuroinflammation and overzealous glial responses are associated with significant neurological damage or even death . the mechanisms by which microglia and astrocytes perceive neurotropic rna and dna viruses are only now becoming apparent with the discovery of a variety of cell surface and cytosolic molecules that serve as sensors for viral components . in this review we discuss the role played by members of the toll - like family of pattern recognition receptors ( prrs ) in the inflammatory responses of glial cells to the principle causative agents of viral encephalitis . importantly , we also describe the evidence for the involvement of a number of newly described intracellular prrs , including retinoic acid - inducible gene i and dna - dependent activator of ifn regulatory factors , that are thought to function as intracellular sensors of rna and dna viruses , respectively . finally , we explore the possibility that cross - talk exists between these disparate viral sensors and their signaling pathways , and describe how glial cytosolic and cell surface / endosomal prrs could act in a cooperative manner to promote the fulminant inflammation associated with acute neurotropic viral infection .

GLIAL CELLS PLAY A CRITICAL ROLE IN THE INITIATION OF VIRALLY INDUCED NEUROINFLAMMATION TLRs MEDIATE GLIAL RESPONSES TO EXTRA CELLULAR OR ENDOSOMAL VIRAL MOTIFS RIG-LIKE RECEPTORS CAN MEDIATE PROTECTIVE IMMUNE RESPONSES AND DAMAGING CNS INFLAMMATION FOLLOWING RNA VIRUS INFECTION NEWLY DISCOVERED CYTOSOLIC VIRAL SENSORS MAY MEDIATE GLIAL RESPONSES TO DNA VIRUSES PRRs ACT IN A COOPERATIVE MANNER TO INDUCE GLIAL RESPONSES TO VIRAL CHALLENGES CONCLUDING REMARKS Conflict of Interest Statement

however , these cell types are now recognized to be key players in the development of protective immune responses or the progression of damaging inflammation during cns disease states ( bauer et al . , 1998 ) astrocytes are the major glial cell type in the brain and may also function as immune effector cells ( dong and benveniste , 2001 ) . stimulated astrocytes can express an array of inflammatory cytokines and chemokines that can initiate leukocyte migration across the blood brain barrier and promote effector functions in these infiltrating cells ( dong and benveniste , 2001 ) . viral infections in the cns are known to broadly trigger glial activation and the release of pro - inflammatory molecules . we have also documented the ability of primary cultured microglia and astrocytes to respond to several rna and dna viruses , including vesicular stomatitis virus ( vsv ) , sendai virus , murine gammaherpesvirus ( mhv)-68 , and hsv-1 , by producing inflammatory mediators such as il-6 , tnf- , and il-1 ( rasley et al . importantly , we confirmed that in vivo vsv administration results in viral infection of both glial cell types in situ , while earlier studies demonstrated that microglia and astrocytes respond to vsv by proliferating , producing inducible nitric oxide synthase , and increasing the expression of cell surface mhc class ii molecules ( bi et al . an immune response to a virus in the cns requires the recognition of this pathogen and the mechanisms by which innate immune cells achieve this have only recently become apparent with the discovery of a variety of cell surface and cytosolic molecules that serve as sensors for viral components . such pattern recognition receptors ( prrs ) are present on and within immune sentinel cells ( furr et al . pattern recognition receptor engagement by pamps can precipitate the production of anti - viral type i interferons ( ifn ) and/or the release of chemotactic and inflammatory molecules that can then direct a subsequent adaptive immune response . three families of prrs have been defined : toll - like receptors ( tlrs ) , nucleotide oligomerization domain ( nod)-like receptors ( nlrs ) , and retinoic acid inducible gene ( rig)-i - like receptors ( rlrs ) . interestingly , similar gene products were found in mammalian cells and these tlrs have since been shown to play an important role in the initiation of innate immune responses to infection ( medzhitov et al . to date , at least 13 tlrs have been discovered in mammals and members of this family of cell surface receptors can elicit inflammatory and/or anti - viral mediator production and can serve to initiate or modify adaptive immune responses ( as reviewed in kawai and akira , 2008 ) . as such , glial activation via tlrs and other prrs is likely to facilitate antigen presentation to , and costimulation of , such cells and the role of these receptors in the antigen - specific activation of t cells following viral challenge has been extensively reviewed elsewhere ( kawai and akira , 2008 ) . while tlr2 is best known to bind a variety of microbial cell wall component including lipoproteins , peptidoglycans , and lipoteichoic acid present in bacterial cell walls and the yeast cell wall component zymosan , this sensor can also recognize as yet unidentified viral motifs . , while these findings suggest that glial cells could become sensitized to the presence of viral pathogens , it is presently unclear whether sustained stimulation via these sensors can alter the pattern of immune mediator production by glial cells to one that favors the resolution of inflammation . upon ligand binding , tlrs dimerize and undergo conformational changes precipitating a complex cascade of intracellular signaling events that ultimately result in activation of transcription factors including nuclear factor-b ( nf-b ) , and interferon - regulatory factors ( irf ) 3 and 7 ( as reviewed in kawai and akira , 2008 ; wilkins and gale , 2010 ) . tlr3 is perhaps the most widely studied prr in the perception of viral pathogens by glial cells . , circumstantial evidence for the involvement of tlr3 in viral encephalitis comes from the finding that the expression of this prr is upregulated following rabv infection prior to the onset of lethal neuroinflammation ( mckimmie and fazakerley , 2005 ; mckimmie et al . , dsrna , a ligand for tlr3 , is produced by wnv during infection and has been shown to trigger the release of cytokines and chemokines from human microglia ( cheeran et al . , interestingly however , this effect appears to be predominantly due to the activation of astrocytes rather than microglia ( butchi et al . however , it should be noted that these studies differ in their assessments of the relative importance of this prr in such responses , and marked elevations in serum il-6 concentrations have been found in tlr2 deficient mice following hsv-1 infection ( kurt - jones et al . , 2010 ) , it appears likely that these sensors act in a cooperative or synergistic manner . for example , the activation of glial cells via tlr2 , 3 , or 9 has been shown to initiate the production of type i ifns ( zhou et al . , 2010 ) which limit viral replication through the induction of proteins such as rnase l and ifn - induced dsrna - activated protein kinase , resulting in mrna degradation and cessation of translation , respectively ( samuel , 2001 ) . however , the mechanisms that keep the balance between damaging and protective glial responses have not been identified and are likely to include a multitude of variables including the number of infectious organisms , the kinetics of glial activation and immune mediator production , the specific identity of the responsive glial cell , and the direct influence of the pathogen itself on the cellular response . while cell - surface receptors like tlrs appear to play an important role in glial responses to extracellular pathogens or those present in intracellular compartments , the ability of such cells to respond to pathogens in the absence of tlr expression suggests that additional mechanisms are present . furthermore , our recent studies employing the model neurotropic rna virus , vsv , demonstrate that viral replication is required to elicit robust immune responses by infected microglia and astrocytes , and indicate that mechanisms exist to sense the presence of replicative viral products within infected cells . these results suggest that vsv - induced glial responses are not predominantly mediated by cell surface and/or endosomal prrs and indicate that active viral replication is a critical requirement for such responses . the recent demonstration that microglia and astrocytes express members of the newly described rlr family of prrs may provide a mechanism underlying the replication - dependent nature of glial responses ( furr et al . rlrs such as rig - i and melanoma differentiation - associated gene 5 ( mda5 ) have been shown to serve as intracellular prrs for viral nucleic acid motifs ( kato et al . , 2006 ; takeuchi and akira , 2008 ) , and their involvement in inflammatory and anti - viral responses in other cell types raises the intriguing possibility that these cytosolic sensors could play an important role in glial responses to viral infection . as shown in figure 1 , helicase interaction with viral rna induces the recruitment of downstream effector molecules including interferon promoter stimulator ( ips)-1 ( also known as visa , cardif , or mavs ) , tnf receptor associated factor ( traf ) 3 ( takeuchi and akira , 2010 ) , and perhaps stimulator of interferon genes ( sting ; ishikawa and barber , 2008 ) , leading to the activation of mitogen activated protein ( map ) kinases , and the ib kinase ( ikk)-related kinases , traf family member - associated nf-b activator ( tank ) binding kinase 1 and ikk - i . retinoic acid inducible gene ( rig)-i - like receptors serve as cytosolic sensors for viral rna motifs and can initiate anti - viral and inflammatory cell responses . helicase interaction with these viral rnas induces the recruitment of downstream effector molecules including mitochondrial- or peroxisomal - associated interferon promoter stimulator ( ips)-1 , tnf receptor associated factor ( traf ) 3 , and/or stimulator of interferon genes ( sting ) , leading to the activation of ib kinase ( ikk)-related kinases , traf family member - associated nf-b activator binding kinase 1 ( tbk1 ) , and ikk - i . signaling by rig - i is triggered during infection by a number of rna viruses and by the presence of synthetic rna transcribed in vitro ( sumpter et al . importantly , we have shown that isolated murine microglia and astrocytes constitutively express rig - i and mda5 transcripts and protein , as well as the critical downstream effector molecule , ips-1 ( furr et al . circumstantial evidence of a role for these rlrs in rna virus infections of the cns comes from our observation that rig - i and mda5 expression is elevated in the brains of mice following intranasal administration of vsv ( furr et al . in mice , intranasal vsv infection results in a severe encephalitis with rapid activation and proliferation of microglia and astrocytes ( huneycutt et al . more direct evidence for the functional nature of rig - i expression in glia comes from our observation that this molecule associates with ips-1 following vsv infection and from the finding that the specific rig - i ligand , 5ppp - ssrna , elicits human astrocyte immune responses ( furr et al . , importantly , we also showed that rig - i knockdown significantly reduces inflammatory cytokine production by vsv - infected human astrocytes and inhibits the production of soluble neurotoxic mediators by virally challenged cells ( furr et al . these findings directly implicate rig - i in the initiation of glial inflammatory immune responses and suggest a potential mechanism underlying the neuronal cell death associated with acute viral cns infections . in contrast to the proposed detrimental role for rlr activation following challenge with vsv , other work suggests that ips-1 is essential for triggering protective innate and adaptive immunity against wnv in the cns . in these studies , mice deficient in the rlr adaptor molecule ips-1 exhibited increased susceptibility to wnv infection , characterized by enhanced inflammation , viral replication , and rapid dissemination into the cns ( suthar et al . however , it is important to note that glial cells were not the focus of this study and ips-1 dependent ifn responses and limiting of viral replication was attributed to effects on systemically generated immune cells and cortical neurons , perhaps via changes in the numbers and/or function of regulatory t cells ( suthar et al . together , these findings raise the exciting possibility that rlr molecules play important roles in the detection of viral cns pathogens and , depending upon the host cell type and viral pathogen , the initiation of protective immune responses or damaging inflammation within the brain . recently , novel cytosolic dna sensors have been discovered that include dna - dependent activator of interferon - regulatory factors ( dai ; also known as z - dna binding protein 1 ; takaoka et al . , 2007 ) and a dna - sensing inflammasome consisting of the hin200 protein , absent in melanoma 2 ( aim2 ; schroder and tschopp , 2010 ) . acting in concert with tlrs , these receptors may provide a diverse repertoire of mechanisms to alert the cell to viral and microbial dna , leading to the activation of the innate immune system in a similar manner to that seen with rlr - mediated responses to viral rna . dna - dependent activator of ifn regulatory factors ( dai ) can serve as a cytosolic sensor for viral or microbial double - stranded dna ( dsdna ) motifs and initiate anti - viral and inflammatory cell responses . dai elicits such immune functions via possible interactions with mitochondrial- or peroxisomal - associated interferon promoter stimulator ( ips)-1 , and/or stimulator of interferon genes ( sting ) , facilitating the activation of the interferon - regulatory factors ( irf ) and nf-b transcription factors . in an additional study , dna - mediated activation of nf-b and the induction of nf-b - dependent genes and their products were also significantly inhibited in cells following dai knockdown ( rebsamen et al . as such , these data are suggestive of a significant role for dai in dna - mediated activation of the nf-b pathway and viral recognition . in the cns , we have shown that microglia and astrocytes constitutively express dai and its effector molecules rip3 and sting , and show that such expression is upregulated following dna virus challenge ( furr et al . interestingly , the ability of viral challenge to augment dai expression by primary glial cells was not limited to this neurotropic alpha herpesvirus , as the lymphotropic gammaherpesvirus , mhv-68 , was also capable of eliciting robust increases in microglial dai expression and caused modest increases in the expression of this molecule in astrocytes . importantly , we showed that transfection with the dai specific ligand b - dna elicits inflammatory cytokine production by isolated glial cells , with induced production of the inflammatory cytokines tnf- and il-6 as rapidly as 6 h post - transfection at levels that matched or exceeded those elicited following a 24-h hsv-1 challenge ( furr et al . to confirm the functional status of dai in glial cells and to begin to determine the relative importance of this innate immune sensor in their responses to neurotropic dna viruses , we also assessed the effect of dai knockdown on inflammatory cytokine production by hsv-1 challenged microglia and astrocytes ( furr et al . finally , we demonstrated that hsv-1 challenged microglia and astrocytes release neurotoxic mediators and showed that such production is significantly attenuated following dai knockdown ( furr et al . , the functional expression of dai by microglia and astrocytes may represent an important innate immune mechanism underlying the rapid and potentially lethal inflammation associated with neurotropic dna virus infection . however , the involvement of dna sensor(s ) other than dai can not be excluded . upon dna ligation , aim2 associates with the apoptosis - associated speck - like protein containing a card ( asc ) leading to nf-b and caspase-1 activation and inflammatory cytokine production . to date , the role of aim2 in innate immune responses to dna viruses in the cns has not been explored . while the preceding sections have focused on the roles of specific individual prrs in the detection of viral pathogens by cns cells , it is highly likely that glial responses represent the sum of the inputs from disparate cell surface , endosomal , and cytosolic viral sensors ( as shown in figure 3 ) . we , and others , have demonstrated that the level of expression of a particular tlr , nlr , or rlr by microglia and astrocytes can be regulated by ligands for dissimilar prrs ( bowman et al . , furthermore , mechanisms of positive and negative regulation of rlr signaling have been identified that include signaling cross - talk between rlr , nlr and dna - sensing pathways , and caspase networks . for example , it has been shown that mice deficient in the expression of the dai downstream effector molecule sting are highly susceptible to infection by negative - stranded viruses including vsv suggesting a role for this signaling molecule in both dai and rig - i mediated cell activation ( ishikawa and barber , 2008 ) . , 2009 ; interestingly , a number of rna viruses including yellow fever virus , dengue virus , and hepatitis c virus , encode proteins with significant structural homology to sting and at least one of these proteins produced by the dengue virus has been shown to inhibit sting function ( ishikawa et al . possible interactions between toll - like receptors ( tlrs ; e.g. , nod2 ) , retinoic acid inducible gene ( rig)-i - like receptors ( rlrs ; e.g. , rig - i and mda5 ) and dna - dependent activator of ifn regulatory factors ( dai ) in the detection of viruses and the initiation of glial immune responses . these disparate cell surface , endosomal , and cytosolic sensors share common signaling components including interferon promoter stimulator ( ips)-1 and stimulator of interferon genes ( sting ) , leading to the activation of the transcription factors nf-b and interferon - regulatory factor ( irf ) 3 , which translocate into the nucleus and initiate anti - viral and inflammatory cytokine production . in addition , the activity of rna polymerase iii ( pol iii ) could provide an additional mechanism for the perception of dna viruses via rlrs . another example of cross - talk between viral sensing pathways is the recent demonstration that rig - i may be important in the production of type i ifns by cells in response to dna viral challenge in a dna - dependent rna polymerase iii manner ( ablasser et al . subsequent activation of rig - i by this ligand has been shown to induce type i ifn production and to activate nf-b , and studies employing rna polymerase iii knockdown or pharmacological inhibitors of this enzyme have shown that this pathway is important in cell responses to the gammaherpesvirus epstein - barr virus ( ablasser et al . together , these studies suggest that the transcription of cytosolic viral dna by rna polymerase iii and subsequent rna recognition by rig - i could provide an additional mechanism by which glial cells perceive dna viruses . interestingly , there has been a recent report of a role for the well - characterized bacterial sensor , nod2 , in the recognition of viral infections and the activation of irf-3 via ips-1 ( sabbah et al . microglia and astrocytes express a diverse array of cell surface , endosomal , and cytosolic molecules that can serve as sensors for rna and dna viruses , either constitutively or following activation . it is also clear that resident cns cells can respond to a diverse array of ligands and that exquisite control of the associated signaling pathways exists leading to the observed complexity in the interactions between the innate and adaptive arms of the host immune response . rather , it appears that tlrs , rlrs , nlrs , and dai function as prrs and/or adaptor molecules that act in a cooperative / synergistic manner to promote glial responses to viral pathogens . in addition to these caveats , it is almost certain that additional prrs await discovery in glial cells and the nature of the interactions between these viral sensors and cellular activation pathways have yet to be determined . finally , it is unclear whether and to what degree viral cns pathogens evade detection or manipulate immune responses to their advantage .

a dgt sampling unit consists of a resin gel , covered by a diffusive gel with a defined thickness and a protective membrane , all of which are enclosed in a sampler housing . the sampling process is simple : once the dgt device gets in contact with the sampled medium , the target solutes diffuse into the sampler where they get bound by the resin gel . on the basis of the 1d steady - state solution of fick s first law of diffusion1the solute concentration at the sampler medium interface can be calculated by eq 2(1)2here , j is the diffusive flux , dgel is the solute diffusion coefficient in the diffusion layer , cb is the concentration in the exterior solution , cr is the concentration in the resin gel ( which is effectively zero as the resin is a strong solute sink ) , g is the diffusion layer thickness , cdgt is the concentration at the sampler medium interface , m is the mass of solute bound by the resin layer , aphys is the physical sampling window surface area and t is the sampling time . when dgt was developed , cdgt was considered a measure of cb , based on the assumptions that either only one diffusing solute species exists or that all species have the same diffusion coefficient and are equally strongly bound by the binding material ( e.g. , differently protonated species ) , that the diffusive boundary layer ( dbl ) forming outside the protective membrane has negligible effect on the sampler s mass uptake in well - stirred laboratory solutions and agitated natural waters , and , implicitly , that diffusion inside the sampler is one - dimensional and has no lateral components . in an investigation of phosphate fluxes in an eutrophic pond the dbl was , however , found to significantly decrease the solute uptake . in this study the dbl thickness , , was estimated by deploying dgt samplers with varying diffusive layer thicknesses and evaluating a plot of the reciprocal mass ( 1/m ) versus g . therefore , separate terms for solute diffusion in the dbl and the filter membrane layer were integrated into the dgt equation3the superscript dgt indicates a cb estimate obtained using dgt . in eq 3 , f represents the thickness of the membrane and df and dwater are the diffusion coefficients in the membrane layer and in water . for standard , apa2-type diffusion gels and supor membranes ( pall coporation , port washington , ny , usa ) , for which diffusion coefficients were reported to be indistinguishable , the membrane thickness can be added to g and the term for the membrane can be omitted . note that eq 3 provides a measure of cb , as the effect of the dbl on solute mass uptake is corrected for . using aphys for evaluating their experimental results , warnken et al . observed systematically higher cbdgt values compared to cb after correcting for the dbl - related mass uptake decrease . high - resolution analysis of dried resin gels using laser ablation inductively coupled plasma mass spectrometry ( la - icpms ) showed that this unexpectedly high mass uptake was caused by lateral solute diffusion inside the dgt samplers . the authors concluded that the dbl - related decrease in mass uptake is balanced by the increase caused by lateral solute diffusion for standard dgt devices ( g = 0.94 mm , aphys = 3.14 cm ) deployed in well - stirred solutions . in this case , the simplification of using the physical sampling window area ( aphys = 3.14 cm ) and the simple dgt equation was found to be acceptable . for cases where no balancing of the two effects can be expected , warnken et al . suggested to use the expanded dgt equation and an effective sampling area ( aeff ) instead of the physical sampling window area to account for the lateral diffusion effect . a general flux increase of 20% was adopted for dgt samplers , translating to aeff = 3.80 cm for standard dgt devices ( aphys = 3.14 cm ) and aeff = 3.08 cm for dgt devices with smaller physical window area ( aphys = 2.54 cm ) . in this and in subsequent studies the relative flux increase due to lateral diffusion was assumed to be constant irrespective of the sampler geometry ( e.g. , g , aphys ) . only very recently , garmo presented modeling data indicating that the previous aeff estimate for samplers with rphys= 0.9 cm is too low , and that there might be an effect of aphys on aeff . a sketch of a dgt sampler shows that lateral diffusion inside the sampler takes place at the edge of the diffusion and resin gels , where the sampler housing extends over the diffusion layer ( figure 1 ) . the solute binding by the underlying resin gel drives the outward , lateral flux of solute in this region . figure 1 indicates that the diffusion layer thickness , but also the size of the covered gel region ( i.e. , the physical sampler window area ) might affect the concentration gradient and thereby the magnitude of the flux increase because of lateral diffusion , hence aeff should depend on the sampler geometry . in this study , we apply numerical modeling for simulating 3d diffusion into and inside dgt samplers to investigate the dependence of the lateral diffusion - induced mass uptake increase on g and aphys . moreover , we strive for identifying additional parameters that might affect the solute flux into and inside dgt samplers . the dgt sampler housing covers a 2.5 mm wide area of the diffusion layer and the resin gel . the direction of the concentration gradient that establishes at the edge of the sampler is oriented toward the resin gel edge , as the solute concentration in this area is lower than in the diffusion layer zone that is in direct contact with the medium . as a consequence , the solute concentration at the diffusion layer dbl interface is lower than in the center of the sampler , leading to a higher solute flux into the sampler at the sampler edge compared to the center . dgt samplers are circular devices , therefore three - dimensional solute diffusion inside and into dgt devices can be reduced to a 2d - axisymmetric problem . we solved the diffusion equation ( eqs s1 , s6 , and s7 in the supporting information ) on two different , 2d - axisymmetric simulation geometries , one representing the diffusion layer without considering the dbl ( supporting information figure s1 top ) and a second including the overlying dbl layer ( supporting information figure s1 bottom ) . in the first case , only the diffusion coefficient in the diffusion gel , dgel , was considered , while in the second case dwater was additionally used as diffusion coefficient in the dbl . the complete description of the mathematical problem is given in the supporting information . the flux profiles into the resin gel that were obtained from the 2d - axisymmetric model were used to create revolution surfaces , across which the flux into the resin gel was integrated to obtain the cumulated solute flux into the dgt sampler . although , strictly , the results reported in this study are based on 2d - axisymmetric simulations , they are a valid representation of the 3d solute flux into the sampler . therefore , our results are addressed as 3d results in the following . the sampling window had radii rphys of 1.0 and 0.9 cm ( standard dgt sampler sizes for solution and soil sampling ) , the resin gel disc radius ( rgel ) was 1.25 cm . the diffusion layer thickness g was varied between 0.001 cm , as used in chemical imaging studies , and 0.5 cm as upper extreme value . the diffusion boundary layer thickness ( ) was varied between 0.0025 and 0.1 cm . a value of dwater = 8.47 10 cm s ( 25 c ) was adopted as diffusion coefficient in water and dgel = 6.05 10 cm s ( 25 c ) was adopted as diffusion coefficient in the diffusion layer . the exterior solution concentration of phosphate ( cb ) was set to 100 nmol l. an overview on model parameters and parameter values is given in supporting information table s1 . a steady solute flux into a dgt sampler is established a few minutes after exposure of the sampler to the sampled medium . throughout this study , stationary solutions of the model were used to evaluate the diffusional behavior after this initial phase of flux establishment . all simulations were done using the earth science module of the comsol 4.0a package ( comsol multiphysics gmbh , gttingen , germany ) . lateral diffusion inside the sampler was evaluated by comparing simulated 3d fluxes into dgt samplers without dbl , which include the lateral diffusion effect , to fluxes calculated using the 1d solution for the dgt flux4disregarding the effect of lateral diffusion at the sampler edges . for investigating the effect of the dbl on the sampler flux these sampler fluxes were compared to 1d fluxes without dbl ( eq 4 ) and to 1d fluxes including the dbl , which were simulated with a simple 1d representation of the dgt geometry . in addition , the effects of different sampling window surface areas and different diffusion coefficients , using literature values for h2po4 , h2aso4 , and cd , ( supporting information table s1 ) were investigated . to date , the constant correction coefficient aeff(7 ) is used to account for lateral diffusion in dgt measurements . here we investigate the effect of applying the nonconstant correction coefficients determined in this study on the results of dbl thickness estimations using ( 1 ) a simulated data set and ( 2 ) literature data . the literature data was digitized from figure 2a in garmo et al . and figure 2b in warnken et al . using the free software the dbl thickness ( along with cb ) can be determined by fitting eq 5,5representing the expanded dgt equation ( eq 3 ) solved for m , to dgt - determined solute masses m. for simplifying the fitting procedure a linearized version of eq 5 , supporting information eq s14 , has been used . parameter estimation using linearized data that might be affected with random measurement errors of constant magnitude may however be biased , as the contribution of the error increases as the data value decreases . proposed to fit m directly to the nonlinearized dgt equation , which is convenient with modern office software packages . furthermore , garmo et al . modified the expanded dgt model ( eqs 3 , 5 ) to include both , the physical sampler window surface area aphys and the effective area aeff6to better account for lateral diffusion . in this study we estimated and cb using both , eqs 5 and 6 . fits of these equations to the simulated and literature data sets were computed using ( 1 ) aeff = 3.8 cm or aeff = 3.08 cm , as appropriate , ( 2 ) correction for lateral diffusion inside the sampler only ( kld , explanation in results and discussion ) , and ( 3 ) correction for lateral diffusion and non-1d diffusion through the dbl ( kld and kdbl , explanation in results and discussion ) . table 1 shows 1d and 3d phosphate fluxes into dgt samplers with different diffusion layer thicknesses . considering only the fluxes computed without dbl it is evident that the 3d fluxes are always higher than the 1d fluxes , with the flux difference increasing with increasing g . the reason for this flux increase , as already outlined in the introduction and visualized in figure 1 , is the lateral diffusion at the sampler edges , which has already been shown experimentally by warnken et al . simulated profiles of the solute flux into the resin gel show that an increasing solute mass fraction is transported toward the edge of the resin gel upon increasing the diffusion layer thickness ( supporting information figure s2 ) . for very small g values ( 0.001 cm , 0.005 cm ) , the outward solute flux is negligible , and the area of resin gel that receives solute corresponds largely to the physical sampler window surface area . when using thicker diffusion layers more solute diffuses outward , increasing the resin gel area that receives solute . it can be seen that the central area of the resin gel receives solute corresponding to the 1d flux for most diffusion layer thicknesses used here , with this central resin gel area becoming smaller with increasing diffusion layer thickness . only for extremely thick diffusion layers ( g = 0.5 cm ) the solute flux to the resin gel decreases to below the 1d situation even in the very center of the sampler . the flux increases predicted by the model range from 0.1% to 34.5% for samplers with rphys = 1.0 cm and from 0.1% to 45.9% for rphys = 0.9 cm ( table 1 ) , showing that not only the diffusion layer thickness , but also the size of the sampler window affects lateral diffusion . only accounting for lateral diffusion ( columns 2 and 3 ) , not for the dbl . it has been suggested to use the effective sampling area ( aeff ) instead of the physical sampling surface area ( aphys ) to correct for lateral diffusion when applying eq 3 , that is , in situations where the flux increase that is brought about by lateral diffusion is not balanced by the flux decrease because of the formation of the dbl . in the special case that both effects balance each other , eq 2 can be used . the result for both situations is that cbdgt / cb 1 , so dgt provides a measurement of the solute concentration in the exterior solution . this mathematical correction of lateral diffusion is necessary because dgt results are evaluated using a 1d solution of fick s law of diffusion , while the sampling process is based on omnidirectional ( 3d ) diffusion . as the standard 1d equation ( eq 2 ) works well unless the dbl and lateral diffusion effects do not balance each other , the need for correcting for lateral diffusion has been recognized relatively late . as lateral diffusion depends on the sampler geometry , the sampler geometry has to be accounted for when correcting for lateral diffusion . the lateral diffusion flux increase coefficient , kld , can be defined as the ratio of the solute flux into the sampler in the 3d situation , f0,3d , and the 1d flux , f0,1d7here , the index 0 indicates a dbl layer thickness of = 0 ( cf . , definition of kdbl , eq 11 ) . a plot of kld versus g for samplers with rphys values of 0.9 and 1.0 cm shows a nonlinear relation of the two parameters ( figure 2 ) . second - order polynomial functions result in excellent fits ( r = 0.9998 and 0.9999 , respectively ) to these data.89therefore , eqs 8 and 9 can be used to determine the flux increase coefficient for a given diffusion layer thickness and correcting cbdgt for lateral diffusion through dividing by the flux increase factor10 the geometry of the model dbl used in this study is idealized with respect to the dbl being equally thick throughout the whole solution - sampler interface ( figure 1 and supporting information figure s1 ) , however , this simplification is consistent with dgt theory . the physical dbl that forms around a dgt sampler in a solution will have a different shape , i.e. a thicker dbl layer toward the edges of the sampling window and a thinner one toward the center can be expected , therefore the dbl thickness estimated using dgt samplers is an average value across the sampler surface . the data points are results of simulation runs , the lines are fits of second - order polynomial functions to the simulated data . table 1 shows simulated dgt fluxes with and without dbl layers ( = 100 and 200 m ) , which indicate that the increased flux because of lateral diffusion into dgt samplers with a common diffusion layer thickness of 0.094 cm ( i.e. , a flux increase of 8.79.7% ) is balanced by dbl layers of 100 m thickness for rphys values of 1 and 0.9 cm . this observation is in line with the general finding that cbdgt / csoln 1 for g = 0.094 cm in well - stirred solutions , as reported in earlier studies . in contrast , warnken et al . reported dbl layer thicknesses of 230 32 m in moderate to well stirred solutions , which did not change upon varying the solution flow velocity ( i.e. , stir rate ) , and matched well with their estimation of the lateral diffusion flux increase of 20% . simulation runs with and without dbl were performed for investigating the effect of solute species ( dwater , dgel ) , rphys , g , and on solute diffusion through the dbl . as we expected the ratio of the diffusion coefficients , dgel / dwater , to determine the effect of the diffusion coefficient , arsenate ( dwater = 9.05 10 cm s , dgel = 5.93 10 cm s ) and cd ( dwater = 7.17 10 cm s , dgel = 6.09 10 cm s ) were chosen as additional solutes to cover a wide range of dgel / dwater ratios . potential differences in the flux decrease due to the dbl in the 1d and 3d situation were evaluated using11where the index dbl denotes a specific dbl thickness , while the index 0 denotes = 0 . fdbl / f0 is the sampler flux decrease caused by the presence of the dbl . by normalizing the sampler flux decrease in the 3d situation for that in 1d , kdbl provides a measure of how much the sampler flux is changed in the 3d geometry compared to the common 1d solutions of the dgt equation . a plot of kdbl vs. dgel / dwater shows that the dbl flux decrease in 3d is 0.950.99 times the flux decrease of the 1d situation ( rphys = 1 cm , g = 0.094 cm ; figure 3 ) and that there is a slight dependence of the magnitude of the flux decrease on the solute species ( i.e. , dgel / dwater ) . kdbl also depends on g and , with extreme values of kdbl = 0.93 ( rphys = 1.0 cm ) and kdbl = 0.92 ( rphys = 0.9 cm ) for g = 0.5 cm and = 0.1 cm . this shows that the flux decrease associated with the formation of the dbl is larger when 3d diffusion is considered , as compared to the 1d situation , and that kdbl is dependent on numerous parameters ( dwater / dgel , rphys , g , ) . parameters affecting the dbl related flux decrease ( kdbl ) . left : dependence of kdbl on dgel / dwater , which is a solute species - specific ratio . the slight difference in the dbl - related flux decrease between 1d and 3d diffusion in the order of a few percent effectively is a propagation of the effect of lateral diffusion inside the sampler to the diffusive regime outside . the concentration gradient that causes the outward diffusion of solutes inside the sampler affects diffusion in the dbl as the solute concentration at the sampler - solution interface at the edge of the sampling window will be smaller than the concentration at the center of the interface . this or similar effects have recently been hypothesized by davison and zhang . in principle , the difference in solute diffusion through the dbl in 1d and 3d can be corrected for in the full dgt equation by introducing kdbl as a further correction factor12however , this would require knowledge , or at least a good estimate , of the dbl thickness before kdbl can be determined . moreover , given the number of controlling parameters , it would be necessary to determine kdbl for each individual dgt deployment by numerical simulation similar to this study . as doing so is rather impractical and as the error by neglecting this correction step is small ( see dbl thickness estimation section ) kdbl could be omitted in most studies . therefore , eq 10 might serve as standard equation to account for lateral diffusion only inside the sampler . the conclusions of the present study and those of warnken et al . on the effect of lateral diffusion and the dbl on dgt measurements differ considerably . in the earlier work a lateral diffusion - related relative flux increase of 20% was estimated , which was considered to be independent of the diffusion layer thickness as well as of the sampling window surface area . this earlier study is an elaborate , extensive treatment of lateral diffusion inside dgt samplers , therefore it is interesting to analyze why its results and conclusions differ from those presented here . direct evidence for lateral diffusion inside dgt samplers was obtained in warnken et al . by measuring the cd distribution across a dgt resin gel disk that was immersed in a cd - containing solution with a diffusion layer thickness of 0.134 cm using la - icpms . the obtained cd profile showed shoulders at the edges of the gel disk , consistent with outward diffusion of cd . from this profile , the authors calculated the effective gel radius , that is , the profile length necessary to explain the additional cd uptake on the basis of 1d diffusion . this resulted in an effective gel radius of 1.08 cm , which corresponds to an effective area of 3.66 cm or a relative flux increase of 17% . the quantification of the lateral diffusion - based flux increase was based on a different approach . therefore , warnken et al . corrected cbdgt values for the dbl effect , which should result in cbdgt / cb > 1 if lateral diffusion increases solute uptake . this was achieved by immersing dgt samplers with different diffusion layer thicknesses in stirred laboratory solutions , plotting 1/m versus g , determining the slope and intercept of the resulting regression line , and calculating cbdgt and using an earlier developed procedure . in one of those experiments , deploying four dgt samplers for 50 h in a cd containing solution , warnken et al . found a dbl thickness of 170 m . the associated cbdgt value , which should correspond to cb , given that the dbl related flux decrease should be accounted for by the estimation procedure , was 20% larger than the solution concentration . the increased mass uptake was considered an effect of lateral diffusion , as this effect is not covered by the applied fitting procedure . this reasoning is sound , provided that the procedure for estimating cb and is correct and accurate . repeating this calculation using data gives cbdgt = 10.9 g l , = 180 m , and cbdgt / cb = 1.16 , that is a lateral diffusion flux increase of 16% . considering the uncertainty associated with data digitalization and that the average cb during the 50 h dgt deployment was not given in the paper ( we estimated a value of 9.4 g l based on the available data ) , the agreement to the published values is very good . the individual cbdgt values for all four samplers have a relative standard deviation ( rsd ) of 1.6% based on = 180 m , which is a minimum compared to lower or higher dbl values . correction for the nonconstant , diffusion layer thickness - dependent flux increase ( kld ) gives an average cbdgt of 9.7 g l and hence cbdgt / cb = 1.03 , consistent with a theoretical cbdgt / cb = 1 after accounting for lateral diffusion . for this case the rsd of the cbdgt values is lowest with 3.0% at = 140 m . the klds used here ranged from 1.014 ( g = 0.014 cm ) to 1.126 ( g = 0.134 cm ) , which is considerably less than the flux increase factor determined by warnken et al . following zhang and davison , warnken et al . assumed a linear relationship of 1/m vs. g , however the assumption of linearity does not hold if the lateral diffusion flux increase is a nonlinear function of g . their procedure for estimating cb and , based on evaluating the slope and intercept of a linear regression line , is therefore not rigorous and leads to different results for the dbl , and , based thereon , aeff , compared to using nonconstant , nonlinear kld values . in conclusion , using a constant flux increase factor ( independent of the sampler geometry , i.e. of g , aphys ) resulted in the adoption of a relative flux increase of 20% . however , even if aeff had been more accurately determined for an individual diffusion layer thickness it would have been very hard to establish nonconstant lateral diffusion induced flux increases based on experimental data , as the flux increases in the commonly used g range ( 0.020.2 cm ) can easily be masked by the measurement uncertainty , which is often in the range of 510% . table 2 shows the results of estimating and cb using simulated and literature dgt measurements with varying g . the fits of the full dgt model to the simulated data resulted in deviations from the initial of up to only 4% for all lateral diffusion correction methods ( aeff , kld or kld and kdbl ) . however , cbdgt converged to the initial value only when kld and kld and kdbl were employed , whereas using aeff resulted in a deviation of 14% . the modified model of garmo et al . resulted in decreased and cbdgt values when using aeff , both in the range of 14% , but performed equally well as the full model when applying kld and kld and kdbl . the full model lead to perfect estimates for both , and cb , when applying kld and kdbl , which is of course expected for a simulated , measurement error - free data set . however , the model of garmo et al . deviated by about 1% from to initial values , showing that their modification is no full representation of the dgt sampling process . moreover , it does not lead to improved parameter estimates when applying aeff , which was the actual aim of introducing their modified model . the estimates obtained by fits of the literature data to the full dgt model with aeff correction are 20% higher than the estimates obtained with kld and kld and kdbl correction . the values obtained from the modified dgt model , irrespective of the correction method , are in the same range as the values obtained with the full dgt model and kld and kld and kdbl correction . the fitted cbdgt values deviate from the measured and estimated solution concentrations by 5% to + 9.6% . these example estimations of and cb show that the lateral diffusion correction methods proposed here , based on the use of kld or , if available , kld and kdbl , result in better estimates than directly fitting the full dgt equation using aeff correction . the modified dgt model proposed by garmo et al . resulted in similar parameter estimates compared to the dgt equation with kld and kld and kdbl correction for the experimental data sets , but not for the simulated data set , indicating that the full dgt equation with kld and kld and kdbl correction provides more reliable parameter estimates . the difference between and cb estimates based on the expanded dgt equation and kld versus kld and kdbl correction was small with 1.0% to 2.4% , therefore the use of kld without kdbl seems acceptable given the effort needed to determine kdbl by numerical simulation for individual dgt applications . the dbl thickness and cb were estimated by fitting eqs 5 and 6 to a simulated and two literature data sets . the effects of lateral diffusion and the dbl were corrected for by using aeff , kld or kld and kdbl . cb nominal is the approximate solute ( cd ) concentration given in the paper ; cb estimated is the estimated , time - averaged cd concentration during this 50 h experiment , based on the data given in the paper , see materials and methods section for details . estimated using the linearized version of the expanded dgt equation ( supporting information eq s14 ) . we show that and cb estimates in dgt applications with samplers of varying diffusion layer thickness vary considerably for different estimation strategies . the expanded dgt equation ( eq 5 ) in combination with the lateral diffusion correction factor kld with or without kdbl resulted in the best cb estimates for simulated and experimental data . moreover , this procedure precisely estimated in a simulated data set where estimates based on aeff were considerably different to initial and measured values in several of the investigated cases . the results obtained using the modified equation ( eq 6 ) were typically within 10% of the best estimates . in the study of garmo et al . , estimates using the linearized version of the full dgt equation were up to 25% ( cb ) and 83% ( ) larger than those based on direct , nonlinearized fits to their modified model , indicating that the use of the linearized estimation procedure may introduce large errors in cb and estimates . therefore , we propose to use direct fits of the nonlinearized form of eq 5 , with kld as the lateral diffusion correction factor . the procedure presented here is expected to increase the accuracy of cb measurements in environmental monitoring applications of labile solute in waterbodies and might furthermore be relevant for obtaining more accurate determinations of complex dissociation kinetics by dgt , where lateral diffusion correction and parameter estimation through data fitting is also applied . so far , the effect of lateral diffusion was only considered for dgt measurements in solutions , but not for measurements of labile solutes in sediments and soils . in solutions diffusion and , if labile complexed species are present , complex dissociation are controlling the mass uptake of dgt samplers . in soils and sediments the porewater concentration of the dissolved species are usually controlled by sorption and dissolution - precipitation equilibria , with potential contributions from the dissociation of complexes , but not by diffusion into the sampler . for these reasons , only up to 3.4-fold increases in the mass uptake of cu , cd , and p by dgt from soil were measured when the concentration gradient into the sampler was 95-fold increased by decreasing the diffusion layer thicknesses from 0.094 to 0.001 cm . given these relatively small mass uptake increases for large increases in solute demand , changes of dgt mass uptake from soil and sediments in response to a potential lateral diffusion flux increase by a factor of up to 1.45 will be hardly measurable as compared to the 1d diffusion situation . mathematical correction of the lateral diffusion flux increases in soil and sediment dgt measurements should therefore not be necessary .

using numerical simulation of diffusion inside diffusive gradients in thin films ( dgt ) samplers , we show that the effect of lateral diffusion inside the sampler on the solute flux into the sampler is a nonlinear function of the diffusion layer thickness and the physical sampling window size . in contrast , earlier work concluded that this effect was constant irrespective of parameters of the sampler geometry . the flux increase caused by lateral diffusion inside the sampler was determined to be 8.8% for standard samplers , which is considerably lower than the previous estimate of 20% . lateral diffusion is also propagated to the diffusive boundary layer ( dbl ) , where it leads to a slightly stronger decrease in the mass uptake than suggested by the common 1d diffusion model that is applied for evaluating dgt results . we introduce a simple correction procedure for lateral diffusion and demonstrate how the effect of lateral diffusion on diffusion in the dbl can be accounted for . these corrections often result in better estimates of the dbl thickness ( ) and the dgt - measured concentration than earlier approaches and will contribute to more accurate concentration measurements in solute monitoring in waters .

Introduction Materials and Methods Results and Discussion

the sampling process is simple : once the dgt device gets in contact with the sampled medium , the target solutes diffuse into the sampler where they get bound by the resin gel . on the basis of the 1d steady - state solution of fick s first law of diffusion1the solute concentration at the sampler medium interface can be calculated by eq 2(1)2here , j is the diffusive flux , dgel is the solute diffusion coefficient in the diffusion layer , cb is the concentration in the exterior solution , cr is the concentration in the resin gel ( which is effectively zero as the resin is a strong solute sink ) , g is the diffusion layer thickness , cdgt is the concentration at the sampler medium interface , m is the mass of solute bound by the resin layer , aphys is the physical sampling window surface area and t is the sampling time . , differently protonated species ) , that the diffusive boundary layer ( dbl ) forming outside the protective membrane has negligible effect on the sampler s mass uptake in well - stirred laboratory solutions and agitated natural waters , and , implicitly , that diffusion inside the sampler is one - dimensional and has no lateral components . therefore , separate terms for solute diffusion in the dbl and the filter membrane layer were integrated into the dgt equation3the superscript dgt indicates a cb estimate obtained using dgt . for standard , apa2-type diffusion gels and supor membranes ( pall coporation , port washington , ny , usa ) , for which diffusion coefficients were reported to be indistinguishable , the membrane thickness can be added to g and the term for the membrane can be omitted . note that eq 3 provides a measure of cb , as the effect of the dbl on solute mass uptake is corrected for . high - resolution analysis of dried resin gels using laser ablation inductively coupled plasma mass spectrometry ( la - icpms ) showed that this unexpectedly high mass uptake was caused by lateral solute diffusion inside the dgt samplers . the authors concluded that the dbl - related decrease in mass uptake is balanced by the increase caused by lateral solute diffusion for standard dgt devices ( g = 0.94 mm , aphys = 3.14 cm ) deployed in well - stirred solutions . in this case , the simplification of using the physical sampling window area ( aphys = 3.14 cm ) and the simple dgt equation was found to be acceptable . suggested to use the expanded dgt equation and an effective sampling area ( aeff ) instead of the physical sampling window area to account for the lateral diffusion effect . a general flux increase of 20% was adopted for dgt samplers , translating to aeff = 3.80 cm for standard dgt devices ( aphys = 3.14 cm ) and aeff = 3.08 cm for dgt devices with smaller physical window area ( aphys = 2.54 cm ) . in this and in subsequent studies the relative flux increase due to lateral diffusion was assumed to be constant irrespective of the sampler geometry ( e.g. a sketch of a dgt sampler shows that lateral diffusion inside the sampler takes place at the edge of the diffusion and resin gels , where the sampler housing extends over the diffusion layer ( figure 1 ) . figure 1 indicates that the diffusion layer thickness , but also the size of the covered gel region ( i.e. , the physical sampler window area ) might affect the concentration gradient and thereby the magnitude of the flux increase because of lateral diffusion , hence aeff should depend on the sampler geometry . in this study , we apply numerical modeling for simulating 3d diffusion into and inside dgt samplers to investigate the dependence of the lateral diffusion - induced mass uptake increase on g and aphys . moreover , we strive for identifying additional parameters that might affect the solute flux into and inside dgt samplers . the dgt sampler housing covers a 2.5 mm wide area of the diffusion layer and the resin gel . the direction of the concentration gradient that establishes at the edge of the sampler is oriented toward the resin gel edge , as the solute concentration in this area is lower than in the diffusion layer zone that is in direct contact with the medium . as a consequence , the solute concentration at the diffusion layer dbl interface is lower than in the center of the sampler , leading to a higher solute flux into the sampler at the sampler edge compared to the center . we solved the diffusion equation ( eqs s1 , s6 , and s7 in the supporting information ) on two different , 2d - axisymmetric simulation geometries , one representing the diffusion layer without considering the dbl ( supporting information figure s1 top ) and a second including the overlying dbl layer ( supporting information figure s1 bottom ) . the flux profiles into the resin gel that were obtained from the 2d - axisymmetric model were used to create revolution surfaces , across which the flux into the resin gel was integrated to obtain the cumulated solute flux into the dgt sampler . although , strictly , the results reported in this study are based on 2d - axisymmetric simulations , they are a valid representation of the 3d solute flux into the sampler . the diffusion boundary layer thickness ( ) was varied between 0.0025 and 0.1 cm . a steady solute flux into a dgt sampler is established a few minutes after exposure of the sampler to the sampled medium . lateral diffusion inside the sampler was evaluated by comparing simulated 3d fluxes into dgt samplers without dbl , which include the lateral diffusion effect , to fluxes calculated using the 1d solution for the dgt flux4disregarding the effect of lateral diffusion at the sampler edges . for investigating the effect of the dbl on the sampler flux these sampler fluxes were compared to 1d fluxes without dbl ( eq 4 ) and to 1d fluxes including the dbl , which were simulated with a simple 1d representation of the dgt geometry . here we investigate the effect of applying the nonconstant correction coefficients determined in this study on the results of dbl thickness estimations using ( 1 ) a simulated data set and ( 2 ) literature data . using the free software the dbl thickness ( along with cb ) can be determined by fitting eq 5,5representing the expanded dgt equation ( eq 3 ) solved for m , to dgt - determined solute masses m. for simplifying the fitting procedure a linearized version of eq 5 , supporting information eq s14 , has been used . fits of these equations to the simulated and literature data sets were computed using ( 1 ) aeff = 3.8 cm or aeff = 3.08 cm , as appropriate , ( 2 ) correction for lateral diffusion inside the sampler only ( kld , explanation in results and discussion ) , and ( 3 ) correction for lateral diffusion and non-1d diffusion through the dbl ( kld and kdbl , explanation in results and discussion ) . the reason for this flux increase , as already outlined in the introduction and visualized in figure 1 , is the lateral diffusion at the sampler edges , which has already been shown experimentally by warnken et al . simulated profiles of the solute flux into the resin gel show that an increasing solute mass fraction is transported toward the edge of the resin gel upon increasing the diffusion layer thickness ( supporting information figure s2 ) . for very small g values ( 0.001 cm , 0.005 cm ) , the outward solute flux is negligible , and the area of resin gel that receives solute corresponds largely to the physical sampler window surface area . it can be seen that the central area of the resin gel receives solute corresponding to the 1d flux for most diffusion layer thicknesses used here , with this central resin gel area becoming smaller with increasing diffusion layer thickness . only for extremely thick diffusion layers ( g = 0.5 cm ) the solute flux to the resin gel decreases to below the 1d situation even in the very center of the sampler . the flux increases predicted by the model range from 0.1% to 34.5% for samplers with rphys = 1.0 cm and from 0.1% to 45.9% for rphys = 0.9 cm ( table 1 ) , showing that not only the diffusion layer thickness , but also the size of the sampler window affects lateral diffusion . only accounting for lateral diffusion ( columns 2 and 3 ) , not for the dbl . it has been suggested to use the effective sampling area ( aeff ) instead of the physical sampling surface area ( aphys ) to correct for lateral diffusion when applying eq 3 , that is , in situations where the flux increase that is brought about by lateral diffusion is not balanced by the flux decrease because of the formation of the dbl . this mathematical correction of lateral diffusion is necessary because dgt results are evaluated using a 1d solution of fick s law of diffusion , while the sampling process is based on omnidirectional ( 3d ) diffusion . as lateral diffusion depends on the sampler geometry , the sampler geometry has to be accounted for when correcting for lateral diffusion . the lateral diffusion flux increase coefficient , kld , can be defined as the ratio of the solute flux into the sampler in the 3d situation , f0,3d , and the 1d flux , f0,1d7here , the index 0 indicates a dbl layer thickness of = 0 ( cf . second - order polynomial functions result in excellent fits ( r = 0.9998 and 0.9999 , respectively ) to these data.89therefore , eqs 8 and 9 can be used to determine the flux increase coefficient for a given diffusion layer thickness and correcting cbdgt for lateral diffusion through dividing by the flux increase factor10 the geometry of the model dbl used in this study is idealized with respect to the dbl being equally thick throughout the whole solution - sampler interface ( figure 1 and supporting information figure s1 ) , however , this simplification is consistent with dgt theory . a thicker dbl layer toward the edges of the sampling window and a thinner one toward the center can be expected , therefore the dbl thickness estimated using dgt samplers is an average value across the sampler surface . table 1 shows simulated dgt fluxes with and without dbl layers ( = 100 and 200 m ) , which indicate that the increased flux because of lateral diffusion into dgt samplers with a common diffusion layer thickness of 0.094 cm ( i.e. , stir rate ) , and matched well with their estimation of the lateral diffusion flux increase of 20% . simulation runs with and without dbl were performed for investigating the effect of solute species ( dwater , dgel ) , rphys , g , and on solute diffusion through the dbl . as we expected the ratio of the diffusion coefficients , dgel / dwater , to determine the effect of the diffusion coefficient , arsenate ( dwater = 9.05 10 cm s , dgel = 5.93 10 cm s ) and cd ( dwater = 7.17 10 cm s , dgel = 6.09 10 cm s ) were chosen as additional solutes to cover a wide range of dgel / dwater ratios . potential differences in the flux decrease due to the dbl in the 1d and 3d situation were evaluated using11where the index dbl denotes a specific dbl thickness , while the index 0 denotes = 0 . fdbl / f0 is the sampler flux decrease caused by the presence of the dbl . by normalizing the sampler flux decrease in the 3d situation for that in 1d , kdbl provides a measure of how much the sampler flux is changed in the 3d geometry compared to the common 1d solutions of the dgt equation . a plot of kdbl vs. dgel / dwater shows that the dbl flux decrease in 3d is 0.950.99 times the flux decrease of the 1d situation ( rphys = 1 cm , g = 0.094 cm ; figure 3 ) and that there is a slight dependence of the magnitude of the flux decrease on the solute species ( i.e. this shows that the flux decrease associated with the formation of the dbl is larger when 3d diffusion is considered , as compared to the 1d situation , and that kdbl is dependent on numerous parameters ( dwater / dgel , rphys , g , ) . the slight difference in the dbl - related flux decrease between 1d and 3d diffusion in the order of a few percent effectively is a propagation of the effect of lateral diffusion inside the sampler to the diffusive regime outside . the concentration gradient that causes the outward diffusion of solutes inside the sampler affects diffusion in the dbl as the solute concentration at the sampler - solution interface at the edge of the sampling window will be smaller than the concentration at the center of the interface . in principle , the difference in solute diffusion through the dbl in 1d and 3d can be corrected for in the full dgt equation by introducing kdbl as a further correction factor12however , this would require knowledge , or at least a good estimate , of the dbl thickness before kdbl can be determined . therefore , eq 10 might serve as standard equation to account for lateral diffusion only inside the sampler . on the effect of lateral diffusion and the dbl on dgt measurements differ considerably . in the earlier work a lateral diffusion - related relative flux increase of 20% was estimated , which was considered to be independent of the diffusion layer thickness as well as of the sampling window surface area . this earlier study is an elaborate , extensive treatment of lateral diffusion inside dgt samplers , therefore it is interesting to analyze why its results and conclusions differ from those presented here . corrected cbdgt values for the dbl effect , which should result in cbdgt / cb > 1 if lateral diffusion increases solute uptake . the associated cbdgt value , which should correspond to cb , given that the dbl related flux decrease should be accounted for by the estimation procedure , was 20% larger than the solution concentration . the increased mass uptake was considered an effect of lateral diffusion , as this effect is not covered by the applied fitting procedure . repeating this calculation using data gives cbdgt = 10.9 g l , = 180 m , and cbdgt / cb = 1.16 , that is a lateral diffusion flux increase of 16% . considering the uncertainty associated with data digitalization and that the average cb during the 50 h dgt deployment was not given in the paper ( we estimated a value of 9.4 g l based on the available data ) , the agreement to the published values is very good . correction for the nonconstant , diffusion layer thickness - dependent flux increase ( kld ) gives an average cbdgt of 9.7 g l and hence cbdgt / cb = 1.03 , consistent with a theoretical cbdgt / cb = 1 after accounting for lateral diffusion . the klds used here ranged from 1.014 ( g = 0.014 cm ) to 1.126 ( g = 0.134 cm ) , which is considerably less than the flux increase factor determined by warnken et al . assumed a linear relationship of 1/m vs. g , however the assumption of linearity does not hold if the lateral diffusion flux increase is a nonlinear function of g . however , even if aeff had been more accurately determined for an individual diffusion layer thickness it would have been very hard to establish nonconstant lateral diffusion induced flux increases based on experimental data , as the flux increases in the commonly used g range ( 0.020.2 cm ) can easily be masked by the measurement uncertainty , which is often in the range of 510% . these example estimations of and cb show that the lateral diffusion correction methods proposed here , based on the use of kld or , if available , kld and kdbl , result in better estimates than directly fitting the full dgt equation using aeff correction . the dbl thickness and cb were estimated by fitting eqs 5 and 6 to a simulated and two literature data sets . the effects of lateral diffusion and the dbl were corrected for by using aeff , kld or kld and kdbl . , estimates using the linearized version of the full dgt equation were up to 25% ( cb ) and 83% ( ) larger than those based on direct , nonlinearized fits to their modified model , indicating that the use of the linearized estimation procedure may introduce large errors in cb and estimates . the procedure presented here is expected to increase the accuracy of cb measurements in environmental monitoring applications of labile solute in waterbodies and might furthermore be relevant for obtaining more accurate determinations of complex dissociation kinetics by dgt , where lateral diffusion correction and parameter estimation through data fitting is also applied . so far , the effect of lateral diffusion was only considered for dgt measurements in solutions , but not for measurements of labile solutes in sediments and soils . for these reasons , only up to 3.4-fold increases in the mass uptake of cu , cd , and p by dgt from soil were measured when the concentration gradient into the sampler was 95-fold increased by decreasing the diffusion layer thicknesses from 0.094 to 0.001 cm . given these relatively small mass uptake increases for large increases in solute demand , changes of dgt mass uptake from soil and sediments in response to a potential lateral diffusion flux increase by a factor of up to 1.45 will be hardly measurable as compared to the 1d diffusion situation .

prostate cancer is the most common cancer in men and the second - leading cause of cancer death in men , with 241,740 new diagnoses and 28,170 deaths projected in the us in 2012.1 advanced prostate cancer has a penchant to metastasize to bone , possibly due to osteomimicry or altered adhesion molecules.2,3 among patients with metastatic , castrate - resistant prostate cancer ( mcrpc ) treated with first - line chemotherapy , almost 90% have radiographic evidence of bone metastasis.4,5 morbidity from complications of osseous metastases , such as pathologic fractures , spinal cord compression , and pain , greatly impairs the quality of life of patients with mcrpc . in addition , pain due to mcrpc is a strong independent prognostic factor for death , and is included in contemporary nomograms for survival in this disease,69 thus agents that can reduce pain and suffering due to metastatic prostate cancer may improve not only quality of life but also quantity of life . mitoxantrone chemotherapy and several radioisotopes , such as samarium-153 ( sm ) and strontium-89 ( sr ) , were the first therapies approved for palliation of bone pain in patients with mcrpc , but without evidence of improvement in overall survival.10,11 since 2004 , four systemic therapies have been approved on the basis of improvements in overall survival , with varying effects on bone pain and skeletal complications.4,5,1214 these agents , particularly the taxanes docetaxel ( taxotere ; sanofi - aventis , bridgewater nj ) , cabazitaxel ( jevtana ; sanofi - aventis ) , and the novel androgen biosynthesis inhibitor abiraterone acetate ( zytiga ; janssen biotech , inc , horsham , pa ) , have direct antitumor effects that may result in pain palliation . contemporaneously , two osteoclast inhibitors ( zoledronic acid [ zometa ; novartis ag , basel , switzerland ] and denosumab [ xgeva ; amgen inc , thousand oaks , ca ] ) have been approved on the basis of delay of skeletal - related events ( sres ; pathologic fracture , spine cord compression , and requirement for radiation or surgery to bone ) , but without improvements in overall survival ( os).15,16 in addition , the novel androgen signaling inhibitor mdv3100 ( enzalutamide , xtandi ; medivation , inc , san francisco , ca ) has demonstrated improvements in os and palliative end points ; its approval was granted august 31 , 2012 , as this article went to press.1719 prior to the development of ra ( alpharadin ; algeta asa , oslo , norway / bayer ag , leverkusen , germany ) , no specifically bone - targeted therapy has been shown to improve median os in the population of patients with mcrpc with osseous metastases . survival - prolonging systemic therapies currently approved for mcrpc include docetaxel , cabazitaxel , sipuleucel - t ( provenge ; dendreon , seattle , wa ) , abiraterone , and mdv3100 ( table 1 ) . pain response is the only sre that has been routinely reported ( table 2 ) , with much fewer data on prevention or delay of sres . a special case to this has been sipuleucel - t , which is specifically indicated for men with mcrpc who have minimal to no symptoms . as a reference , the palliative chemotherapy regimen of mitoxantrone / prednisone demonstrated a 29% response on the present pain intensity ( ppi ) scale versus 12% with prednisone alone ( p = 0.01 ) , with a duration of 43 versus 18 weeks ( p < 0.0001).10 the landmark tax-327 study showed a pain response of 35% versus 22% ( p = 0.01 ) on the ppi scale for docetaxel / prednisone versus mitoxantrone / prednisone , with a duration of 3.5 versus 4.8 months ( p not significant).5 patients who experienced a pain response in this trial were found to have improved overall survival as compared with those men without such a response.7 in the post - docetaxel mcrpc setting , cabazitaxel / prednisone showed no improvement over mitoxantrone / prednisone on the ppi scale ( 9.2% vs 7.7% ; p = 0.63 ; duration not reached ) in the tropic trial.13 in a similar post - docetaxel setting , the cou - aa-301 study demonstrated a 44% versus 27% pain response on the brief pain inventory ( bpi ) scale for abiraterone / prednisone vs placebo / prednisone , with a time to progression of approximately 8 versus 5 months ( 25th percentile ; p = 0.0056).20 the great variability in pain response for the control arms ( 7.7% for mitoxantrone / prednisone in the tropic study , and 27% for prednisone alone in the cou - aa-301 study ) suggests great variability in the reporting methods . notably , in the cou - aa-301 study , the time to sre was also delayed to approximately 10 versus 5 months ( p = 0.0006 ) . there were previously no other data regarding prevention or delay of sres with the other survival - prolonging systemic therapies . bone - targeted osteoclast inhibitors and bone - seeking radiopharmaceuticals have been approved for mcrpc on the basis of prevention or delay of sres and palliation of pain , respectively . zoledronic acid , a bisphosphonate that inhibits osteoclasts , significantly delayed the time to sre versus placebo ( approximately 16 versus 11 months , p = 0.009 ) and also improved pain and analgesia scores.15,21 more recently , a monoclonal antibody to the receptor activator of nf-b ligand ( rankl ) , denosumab , has also been approved . in a placebo - controlled phase iii trial , denosumab showed a 3.6-month improvement in time to first sre over zoledronic acid ( 20.7 versus 17.1 months ; p = 0.0002 [ noninferiority ] , p = 0.008 [ superiority]).16 of note , neither zoledronic acid nor denosumab demonstrated improvements in prostate - specific antigen ( psa ) levels , overall disease progression , or overall survival . thus , while these are bone - targeted agents , they may have a greater effect on bone health and quality than on the tumor microenvironment , which supports metastatic cancer and promotes treatment resistance . an ideal tumor microenvironment targeting agent would thus not only prevent symptomatic deterioration but also improve upon disease - related outcomes , particularly os . in addition to osteoclast inhibitors , therapeutic radioisotopes that have a predilection to accumulate in bone turnover sites can be administered . an alpha particle , which is ejected from a heavy nucleus during alpha decay , consists of two neutrons and two protons ( ie , a helium nucleus).22 a beta particle is an electron released from a nucleus containing excess neutrons during beta decay , in which one neutron is converted to a proton , an electron , and a neutrino.22 both - and -particles can deliver damaging radiation locally to cancerous cells . the most commonly used radiopharmaceuticals , both -emitters , currently approved in the us for treatment of bone metastases are sr ( metastron ; ge healthcare , arlington heights , il ) and sm ( quadramet ; eusa pharma , oxford , uk ) . there are key differences in the physical properties of these radioisotopes , which have important clinical implications ( table 3 ) . sr is a pure -emitter with a long half - life ( 50 days ) , whereas sm has a much shorter half - life ( 1.9 days ) and is also a -emitter , which allows posttreatment scintigraphic imaging . in addition , sr emits higher - energy beta particles , resulting in greater tissue penetration and consequently higher bone marrow toxicity . multiple randomized trials have been conducted with sr and sm in patients with mcrpc.2329 there was no demonstration of improvement in overall survival in phase iii trials , although palliative benefits were seen that formed the basis of us fda approval . one recent meta - analysis concluded that , although there is some evidence that these beta - emitting radioisotopes might provide a small benefit with complete reduction in pain over 16 months and no increase in analgesic use , severe adverse effects ( mainly leukopenia and thrombocytopenia ) are relatively frequent.11 nonetheless , sm can be administered in repeat doses , once hematologic toxicities have recovered , for persistent pain.30 other limitations to sr and sm include the fact that they are renally excreted ; this is not ideal in patients with genitourinary cancers . overall , sr and sm might provide some palliative of pain , at the potential expense of significant hematologic toxicities and without demonstrated os benefit . ongoing work of these agents in combination with chemotherapy may optimize their efficacy.3134 in comparison to beta - emitting radioisotopes , radium- 223 ( ra ; alpharadin , algeta ) is an -emitter that delivers radiation with a higher biologic effect to a more localized area . in a large randomized phase iii trial , ra has recently demonstrated improvements in os , time to first sre , and biochemical parameters , with a remarkably tolerable adverse - event profile , in men with bone - metastatic crpc.35 while ra has not yet been approved for use in the us , and there is little experience in the us with the use of this agent , it represents the first bone - targeted therapeutic radiopharmaceutical to demonstrate a survival benefit , which has been the benchmark for recent fda approvals in this disease setting . an expanded access trial of ra in men with mcrpc with bone metastases is planned.36 many questions have arisen as to the appropriate sequencing of existing and novel therapies with ra and if / how to combine ra with these systemic therapies . this manuscript will review the preclinical and clinical development of ra , and comment on its potential role and use in the armamentarium of therapy for mcrpc . bone - seeking therapeutic radiopharmaceuticals are unique among anticancer therapies in that they actually target the stroma rather than the tumor itself : the target is calcium hydroxyapatite in bone . ionizing radiation can thus be selectively delivered to areas of increased osteoblastic activity , allowing the targeting of multiple metastases simultaneously , including both symptomatic as well as asymptomatic lesions . as mentioned above , the commonly used clinically available radiopharmaceuticals are -emitters , with sr and sm the most well studied.37 following injection of sr , radiation doses are delivered to the osseous target lesions at low dose rates.38 between 16 weeks and 1 year , absorbed doses in the 2040 gy range are deposited as a steadily declining dose rate.39,40 because of its higher dose rate and the shorter range ( ie , tissue penetration ) , sm may have an improved therapeutic index compared with that of sr ( table 3).38 due to its relatively shorter half - life , repeated administration of sm is feasible for persistent or recurrent bone pain provided adequate hematologic function returns ; however , effects on bone marrow remain a limitation.30 this suggests that an agent with less depth of tissue penetration but similar or higher biologic effect is needed to facilitate repeated dosing . one potential reason for difficulty demonstrating antitumor and survival benefits with -emitters could be related to this dosing limitation . cationic radium is a calcium mimetic ( located in the same column of the periodic table as calcium ) , and as such naturally targets areas of bone turnover without the need for a carrier . ( recall that the bone stores 99% of the human body s calcium and 85% of the phosphorous . blood levels of calcium and bone resorption / formation are both tightly regulated by various hormones.41 ) for example , radium-224 ( ra ) has been used extensively for treatment of ankylosing spondylitis , a chronic inflammatory disease of the axial skeleton characterized by new bone formation.42 ra has a half - life of 11.4 days , produces four alpha particles from decay through short - lived daughter radionuclides , and has advantages over ra in terms of the decay chain.43 ra was selected for biomedical applications based on its favorable decay chain and half - life.43 in contrast to -emitters , which have a low linear energy transfer ( let ) and track lengths of up to several millimeters , -emitters deliver a much more densely ionizing ( high let ) radiation with an immense quantity of energy per track length and much shorter tissue penetration ( < 100 m , or 210 cell diameters ) . the key physical differences in selected - and -emitters are summarized in table 3 , and the mechanism of action of ra is summarized in figure 1 . because alpha - particle irradiation induces mostly double - stranded dna breaks , cellular repair mechanisms may be less effective.44 lesions with clones of tumor cells remaining dormant in g0 of the cell cycle may be eradicated by high let radiation from -emitters.38,45 in addition , the limited penetration depth also results in lower radiation dose to the marrow and other adjacent normal tissues . , the risk of radiation exposure to others is very small , with the main recommendation being to maintain good hygiene . while sm and sr are excreted primarily by the kidneys , the major route of excretion of ra is through the feces , with only a small fraction of renal excretion ; this reduces the chances of contamination . the patient s external radiation exposure to others is low enough to allow patients to be immediately released under 10 cfr 35.75 following ra administration . preclinical studies of ra demonstrated its potential to be bone marrow sparing , with promising antitumor activity and favorable biodistribution . in mice , bone uptake of ra compared with sr was similar ; however , estimates of dose to marrow cavities showed that ra could be less toxic to bone marrow than -emitters.46 in a nude rat model of osseous metastases resistant to chemotherapy and biphosphonates , rats treated with ra had superior symptom - free survival.47 a biodistribution study in a dog with bone cancer injected with ra showed elimination via intestinal clearance , with low activity in the intestinal wall that was similar to other soft tissues.38 in addition , examination of these canine specimens using -track microautoradiography demonstrated some concentration of the -emitter on the surfaces of trabecular bone and a very high accumulation in the osteoblastic bone matrix . based on the promising preclinical results , a phase i dose - escalation and safety study was conducted in 25 men and women with prostate ( n = 15 ) and breast ( n = 10 ) cancers ( at1-bc-1).43 five patients were treated with a single dose of ra at each of the following dose levels and followed for 8 weeks : 46 , 93 , 163 , 213 , or 250 kbq / kg . subjects were followed for 8 weeks . the patients were observed for dose - limiting toxicity ( defined as platelets < 20 10/l or neutrophils < 0.5 10/l ) and adverse events ( aes ) . in addition , the blood - clearance profile of ra was evaluated at 10 minutes and at 1 , 4 , 24 , and 48 hours and 7 days postinjection . gamma camera scintigraphy was performed in six patients , using the 271 kev peak of the rn daughter to indicate the position of its mother nuclide ; this required long acquisition times due to the low number of events . pain was assessed using the european organization for research and treatment of cancer qlq - c 30 questionnaire at baseline , 1 , 4 , and 8 weeks . overall , ra was well - tolerated , and no dose - limiting hematologic toxicity was observed.43 reversible myelosuppression was observed , with nadir counts occurring 24 weeks after administration of ra . interestingly , there was more neutropenia than thrombocytopenia ( in contrast to -emitters , with which thrombocytopenia is often more frequent ) . grade 3 leukopenia and neutropenia were only seen at the 163 kbq / kg doses in the same two patients , in addition to one isolated instance of leukopenia . the most common aes were : transient diarrhea ( 40% of subjects ) ; bone pain , including flare ( 36% ) ; fatigue ( 25% ) ; nausea ( 25% ) ; and vomiting ( 25% ) . whereas transient diarrhea was observed at all dose levels , nausea and vomiting occurred at the higher dose levels . aes were generally mild , reversible , and manageable with supportive care ( ie , antidiarrheal medication ) . blood radioactivity levels decreased from 12% of the initial at 10 minutes postinjection to 6% at 1 hour and < 1% after 24 hours . by gamma scintigraphy , ra appeared to accumulate in osteoblastic metastases , correlating well with conventional diagnostic tc - mdp bone scans . serum alkaline phosphatase decreased in both groups of patients , but was greatest in the patients with prostate cancer ( 52.1 14.8 , mean standard deviation ) and significantly different between the groups ( p = 0.0028 ) . the 16 patients with an elevated serum alkaline phosphatase at baseline ( of which eleven had prostate cancer ) had a particularly robust decline , which was significantly different than patients with normal - range alkaline phosphatase at baseline . pain palliation was noted in more than half of the patients , with 52% reporting improvement at 1 week , 60% at 4 weeks , and 56% at 8 weeks . of note , response ( transient increase in bone pain ) was observed in about one - fifth of patients ( seven of 25 ) during the first week of treatment . this was the first clinical trial of an -emitter for treatment of cancer patients with osseous metastases . because of a very tolerable toxicity profile ( including mild myelosuppression and gastrointestinal side effects ) , pharmaco - dynamic effects consistent with the hypothesized mechanism of action ( decline in serum alkaline phosphatase and correlation of gamma scintigraphy with bone scans ) , and evidence of clinical benefit ( improved pain scores compared with baseline in up to 60% ) , ra was deemed to be promising for further study . also of note , survival in this phase i study was observed to be greater than 20 months ( with > 20 months follow - up ) , which compared favorably to a contemporary scandinavian randomized trial with sr in a similar population.48 an additional phase ib study ( bc1 - 05 ) of six patients with advanced prostate cancer demonstrated the feasibility of repeat dosing of ra , but has only been reported in abstract form.38,49 finally , a separate phase ib study ( bc1 - 08 ) in men with progressive crpc and two or more bone metastases on bone scan ( n = 10 ) did not reach the maximum tolerated dose with escalation up to 200 kbq / kg and demonstrated targeting of osseous lesions , rapid blood clearance , and excretion through the small intestine followed by transit through the large intestine.50 two randomized , multicenter phase ii studies in patients with mcrpc have been published . in the first double - blind , placebo - controlled study ( bc1 - 02 ) , patients were randomized to be given either four intravenous injections of ra at 50 kbq / kg or placebo every 4 weeks.51 in the second double - blind , dose - response study ( bc1 - 04 ) , patients were randomized to a single intravenous injection of ra at 5 , 25 , 50 , or 100 kbq / kg.52 the first study ( bc1 - 02 ) aimed to evaluate the effects of repeated ra dosing in men with symptomatic crpc.51 patients were required to have multiple osseous metastatic lesions or one painful lesion with two consecutive rising psa values . in addition , all patients had bone pain requiring external - beam radiotherapy ( ebrt ) and either had bilateral orchiectomy or else were maintained on a luteinizing hormone - releasing hormone agonist during the study . patients were randomized to receive either four consecutive monthly injections of ra at 50 kbq / kg or saline . ebrt was given to the most painful bony lesion starting no later than 7 days after the first injection . the primary end points were the mean change in bone alkaline phosphatase ( alp ) from baseline to 4 weeks after the last injection and time to occurrence of sres . secondary end points included evaluation of safety , serum markers of bone turnover , psa , and overall survival . sixty - four patients were enrolled from eleven centers in sweden , norway , and the uk : 33 patients were randomized to ebrt with ra and 31 patients to ebrt with placebo.51 no difference in ebrt dose fractionation at baseline was observed between the groups . hematological toxicity was minimal , with no thrombocytopenia observed in the ra arm compared with one event in a placebo - arm patient . grade 2 neutropenia occurred in three patients treated with ra compared with none treated with placebo . no patient discontinued ra because of treatment - emergent aes , and there were more serious aes in the placebo arm . the only statistically significant difference in aes between the arms was constipation in twelve patients treated with ra ( mild moderate in all but one case ) versus two with placebo . in addition to a tolerable safety profile , evidence of biologic effects and efficacy were demonstrated.51 the median change in bone alp from baseline to 4 weeks after the last study injection was 65.6% in the ra group compared with 9.3% in the placebo group ( p < 0.0001 ) . median time to first sre was 14 weeks in the ra arm versus 11 weeks in the placebo arm ( p = 0.257 ) , with a hazard ratio ( hr ) of 1.75 ( 95% confidence interval [ ci ] , 0.963.19 ; p = 0.065 ) favoring ra when adjusted for baseline covariates . the median relative change in psa from baseline to 4 weeks after the last injection was 23.8% in the ra group versus + 44.9% in the placebo group ( p = 0.003 ) . confirmed psa responses by psa working group criteria53 ( 50% decline ) were observed in eleven of 31 ( 35% ) patients on the ra arm and five of 28 ( 18% ) on the placebo arm ( p = 0.153 ) , with median time to psa progression of 26 weeks and 8 weeks , respectively , on each arm ( p = 0.048 ) . the median overall survival was 65.3 weeks for the ra group versus 46.4 weeks for placebo ( p = 0.066 ) , with an adjusted hr of 2.12 ( 95% ci , 1.133.98 ; p = 0.020 ) favoring ra . these survival results have been updated and remain similar with longer follow - up.54 post hoc analyses showed os of all patients to be 102.4 versus 42.6 weeks ( p < 0.001 ) for posttreatment normalized versus nonnormalized from baseline alp values , respectively ; patients on the ra arm had os of 102.1 versus 42.5 weeks ( p < 0.001 ) for posttreatment normalized versus nonnormalized from baseline alp values , respectively.55 in summary , this randomized , placebo - controlled study showed that repeat dosing of ra every 4 weeks is well tolerated in men with mcrpc and has a significant effect on bone alp 4 weeks after finishing treatment , in addition to potential beneficial efficacy in terms of sres , psa , and overall survival end points . the second study ( bc1 - 03 ) , a double - blind , randomized , dose - ranging study , was designed to examine the effect of a single injection of ra at 5 , 25 , 50 , or 100 kbq / kg in patients with progressive mcrpc and pain.52 patients were required to have testosterone levels < 50 ng / dl after castration therapy , a score of 2 on the bpi,56 progression of disease based on rising psa levels , and sites of clinical pain correlating with multifocal osteoblastic disease on bone scintigraphy . the primary end point was the change in pain index ( derived from a combination of the visual analogue scale and analgesic consumption categorized according to the world health organization [ who ] analgesic ladder57 ) at weeks 2 , 4 , 8 , 12 , and 16 . secondary end points included change from baseline in the bpi severity index and functional index , overall survival , duration of pain relief , relative change in bone alp and psa , and assessment of aes . one hundred patients were randomized and treated at 16 centers in sweden , germany , france , and the uk.52 over half of patients had > 20 bone lesions or a superscan , 81% had performance status of 01 , 36% had prior docetaxel , and 48% had a baseline who level of analgesia of 3 ; median baseline visual analogue scale was 42 mm . a statistically significant dose response for pain index was observed at week 2 only ( p = 0.035 ) . at week 8 , the percentage of pain responders was 40% , 63% , 56% and 71% on the 5 , 25 , 50 , and 100 kbq / kg arms , respectively . the bpi data also showed a significant dose response at week 8 for the pain severity index ( p = 0.040 ) . in a post hoc analysis of pain responders , pain decreased by a mean of 30 , 31 , 27 , and 28 mm ( p = 0.008 , p = 0.0005 , p = 0.002 , and p < 0.0001 ) . furthermore , these responders showed an improvement in the bpi functional interference index in all groups . there were no differences in aes among the dose groups , with the most frequent nonhematologic aes being nausea , fatigue , vomiting , diarrhea , constipation , bone pain , urinary tract infection , and peripheral edema . in the two highest - dose groups , there appeared to be slightly greater reductions in platelet , leukocyte , and neutrophil counts . changes in bone alp were significant only in the 100-kbq / kg dose group at weeks 4 and 8 ( p < 0.0001 and p = 0.0067 , respectively ) , whereas psa increased from baseline to week 16 in all dose groups . the median overall survival for the study population was 50 weeks and did not significantly differ among groups . overall , this study focusing on the pain - relieving effects of a single injection of ra in escalating doses demonstrated an early dose response at week 2 and a maximum of 71% pain response at week 8 at the highest dose level . a well - tolerated safety profile was observed , although there were no effects on psa levels and only a significant effect on bone alp at the highest dose level . notably , there may have been a problem with dropout bias , as 17 patients had dropped out by week 8 and there was differential dropout in the low- versus high - dose groups ( eleven versus six patients ) . a third randomized , double - blind dose - finding phase ii study of ra ( bc1 - 04 ) has only been reported in abstract form.54,57 patients were randomized to 25 , 50 , or 80 kbq / kg every 6 weeks for 12 weeks ( three total doses ) . sixty - one percent of patients had an elevated alp at baseline . in these patients , normalization of alp was associated with a significantly better survival compared with those who did not have normalization of alp . the results of the international , randomized , double - blind alpharadin in symptomatic prostate cancer ( alsympca ) phase iii trial were recently presented.35 eligible patients had confirmed symptomatic crpc , two or more bone metastases , no known visceral metastases , and were either docetaxel - pretreated or unfit for docetaxel . patients were randomized 2:1 to either ra injections at 50 kbq / kg with best standard of care or saline injections with best standard of care . could include secondary hormonal therapies ( antiandrogens , androgen biosynthesis inhibitors , steroids ) or ebrt , but not cytotoxic chemotherapy or radioisotopes . subjects were stratified according to total alp , bisphosphonate use , and prior docetaxel treatment . the primary end point was overall survival , with secondary end points of time to first sre , time to total alp progression , total alp response , total alp normalization , time to psa progression , safety , and quality of life . the trial was designed with 90% power to detect an hr of 0.76 with a two - sided alpha of 0.05 . data from a planned interim analysis after 314 events from 809 randomized patients were presented . the trial was stopped based on the recommendation of an independent data - monitoring committee , due to early evidence of benefit in terms of overall survival ( predetermined boundary crossed ) . from june 2008 through february 2011 , the investigators randomized 541 patients to ra and 268 patients to placebo ( intention - to - treat group).35 the baseline characteristics between the arms appeared well matched , with baseline eastern cooperative oncology group ( ecog ) 1 in 86% , 620 metastases in 44% , and > 20 metastases / superscan in 40% , and who ladder cancer pain index 2 in 54% on the ra arm . median overall survival was significantly improved in the ra group compared with placebo : 14.0 versus 11.2 months ( hr 0.695 [ 95% ci , 0.5520.875 ] ; p = 0.00185 ) . in addition , time to first sre was significantly improved in the ra arm : 13.6 versus 8.4 months ( hr 0.610 [ 95% ci , 0.4610.807 ] ; p = 0.00046 ) . significant improvements in the biochemical end points of time to total alp progression ( hr 0.163 [ 95% ci , 0.1210.221 ] ; p < 0.00001 ) , time to psa progression ( hr 0.671 [ 95% ci , 0.5460.826 ] ; p = 0.00015 ) , total alp response ( 43% vs 3% ; p < 0.001 ) , and total alp normalization ( 33% vs 1% ; p < notably , there were fewer aes in the ra group than the placebo group as measured by all - grade aes ( 88% vs 94% ) , grade 3 or 4 aes ( 51% vs 59% ) , serious aes ( 43% vs 55% ) , and discontinuation due to aes ( 13% vs 20% ) . the incidence of grade 3 or 4 neutropenia in the ra group was 2% versus 1% in the placebo arm . grade 3 or 4 thrombocytopenia was observed in 4% of subjects on the ra arm versus 2% on the placebo arm . all - grade aes and grade 3 or 4 anemia were similar between arms . in terms of all - grade nonhematologic aes , diarrhea ( 22% vs 13% ) and vomiting ( 17% vs 13% ) appeared to be more frequent in the ra group , whereas nausea ( 34% vs 32% ) and constipation ( 18% vs 18% ) appeared similar between groups . subgroup analysis demonstrated survival benefits across most clinically important patient subgroups , regardless of current use of bisphophonates , prior use of docetaxel , or ecog performance status.35 one interesting result was that the hr appeared better in patients currently using bisphosphonates compared with those not on bisphosphonates . there is some biologic rationale for this effect , since the mechanism of action for bisphosphonates is inhibition of osteoclast activity.59 in theory , this decreased osteoclastic activity could result in ra having increased effect at sites of osteoblastic activity , due to longer binding times.35 it remains to be seen whether this effect would also be seen with denosumab , a monoclonal antibody against rankl , but one might expect similar results . it would also be interesting to evaluate ra with agents known to elicit osteoblastic responses on bone scan , such as abiraterone acetate.60 more detailed results of the impact of ra on sres were presented recently as well.61 in contrast to other trials of bone - targeted agents,16 no skeletal surveys were routinely performed during the alsympca trial ; all imaging was performed only as clinically indicated.35 sre components included pathologic bone fracture , spinal cord compression , ebrt , and surgical intervention . in addition to delaying time to first sre ( as mentioned above ) , ra significantly delayed time to all sre components except surgical intervention.61 ebrt was the most common sre component : 23% vs 27% for the ra and placebo arms , respectively ( hr 0.65 [ 95% ci , 0.480.87 ] ; p = 0.0038 ) . pathologic bone fracture was observed in 4% vs 7% , respectively ( hr 0.45 [ 95% ci , 0.240.86 ] ; p = 0.013 ) . of particular interest , spinal cord compression occurred in 3% of patients on the ra arm compared with 6% on placebo ( hr 0.44 [ 95% ci , 0.220.88 ] ; p = 0.016 ) . although not common , this dreaded complication results in significant morbidity in men with advanced prostate cancer.62 this is the first study to demonstrate a significant effect on delay of spinal cord compression in this population . at the time of this writing , no quality - of - life or pain - response data from the alsympca trial have been reported . ideally , a therapy that prolongs os and delays sres , besides having biochemical effects , should do so with minimal compromise of quality of life and with improvements in pain . these results are eagerly awaited in order to confirm the benefit of ra in terms of patient - reported outcomes . based on the promising preclinical results , a phase i dose - escalation and safety study was conducted in 25 men and women with prostate ( n = 15 ) and breast ( n = 10 ) cancers ( at1-bc-1).43 five patients were treated with a single dose of ra at each of the following dose levels and followed for 8 weeks : 46 , 93 , 163 , 213 , or 250 kbq / kg . subjects were followed for 8 weeks . the patients were observed for dose - limiting toxicity ( defined as platelets < 20 10/l or neutrophils < 0.5 10/l ) and adverse events ( aes ) . in addition , the blood - clearance profile of ra was evaluated at 10 minutes and at 1 , 4 , 24 , and 48 hours and 7 days postinjection . gamma camera scintigraphy was performed in six patients , using the 271 kev peak of the rn daughter to indicate the position of its mother nuclide ; this required long acquisition times due to the low number of events . pain was assessed using the european organization for research and treatment of cancer qlq - c 30 questionnaire at baseline , 1 , 4 , and 8 weeks . overall , ra was well - tolerated , and no dose - limiting hematologic toxicity was observed.43 reversible myelosuppression was observed , with nadir counts occurring 24 weeks after administration of ra . interestingly , there was more neutropenia than thrombocytopenia ( in contrast to -emitters , with which thrombocytopenia is often more frequent ) . grade 3 leukopenia and neutropenia were only seen at the 163 kbq / kg doses in the same two patients , in addition to one isolated instance of leukopenia . the most common aes were : transient diarrhea ( 40% of subjects ) ; bone pain , including flare ( 36% ) ; fatigue ( 25% ) ; nausea ( 25% ) ; and vomiting ( 25% ) . whereas transient diarrhea was observed at all dose levels , nausea and vomiting occurred at the higher dose levels . aes were generally mild , reversible , and manageable with supportive care ( ie , antidiarrheal medication ) . blood radioactivity levels decreased from 12% of the initial at 10 minutes postinjection to 6% at 1 hour and < 1% after 24 hours . by gamma scintigraphy , ra appeared to accumulate in osteoblastic metastases , correlating well with conventional diagnostic tc - mdp bone scans . serum alkaline phosphatase decreased in both groups of patients , but was greatest in the patients with prostate cancer ( 52.1 14.8 , mean standard deviation ) and significantly different between the groups ( p = 0.0028 ) . the 16 patients with an elevated serum alkaline phosphatase at baseline ( of which eleven had prostate cancer ) had a particularly robust decline , which was significantly different than patients with normal - range alkaline phosphatase at baseline . pain palliation was noted in more than half of the patients , with 52% reporting improvement at 1 week , 60% at 4 weeks , and 56% at 8 weeks . of note , response ( transient increase in bone pain ) was observed in about one - fifth of patients ( seven of 25 ) during the first week of treatment . this was the first clinical trial of an -emitter for treatment of cancer patients with osseous metastases . because of a very tolerable toxicity profile ( including mild myelosuppression and gastrointestinal side effects ) , pharmaco - dynamic effects consistent with the hypothesized mechanism of action ( decline in serum alkaline phosphatase and correlation of gamma scintigraphy with bone scans ) , and evidence of clinical benefit ( improved pain scores compared with baseline in up to 60% ) , ra was deemed to be promising for further study . also of note , survival in this phase i study was observed to be greater than 20 months ( with > 20 months follow - up ) , which compared favorably to a contemporary scandinavian randomized trial with sr in a similar population.48 an additional phase ib study ( bc1 - 05 ) of six patients with advanced prostate cancer demonstrated the feasibility of repeat dosing of ra , but has only been reported in abstract form.38,49 finally , a separate phase ib study ( bc1 - 08 ) in men with progressive crpc and two or more bone metastases on bone scan ( n = 10 ) did not reach the maximum tolerated dose with escalation up to 200 kbq / kg and demonstrated targeting of osseous lesions , rapid blood clearance , and excretion through the small intestine followed by transit through the large intestine.50 two randomized , multicenter phase ii studies in patients with mcrpc have been published . in the first double - blind , placebo - controlled study ( bc1 - 02 ) , patients were randomized to be given either four intravenous injections of ra at 50 kbq / kg or placebo every 4 weeks.51 in the second double - blind , dose - response study ( bc1 - 04 ) , patients were randomized to a single intravenous injection of ra at 5 , 25 , 50 , or 100 kbq / kg.52 the first study ( bc1 - 02 ) aimed to evaluate the effects of repeated ra dosing in men with symptomatic crpc.51 patients were required to have multiple osseous metastatic lesions or one painful lesion with two consecutive rising psa values . in addition , all patients had bone pain requiring external - beam radiotherapy ( ebrt ) and either had bilateral orchiectomy or else were maintained on a luteinizing hormone - releasing hormone agonist during the study . patients were randomized to receive either four consecutive monthly injections of ra at 50 kbq / kg or saline . ebrt was given to the most painful bony lesion starting no later than 7 days after the first injection . the primary end points were the mean change in bone alkaline phosphatase ( alp ) from baseline to 4 weeks after the last injection and time to occurrence of sres . secondary end points included evaluation of safety , serum markers of bone turnover , psa , and overall survival . sixty - four patients were enrolled from eleven centers in sweden , norway , and the uk : 33 patients were randomized to ebrt with ra and 31 patients to ebrt with placebo.51 no difference in ebrt dose fractionation at baseline was observed between the groups . hematological toxicity was minimal , with no thrombocytopenia observed in the ra arm compared with one event in a placebo - arm patient . grade 2 neutropenia occurred in three patients treated with ra compared with none treated with placebo . no patient discontinued ra because of treatment - emergent aes , and there were more serious aes in the placebo arm . the only statistically significant difference in aes between the arms was constipation in twelve patients treated with ra ( mild moderate in all but one case ) versus two with placebo . in addition to a tolerable safety profile , evidence of biologic effects and efficacy were demonstrated.51 the median change in bone alp from baseline to 4 weeks after the last study injection was 65.6% in the ra group compared with 9.3% in the placebo group ( p < 0.0001 ) . median time to first sre was 14 weeks in the ra arm versus 11 weeks in the placebo arm ( p = 0.257 ) , with a hazard ratio ( hr ) of 1.75 ( 95% confidence interval [ ci ] , 0.963.19 ; p = 0.065 ) favoring ra when adjusted for baseline covariates . the median relative change in psa from baseline to 4 weeks after the last injection was 23.8% in the ra group versus + 44.9% in the placebo group ( p = 0.003 ) . confirmed psa responses by psa working group criteria53 ( 50% decline ) were observed in eleven of 31 ( 35% ) patients on the ra arm and five of 28 ( 18% ) on the placebo arm ( p = 0.153 ) , with median time to psa progression of 26 weeks and 8 weeks , respectively , on each arm ( p = 0.048 ) . the median overall survival was 65.3 weeks for the ra group versus 46.4 weeks for placebo ( p = 0.066 ) , with an adjusted hr of 2.12 ( 95% ci , 1.133.98 ; p = 0.020 ) favoring ra . these survival results have been updated and remain similar with longer follow - up.54 post hoc analyses showed os of all patients to be 102.4 versus 42.6 weeks ( p < 0.001 ) for posttreatment normalized versus nonnormalized from baseline alp values , respectively ; patients on the ra arm had os of 102.1 versus 42.5 weeks ( p < 0.001 ) for posttreatment normalized versus nonnormalized from baseline alp values , respectively.55 in summary , this randomized , placebo - controlled study showed that repeat dosing of ra every 4 weeks is well tolerated in men with mcrpc and has a significant effect on bone alp 4 weeks after finishing treatment , in addition to potential beneficial efficacy in terms of sres , psa , and overall survival end points . the second study ( bc1 - 03 ) , a double - blind , randomized , dose - ranging study , was designed to examine the effect of a single injection of ra at 5 , 25 , 50 , or 100 kbq / kg in patients with progressive mcrpc and pain.52 patients were required to have testosterone levels < 50 ng / dl after castration therapy , a score of 2 on the bpi,56 progression of disease based on rising psa levels , and sites of clinical pain correlating with multifocal osteoblastic disease on bone scintigraphy . the primary end point was the change in pain index ( derived from a combination of the visual analogue scale and analgesic consumption categorized according to the world health organization [ who ] analgesic ladder57 ) at weeks 2 , 4 , 8 , 12 , and 16 . secondary end points included change from baseline in the bpi severity index and functional index , overall survival , duration of pain relief , relative change in bone alp and psa , and assessment of aes . one hundred patients were randomized and treated at 16 centers in sweden , germany , france , and the uk.52 over half of patients had > 20 bone lesions or a superscan , 81% had performance status of 01 , 36% had prior docetaxel , and 48% had a baseline who level of analgesia of 3 ; median baseline visual analogue scale was 42 mm . a statistically significant dose response for pain index was observed at week 2 only ( p = 0.035 ) . at week 8 , the percentage of pain responders was 40% , 63% , 56% and 71% on the 5 , 25 , 50 , and 100 kbq / kg arms , respectively . the bpi data also showed a significant dose response at week 8 for the pain severity index ( p = 0.040 ) . in a post hoc analysis of pain responders , pain decreased by a mean of 30 , 31 , 27 , and 28 mm ( p = 0.008 , p = 0.0005 , p = 0.002 , and p < 0.0001 ) . furthermore , these responders showed an improvement in the bpi functional interference index in all groups . there were no differences in aes among the dose groups , with the most frequent nonhematologic aes being nausea , fatigue , vomiting , diarrhea , constipation , bone pain , urinary tract infection , and peripheral edema . in the two highest - dose groups , there appeared to be slightly greater reductions in platelet , leukocyte , and neutrophil counts . changes in bone alp were significant only in the 100-kbq / kg dose group at weeks 4 and 8 ( p < 0.0001 and p = 0.0067 , respectively ) , whereas psa increased from baseline to week 16 in all dose groups . the median overall survival for the study population was 50 weeks and did not significantly differ among groups . overall , this study focusing on the pain - relieving effects of a single injection of ra in escalating doses demonstrated an early dose response at week 2 and a maximum of 71% pain response at week 8 at the highest dose level . a well - tolerated safety profile was observed , although there were no effects on psa levels and only a significant effect on bone alp at the highest dose level . notably , there may have been a problem with dropout bias , as 17 patients had dropped out by week 8 and there was differential dropout in the low- versus high - dose groups ( eleven versus six patients ) . a third randomized , double - blind dose - finding phase ii study of ra ( bc1 - 04 ) has only been reported in abstract form.54,57 patients were randomized to 25 , 50 , or 80 kbq / kg every 6 weeks for 12 weeks ( three total doses ) . sixty - one percent of patients had an elevated alp at baseline . in these patients , normalization of alp was associated with a significantly better survival compared with those who did not have normalization of alp . the results of the international , randomized , double - blind alpharadin in symptomatic prostate cancer ( alsympca ) phase iii trial were recently presented.35 eligible patients had confirmed symptomatic crpc , two or more bone metastases , no known visceral metastases , and were either docetaxel - pretreated or unfit for docetaxel . patients were randomized 2:1 to either ra injections at 50 kbq / kg with best standard of care or saline injections with best standard of care . could include secondary hormonal therapies ( antiandrogens , androgen biosynthesis inhibitors , steroids ) or ebrt , but not cytotoxic chemotherapy or radioisotopes . subjects were stratified according to total alp , bisphosphonate use , and prior docetaxel treatment . the primary end point was overall survival , with secondary end points of time to first sre , time to total alp progression , total alp response , total alp normalization , time to psa progression , safety , and quality of life . the trial was designed with 90% power to detect an hr of 0.76 with a two - sided alpha of 0.05 . data from a planned interim analysis after 314 events from 809 randomized patients were presented . the trial was stopped based on the recommendation of an independent data - monitoring committee , due to early evidence of benefit in terms of overall survival ( predetermined boundary crossed ) . from june 2008 through february 2011 , the investigators randomized 541 patients to ra and 268 patients to placebo ( intention - to - treat group).35 the baseline characteristics between the arms appeared well matched , with baseline eastern cooperative oncology group ( ecog ) 1 in 86% , 620 metastases in 44% , and > 20 metastases / superscan in 40% , and who ladder cancer pain index 2 in 54% on the ra arm . median overall survival was significantly improved in the ra group compared with placebo : 14.0 versus 11.2 months ( hr 0.695 [ 95% ci , 0.5520.875 ] ; p = 0.00185 ) . in addition , time to first sre was significantly improved in the ra arm : 13.6 versus 8.4 months ( hr 0.610 [ 95% ci , 0.4610.807 ] ; p = 0.00046 ) . significant improvements in the biochemical end points of time to total alp progression ( hr 0.163 [ 95% ci , 0.1210.221 ] ; p < 0.00001 ) , time to psa progression ( hr 0.671 [ 95% ci , 0.5460.826 ] ; p = 0.00015 ) , total alp response ( 43% vs 3% ; p < 0.001 ) , and total alp normalization ( 33% vs 1% ; p < notably , there were fewer aes in the ra group than the placebo group as measured by all - grade aes ( 88% vs 94% ) , grade 3 or 4 aes ( 51% vs 59% ) , serious aes ( 43% vs 55% ) , and discontinuation due to aes ( 13% vs 20% ) . the incidence of grade 3 or 4 neutropenia in the ra group was 2% versus 1% in the placebo arm . grade 3 or 4 thrombocytopenia was observed in 4% of subjects on the ra arm versus 2% on the placebo arm . all - grade aes and grade 3 or 4 anemia were similar between arms . in terms of all - grade nonhematologic aes , diarrhea ( 22% vs 13% ) and vomiting ( 17% vs 13% ) appeared to be more frequent in the ra group , whereas nausea ( 34% vs 32% ) and constipation ( 18% vs 18% ) appeared similar between groups . subgroup analysis demonstrated survival benefits across most clinically important patient subgroups , regardless of current use of bisphophonates , prior use of docetaxel , or ecog performance status.35 one interesting result was that the hr appeared better in patients currently using bisphosphonates compared with those not on bisphosphonates . there is some biologic rationale for this effect , since the mechanism of action for bisphosphonates is inhibition of osteoclast activity.59 in theory , this decreased osteoclastic activity could result in ra having increased effect at sites of osteoblastic activity , due to longer binding times.35 it remains to be seen whether this effect would also be seen with denosumab , a monoclonal antibody against rankl , but one might expect similar results . it would also be interesting to evaluate ra with agents known to elicit osteoblastic responses on bone scan , such as abiraterone acetate.60 more detailed results of the impact of ra on sres were presented recently as well.61 in contrast to other trials of bone - targeted agents,16 no skeletal surveys were routinely performed during the alsympca trial ; all imaging was performed only as clinically indicated.35 sre components included pathologic bone fracture , spinal cord compression , ebrt , and surgical intervention . in addition to delaying time to first sre ( as mentioned above ) , ra significantly delayed time to all sre components except surgical intervention.61 ebrt was the most common sre component : 23% vs 27% for the ra and placebo arms , respectively ( hr 0.65 [ 95% ci , 0.480.87 ] ; p = 0.0038 ) . pathologic bone fracture was observed in 4% vs 7% , respectively ( hr 0.45 [ 95% ci , 0.240.86 ] ; p = 0.013 ) . of particular interest , spinal cord compression occurred in 3% of patients on the ra arm compared with 6% on placebo ( hr 0.44 [ 95% ci , 0.220.88 ] ; p = 0.016 ) . although not common , this dreaded complication results in significant morbidity in men with advanced prostate cancer.62 this is the first study to demonstrate a significant effect on delay of spinal cord compression in this population . at the time of this writing , no quality - of - life or pain - response data from the alsympca trial have been reported . ideally , a therapy that prolongs os and delays sres , besides having biochemical effects , should do so with minimal compromise of quality of life and with improvements in pain . these results are eagerly awaited in order to confirm the benefit of ra in terms of patient - reported outcomes . the pivotal phase iii randomized double - blind placebo controlled alsympca trial of ra demonstrated a highly significant os improvement in docetaxel - pretreated or docetaxel - unfit men with symptomatic bone - metastatic crpc and a very tolerable side - effect profile . a large number of men in the us with mcrpc are unfit for systemic chemotherapy due to toxicity concerns or comorbidities , and many men with mcrpc decline chemotherapy and all eventually fail first - line docetaxel . ra is pending regulatory approval in the us and abroad ; however , based on recent fda approvals in mcrpc based on overall survival benefits,5,1214 approval of ra appears likely . although time to first sre was not the primary end point of the alsympca trial , the effects of ra on delaying sres are important , and this end point has led to the regulatory approval of other bone - targeted agents even in the absence of beneficial effects on overall survival and disease progression.15,16 based on eligibility criteria from this pivotal trial , it is likely that the label will be broad , encompassing both men with symptomatic bone - metastatic crpc who have failed docetaxel and those who are not candidates for docetaxel chemotherapy . the population of the alsympca trial was defined somewhat differently compared with prior trials in mcrpc . because docetaxel / prednisone was the first therapy proven to extend survival in men with mcrpc and therefore became the first - line standard of care , many contemporary trials have focused on either pre - docetaxel or post - docetaxel populations . patients ( eg , impact,12 cou - aa-302,63 prevail64 ) . however , this is the first phase iii study of a survival - prolonging therapy to concentrate on the symptomatic , bone - metastatic subpopulation of mcrpc patients . ra occupies a unique niche in that it prolongs os , is a bone - targeted agent that delays sres , and is also a radiopharmaceutical that might be expected to provide pain palliation ( phase iii data not yet reported ) . ra has not been compared head - to - head against other approved therapies in mcrpc . however , with this caveat and based on what is known from the fairly extensive study of ra in this population to date , the following observations can be made . a large and significant improvement in os was observed when compared with placebo in men with mcrpc and symptomatic bony metastases . the concurrent demonstration of benefit in terms of delayed sres and improvement in all biochemical end points ( ie , psa , total alp ) , which would be expected with a therapy of ra s mechanism of action , adds to the robustness of this finding . importantly , these benefits were seen in prespecified groups ( strata ) , including total alp < 220 u / l and 220 u / l ; current bisphosphonate use ( yes / no ) ; or prior use of docetaxel ( yes / no ) . the population of patients in the alsympca trial included both docetaxel - unfit and docetaxel - pretreated men . this makes cross - trial comparisons more difficult , since eligibility criteria for trials in mcrpc have historically focused on either chemonaive or chemo - pretreated populations . men who are unfit for docetaxel - based chemotherapy may have a poorer survival relative to those who are chemotherapy candidates.65 the reason for the choice of treatment with six total injections ( or until progression of disease ) is not clear , and could be an area for future study . overall survival ( table 1 ) : cytotoxic chemotherapy : the absolute median os difference of 2.8 months observed on the alsympca trial compares favorably with the 2.9-month difference seen in the chemonaive ( but docetaxel - fit ) population in the tax-327 study66 and the 2.4-month difference in the docetaxel - pretreated population in the tropic study.13 however , it should be noted that both of these latter two trials used an active comparator ( mitoxantrone plus prednisone10 ) . ar - directed therapies : trials with androgen receptor ( ar)-directed therapies , such as abiraterone14 and mdv3100,17 in docetaxel - pretreated populations have demonstrated median os differences of 4.6 and 4.8 months , respectively . notably , these trials used the less active comparator of placebo plus prednisone . however , there is a strong rationale for combining these agents with ra based on the allowance of these classes of agents in the alsympca trial and the nonoverlapping toxicity profiles . none of the currently fda - approved radiopharmaceuticals ( eg , sr , sm ) have demonstrated an os benefit in a randomized phase iii study in men with mcrpc.11 antiresorptive agents : none of the currently fda - approved osteoclast inhibitors ( eg , zoledronic acid , denosumab ) have demonstrated os benefits over placebo . however , their use with ra is likely to be beneficial without overlapping toxicity based on the phase iii evidence to date . skeletal - related events ( table 4 ) : cytotoxic chemotherapy : there is insufficient evidence to demonstrate that docetaxel and cabazitaxel prevent or delay sres , although they are known to result in pain palliation and to prolong time to pain progression , disease progression , and overall survival . ar - directed therapies : abiraterone has demonstrated delay in time to first sre ( 25th percentile , 301 vs 150 days ; p < 0.0001 ) in the cou - aa-301 trial.20 time to first sre was a secondary end point in the affirm trial of mdv3100 ( nct00974311 ) , but this result has not yet been reported.17 radiopharmaceuticals : there is insufficient evidence to demonstrate that sr or sm prevent or delay sres.11 antiresorptive agents : zoledronic acid delays sres compared with placebo , and denosumab is superior to zoledronic acid in delaying sres in men with mcrpc . notably , subgroup analysis of the alsympca trial suggested that bisphosphonates may potentiate the activity of ra ; a similar effect would be expected with denosumab , though there is a lack of data regarding this . pain palliation was demonstrated in phase iii trials of docetaxel and abiraterone , but not cabazitaxel ( table 2 ) . a key indication for the use of radioisotopes for men with mcrpc has been pain palliation , although there is likely only a small benefit in complete reduction of pain over 16 months and no increase in analgesic use ( table 5).11 data on pain palliation from the ra phase iii alsympca trial have not been reported ; however , the phase i and ii experience with ra would suggest a benefit in terms of pain palliation . the most common nonhematologic side effects of ra in the alsympca trial include diarrhea , nausea , vomiting , and constipation . notably , nausea and constipation did not appear to be more frequent on the ra arm versus placebo . two percent or fewer of these nonhematologic , gastrointestinal side effects were grade 3 or 4 . overall , only about 10% of patients on the ra arm had grade 3 aes , fewer than on the placebo arm . neutropenia and thrombocytopenia are side effects of ra and occurred overall in 4% and 8% , respectively , of patients in the alsympca trial . grade 3 or 4 neutropenia and thrombocytopenia were observed in 2% and 4% , respectively . this property allows consecutive doses to be administered safely , and may be the basis for its more substantial antitumor effects and os compared with -emitters . additionally , with ra , neutropenia predominates over thrombocytopenia , which is the inverse of the pattern seen with -emitters . judged against cytotoxic chemotherapies , ra is likely to have a significantly better toxicity profile in terms of myelosuppression and gastrointestinal and other side effects . in an extreme example , in the tropic trial of cabazitaxel , grade 3 or 4 diarrhea was observed in 6.2% and grade 3 or 4 neutropenia in 81.7% ( febrile neutropenia 7.5% ) . in comparison to ar - directed therapies patients treated with abiraterone acetate have demonstrated side effects related to mineralocorticoid excess due to the drug s mechanism of action , such as fluid retention , hypertension , and hypokalemia.14 the most common side effect overall and grade 3 or 4 side effect observed with mdv3100 was fatigue , with patients rarely experiencing seizures ( 0.6% ; all grade 3).17 antiresorptive agents are likely to be used in combination with ra , as in the phase iii trial , and side effects generally appear to be nonoverlapping . data on quality of life with ra from the alsympca trial are eagerly awaited . in comparison to -emitters , which are renally excreted , however , there is extensive experience with ra in patients with ankylosing spondylitis.42 an increased risk for later cancers has been observed , but only in individuals treated with ra as children and , importantly , not in those treated as adults.67 in comparison , there have been reports of acute leukemia with sr but not sm.68 a large and significant improvement in os was observed when compared with placebo in men with mcrpc and symptomatic bony metastases . the concurrent demonstration of benefit in terms of delayed sres and improvement in all biochemical end points ( ie , psa , total alp ) , which would be expected with a therapy of ra s mechanism of action , adds to the robustness of this finding . importantly , these benefits were seen in prespecified groups ( strata ) , including total alp < 220 u / l and 220 u / l ; current bisphosphonate use ( yes / no ) ; or prior use of docetaxel ( yes / no ) . the population of patients in the alsympca trial included both docetaxel - unfit and docetaxel - pretreated men . this makes cross - trial comparisons more difficult , since eligibility criteria for trials in mcrpc have historically focused on either chemonaive or chemo - pretreated populations . men who are unfit for docetaxel - based chemotherapy may have a poorer survival relative to those who are chemotherapy candidates.65 the reason for the choice of treatment with six total injections ( or until progression of disease ) is not clear , and could be an area for future study . overall survival ( table 1 ) : cytotoxic chemotherapy : the absolute median os difference of 2.8 months observed on the alsympca trial compares favorably with the 2.9-month difference seen in the chemonaive ( but docetaxel - fit ) population in the tax-327 study66 and the 2.4-month difference in the docetaxel - pretreated population in the tropic study.13 however , it should be noted that both of these latter two trials used an active comparator ( mitoxantrone plus prednisone10 ) . ar - directed therapies : trials with androgen receptor ( ar)-directed therapies , such as abiraterone14 and mdv3100,17 in docetaxel - pretreated populations have demonstrated median os differences of 4.6 and 4.8 months , respectively . notably , these trials used the less active comparator of placebo plus prednisone . however , there is a strong rationale for combining these agents with ra based on the allowance of these classes of agents in the alsympca trial and the nonoverlapping toxicity profiles . radiopharmaceuticals : none of the currently fda - approved radiopharmaceuticals ( eg , sr , sm ) have demonstrated an os benefit in a randomized phase iii study in men with mcrpc.11 antiresorptive agents : none of the currently fda - approved osteoclast inhibitors ( eg , zoledronic acid , denosumab ) have demonstrated os benefits over placebo . however , their use with ra is likely to be beneficial without overlapping toxicity based on the phase iii evidence to date . skeletal - related events ( table 4 ) : cytotoxic chemotherapy : there is insufficient evidence to demonstrate that docetaxel and cabazitaxel prevent or delay sres , although they are known to result in pain palliation and to prolong time to pain progression , disease progression , and overall survival . ar - directed therapies : abiraterone has demonstrated delay in time to first sre ( 25th percentile , 301 vs 150 days ; p < 0.0001 ) in the cou - aa-301 trial.20 time to first sre was a secondary end point in the affirm trial of mdv3100 ( nct00974311 ) , but this result has not yet been reported.17 radiopharmaceuticals : there is insufficient evidence to demonstrate that sr or sm prevent or delay sres.11 antiresorptive agents : zoledronic acid delays sres compared with placebo , and denosumab is superior to zoledronic acid in delaying sres in men with mcrpc . notably , subgroup analysis of the alsympca trial suggested that bisphosphonates may potentiate the activity of ra ; a similar effect would be expected with denosumab , though there is a lack of data regarding this . pain palliation was demonstrated in phase iii trials of docetaxel and abiraterone , but not cabazitaxel ( table 2 ) . a key indication for the use of radioisotopes for men with mcrpc has been pain palliation , although there is likely only a small benefit in complete reduction of pain over 16 months and no increase in analgesic use ( table 5).11 data on pain palliation from the ra phase iii alsympca trial have not been reported ; however , the phase i and ii experience with ra would suggest a benefit in terms of pain palliation . the most common nonhematologic side effects of ra in the alsympca trial include diarrhea , nausea , vomiting , and constipation . notably , nausea and constipation did not appear to be more frequent on the ra arm versus placebo . two percent or fewer of these nonhematologic , gastrointestinal side effects were grade 3 or 4 . overall , only about 10% of patients on the ra arm had grade 3 aes , fewer than on the placebo arm . neutropenia and thrombocytopenia are side effects of ra and occurred overall in 4% and 8% , respectively , of patients in the alsympca trial . grade 3 or 4 neutropenia and thrombocytopenia were observed in 2% and 4% , respectively . this property allows consecutive doses to be administered safely , and may be the basis for its more substantial antitumor effects and os compared with -emitters . additionally , with ra , neutropenia predominates over thrombocytopenia , which is the inverse of the pattern seen with -emitters . judged against cytotoxic chemotherapies , ra is likely to have a significantly better toxicity profile in terms of myelosuppression and gastrointestinal and other side effects . in an extreme example , in the tropic trial of cabazitaxel , grade 3 or 4 diarrhea was observed in 6.2% and grade 3 or 4 neutropenia in 81.7% ( febrile neutropenia 7.5% ) . in comparison to ar - directed therapies , ra has a distinct toxicity profile but may be similarly tolerable . patients treated with abiraterone acetate have demonstrated side effects related to mineralocorticoid excess due to the drug s mechanism of action , such as fluid retention , hypertension , and hypokalemia.14 the most common side effect overall and grade 3 or 4 side effect observed with mdv3100 was fatigue , with patients rarely experiencing seizures ( 0.6% ; all grade 3).17 antiresorptive agents are likely to be used in combination with ra , as in the phase iii trial , and side effects generally appear to be nonoverlapping . data on quality of life with ra from the alsympca trial are eagerly awaited . in comparison to -emitters , which are renally excreted , however , there is extensive experience with ra in patients with ankylosing spondylitis.42 an increased risk for later cancers has been observed , but only in individuals treated with ra as children and , importantly , not in those treated as adults.67 in comparison , there have been reports of acute leukemia with sr but not sm.68 emerging therapies aimed at preventing skeletal morbidity in men with prostate cancer have recently been reviewed in detail.69 notable bone - targeted therapies in clinical development for mcrpc include src - targeted therapies ( dasatinib ) , and met - targeted therapies . while the oral endothelin ( eta ) receptor antagonists atrasentan and zibotentan ( zd4054 ) were not successful in phase iii trials when combined with docetaxel in mcrpc , it is possible that these agents may combine favorably with other bone - targeted strategies . the oral src tyrosine kinase inhibitor dasatinib ( sprycel ; bristol- myers squibb , new york , ny ) is being evaluated in a phase iii mcrpc trial of docetaxel / prednisone with dasatinib or placebo ( nct00744497),70 with results anticipated in 2012 . finally , the multitargeted tyrosine kinase inhibitor xl-184 ( cabozantinib ) , which inhibits met , vefgr2 , ret , and kit , has shown impressive results in a phase ii study71 and will enter phase iii testing soon.72,73 beta emitters have been combined with chemotherapy in several studies , including two randomized studies , demonstrating the possible feasibility and safety of this approach.3134 for example , one recent phase i study by morris et al demonstrated that docetaxel and sm could be administered at full doses over repeated cycles.31 given the improved toxicity profile with ra over -emitters , it seems likely that ra could be combined with chemotherapy as well . this concept is currently being investigated in a phase i / ii trial of ra with docetaxel chemotherapy in men with bone metastasis from crpc ( nct01106352).74 as a synergistic interaction between androgen - deprivation therapy and radiotherapy that increases apoptosis is known to exist in prostate cancer,75 it is conceivable that this synergy could exist between ra and the newer ar - directed therapies ( eg , abiraterone , mdv3100 ) as well . finally , it is possible that ra and bone - targeted agents in development could have complementary mechanisms of action . all of these remain important research questions that should be addressed in the near future . the available data demonstrate that ra has activity in men with symptomatic bone - metastatic crpc who are either docetaxel - unfit or docetaxel - pretreated . the significant improvement in median os is an acceptable surrogate of clinical benefit , and is bolstered by delay of sres and improvement in biochemical end points . these benefits are in the range observed with other approved therapies in this setting . when examined in the context of ra - associated toxicity , these benefits appear well worth the risk in the subpopulation of patients included in the alsympca trial . toxicities appear to be less than those with cytotoxic chemotherapies and are similarly mild , though distinct , when compared to ar - directed therapies . the effects of ra on quality of life and pain palliation from the randomized phase iii trial have not been reported . in clinical practice , it is likely that ra would be used in conjunction with antiresorptive agents . compared with approved cytotoxic chemotherapies , ar - directed therapies , and other radioisotopes , ra could occupy a unique niche in the treatment of mcrpc .

backgroundradium-223 chloride ( 223ra ; alpharadin ) is an alpha - emitting radioisotope that targets areas of osteoblastic metastasis and is excreted by the small intestine . when compared with beta - emitters ( eg , strontium-89 , samarium-153 ) , 223ra delivers a high quantity of energy per track length with short tissue penetration.objectivethis review describes the mechanism , radiobiology , and preclinical development of 223ra and discusses the clinical data currently available regarding its safety and efficacy profile.methodsdata from clinical trials including abstracts were collected and reviewed using the pubmed database , as well as the american society of clinical oncology abstract database.conclusioncurrent bone - targeted therapies fall into two main categories : antiresorptive agents ( eg , zoledronic acid , denosumab ) , which have been shown to delay skeletal - related events , and radiopharmaceuticals ( eg , samarium-153 ) , which may have a role in pain palliation . historically , neither antiresorptive agents nor radiopharmaceuticals have shown definitive evidence of improved overall survival or other antitumor effects in metastatic castrate - resistant prostate cancer ( mcrpc ) . radiopharmaceuticals are limited by myelosuppresion , thrombocytopenia , and renal excretion . in a recently reported randomized phase iii trial in men with symptomatic bone - metastatic crpc who had received or were ineligible for docetaxel chemotherapy , 223ra treatment resulted in improved overall survival and delayed skeletal - related events . toxicity consisted of minor gastrointestinal side effects and mild neutropenia and thrombocytopenia that were rarely severe . pending regulatory approval , 223ra may represent a unique and distinct option for an important subgroup of patients with mcrpc ; future trials should address its use in combination or in sequence with existing and novel agents .

Introduction Radiobiology, mechanism of action, and preclinical development of Clinical evaluation of Phase I studies Phase II studies Phase III study Critical appraisal and potential role of Efficacy of Safety and toxicity profile Future directions Conclusion

prostate cancer is the most common cancer in men and the second - leading cause of cancer death in men , with 241,740 new diagnoses and 28,170 deaths projected in the us in 2012.1 advanced prostate cancer has a penchant to metastasize to bone , possibly due to osteomimicry or altered adhesion molecules.2,3 among patients with metastatic , castrate - resistant prostate cancer ( mcrpc ) treated with first - line chemotherapy , almost 90% have radiographic evidence of bone metastasis.4,5 morbidity from complications of osseous metastases , such as pathologic fractures , spinal cord compression , and pain , greatly impairs the quality of life of patients with mcrpc . mitoxantrone chemotherapy and several radioisotopes , such as samarium-153 ( sm ) and strontium-89 ( sr ) , were the first therapies approved for palliation of bone pain in patients with mcrpc , but without evidence of improvement in overall survival.10,11 since 2004 , four systemic therapies have been approved on the basis of improvements in overall survival , with varying effects on bone pain and skeletal complications.4,5,1214 these agents , particularly the taxanes docetaxel ( taxotere ; sanofi - aventis , bridgewater nj ) , cabazitaxel ( jevtana ; sanofi - aventis ) , and the novel androgen biosynthesis inhibitor abiraterone acetate ( zytiga ; janssen biotech , inc , horsham , pa ) , have direct antitumor effects that may result in pain palliation . contemporaneously , two osteoclast inhibitors ( zoledronic acid [ zometa ; novartis ag , basel , switzerland ] and denosumab [ xgeva ; amgen inc , thousand oaks , ca ] ) have been approved on the basis of delay of skeletal - related events ( sres ; pathologic fracture , spine cord compression , and requirement for radiation or surgery to bone ) , but without improvements in overall survival ( os).15,16 in addition , the novel androgen signaling inhibitor mdv3100 ( enzalutamide , xtandi ; medivation , inc , san francisco , ca ) has demonstrated improvements in os and palliative end points ; its approval was granted august 31 , 2012 , as this article went to press.1719 prior to the development of ra ( alpharadin ; algeta asa , oslo , norway / bayer ag , leverkusen , germany ) , no specifically bone - targeted therapy has been shown to improve median os in the population of patients with mcrpc with osseous metastases . as a reference , the palliative chemotherapy regimen of mitoxantrone / prednisone demonstrated a 29% response on the present pain intensity ( ppi ) scale versus 12% with prednisone alone ( p = 0.01 ) , with a duration of 43 versus 18 weeks ( p < 0.0001).10 the landmark tax-327 study showed a pain response of 35% versus 22% ( p = 0.01 ) on the ppi scale for docetaxel / prednisone versus mitoxantrone / prednisone , with a duration of 3.5 versus 4.8 months ( p not significant).5 patients who experienced a pain response in this trial were found to have improved overall survival as compared with those men without such a response.7 in the post - docetaxel mcrpc setting , cabazitaxel / prednisone showed no improvement over mitoxantrone / prednisone on the ppi scale ( 9.2% vs 7.7% ; p = 0.63 ; duration not reached ) in the tropic trial.13 in a similar post - docetaxel setting , the cou - aa-301 study demonstrated a 44% versus 27% pain response on the brief pain inventory ( bpi ) scale for abiraterone / prednisone vs placebo / prednisone , with a time to progression of approximately 8 versus 5 months ( 25th percentile ; p = 0.0056).20 the great variability in pain response for the control arms ( 7.7% for mitoxantrone / prednisone in the tropic study , and 27% for prednisone alone in the cou - aa-301 study ) suggests great variability in the reporting methods . in a placebo - controlled phase iii trial , denosumab showed a 3.6-month improvement in time to first sre over zoledronic acid ( 20.7 versus 17.1 months ; p = 0.0002 [ noninferiority ] , p = 0.008 [ superiority]).16 of note , neither zoledronic acid nor denosumab demonstrated improvements in prostate - specific antigen ( psa ) levels , overall disease progression , or overall survival . ongoing work of these agents in combination with chemotherapy may optimize their efficacy.3134 in comparison to beta - emitting radioisotopes , radium- 223 ( ra ; alpharadin , algeta ) is an -emitter that delivers radiation with a higher biologic effect to a more localized area . in a large randomized phase iii trial , ra has recently demonstrated improvements in os , time to first sre , and biochemical parameters , with a remarkably tolerable adverse - event profile , in men with bone - metastatic crpc.35 while ra has not yet been approved for use in the us , and there is little experience in the us with the use of this agent , it represents the first bone - targeted therapeutic radiopharmaceutical to demonstrate a survival benefit , which has been the benchmark for recent fda approvals in this disease setting . an expanded access trial of ra in men with mcrpc with bone metastases is planned.36 many questions have arisen as to the appropriate sequencing of existing and novel therapies with ra and if / how to combine ra with these systemic therapies . as mentioned above , the commonly used clinically available radiopharmaceuticals are -emitters , with sr and sm the most well studied.37 following injection of sr , radiation doses are delivered to the osseous target lesions at low dose rates.38 between 16 weeks and 1 year , absorbed doses in the 2040 gy range are deposited as a steadily declining dose rate.39,40 because of its higher dose rate and the shorter range ( ie , tissue penetration ) , sm may have an improved therapeutic index compared with that of sr ( table 3).38 due to its relatively shorter half - life , repeated administration of sm is feasible for persistent or recurrent bone pain provided adequate hematologic function returns ; however , effects on bone marrow remain a limitation.30 this suggests that an agent with less depth of tissue penetration but similar or higher biologic effect is needed to facilitate repeated dosing . blood levels of calcium and bone resorption / formation are both tightly regulated by various hormones.41 ) for example , radium-224 ( ra ) has been used extensively for treatment of ankylosing spondylitis , a chronic inflammatory disease of the axial skeleton characterized by new bone formation.42 ra has a half - life of 11.4 days , produces four alpha particles from decay through short - lived daughter radionuclides , and has advantages over ra in terms of the decay chain.43 ra was selected for biomedical applications based on its favorable decay chain and half - life.43 in contrast to -emitters , which have a low linear energy transfer ( let ) and track lengths of up to several millimeters , -emitters deliver a much more densely ionizing ( high let ) radiation with an immense quantity of energy per track length and much shorter tissue penetration ( < 100 m , or 210 cell diameters ) . because of a very tolerable toxicity profile ( including mild myelosuppression and gastrointestinal side effects ) , pharmaco - dynamic effects consistent with the hypothesized mechanism of action ( decline in serum alkaline phosphatase and correlation of gamma scintigraphy with bone scans ) , and evidence of clinical benefit ( improved pain scores compared with baseline in up to 60% ) , ra was deemed to be promising for further study . also of note , survival in this phase i study was observed to be greater than 20 months ( with > 20 months follow - up ) , which compared favorably to a contemporary scandinavian randomized trial with sr in a similar population.48 an additional phase ib study ( bc1 - 05 ) of six patients with advanced prostate cancer demonstrated the feasibility of repeat dosing of ra , but has only been reported in abstract form.38,49 finally , a separate phase ib study ( bc1 - 08 ) in men with progressive crpc and two or more bone metastases on bone scan ( n = 10 ) did not reach the maximum tolerated dose with escalation up to 200 kbq / kg and demonstrated targeting of osseous lesions , rapid blood clearance , and excretion through the small intestine followed by transit through the large intestine.50 two randomized , multicenter phase ii studies in patients with mcrpc have been published . these survival results have been updated and remain similar with longer follow - up.54 post hoc analyses showed os of all patients to be 102.4 versus 42.6 weeks ( p < 0.001 ) for posttreatment normalized versus nonnormalized from baseline alp values , respectively ; patients on the ra arm had os of 102.1 versus 42.5 weeks ( p < 0.001 ) for posttreatment normalized versus nonnormalized from baseline alp values , respectively.55 in summary , this randomized , placebo - controlled study showed that repeat dosing of ra every 4 weeks is well tolerated in men with mcrpc and has a significant effect on bone alp 4 weeks after finishing treatment , in addition to potential beneficial efficacy in terms of sres , psa , and overall survival end points . the results of the international , randomized , double - blind alpharadin in symptomatic prostate cancer ( alsympca ) phase iii trial were recently presented.35 eligible patients had confirmed symptomatic crpc , two or more bone metastases , no known visceral metastases , and were either docetaxel - pretreated or unfit for docetaxel . because of a very tolerable toxicity profile ( including mild myelosuppression and gastrointestinal side effects ) , pharmaco - dynamic effects consistent with the hypothesized mechanism of action ( decline in serum alkaline phosphatase and correlation of gamma scintigraphy with bone scans ) , and evidence of clinical benefit ( improved pain scores compared with baseline in up to 60% ) , ra was deemed to be promising for further study . also of note , survival in this phase i study was observed to be greater than 20 months ( with > 20 months follow - up ) , which compared favorably to a contemporary scandinavian randomized trial with sr in a similar population.48 an additional phase ib study ( bc1 - 05 ) of six patients with advanced prostate cancer demonstrated the feasibility of repeat dosing of ra , but has only been reported in abstract form.38,49 finally , a separate phase ib study ( bc1 - 08 ) in men with progressive crpc and two or more bone metastases on bone scan ( n = 10 ) did not reach the maximum tolerated dose with escalation up to 200 kbq / kg and demonstrated targeting of osseous lesions , rapid blood clearance , and excretion through the small intestine followed by transit through the large intestine.50 two randomized , multicenter phase ii studies in patients with mcrpc have been published . these survival results have been updated and remain similar with longer follow - up.54 post hoc analyses showed os of all patients to be 102.4 versus 42.6 weeks ( p < 0.001 ) for posttreatment normalized versus nonnormalized from baseline alp values , respectively ; patients on the ra arm had os of 102.1 versus 42.5 weeks ( p < 0.001 ) for posttreatment normalized versus nonnormalized from baseline alp values , respectively.55 in summary , this randomized , placebo - controlled study showed that repeat dosing of ra every 4 weeks is well tolerated in men with mcrpc and has a significant effect on bone alp 4 weeks after finishing treatment , in addition to potential beneficial efficacy in terms of sres , psa , and overall survival end points . the results of the international , randomized , double - blind alpharadin in symptomatic prostate cancer ( alsympca ) phase iii trial were recently presented.35 eligible patients had confirmed symptomatic crpc , two or more bone metastases , no known visceral metastases , and were either docetaxel - pretreated or unfit for docetaxel . the pivotal phase iii randomized double - blind placebo controlled alsympca trial of ra demonstrated a highly significant os improvement in docetaxel - pretreated or docetaxel - unfit men with symptomatic bone - metastatic crpc and a very tolerable side - effect profile . although time to first sre was not the primary end point of the alsympca trial , the effects of ra on delaying sres are important , and this end point has led to the regulatory approval of other bone - targeted agents even in the absence of beneficial effects on overall survival and disease progression.15,16 based on eligibility criteria from this pivotal trial , it is likely that the label will be broad , encompassing both men with symptomatic bone - metastatic crpc who have failed docetaxel and those who are not candidates for docetaxel chemotherapy . ra occupies a unique niche in that it prolongs os , is a bone - targeted agent that delays sres , and is also a radiopharmaceutical that might be expected to provide pain palliation ( phase iii data not yet reported ) . none of the currently fda - approved radiopharmaceuticals ( eg , sr , sm ) have demonstrated an os benefit in a randomized phase iii study in men with mcrpc.11 antiresorptive agents : none of the currently fda - approved osteoclast inhibitors ( eg , zoledronic acid , denosumab ) have demonstrated os benefits over placebo . skeletal - related events ( table 4 ) : cytotoxic chemotherapy : there is insufficient evidence to demonstrate that docetaxel and cabazitaxel prevent or delay sres , although they are known to result in pain palliation and to prolong time to pain progression , disease progression , and overall survival . ar - directed therapies : abiraterone has demonstrated delay in time to first sre ( 25th percentile , 301 vs 150 days ; p < 0.0001 ) in the cou - aa-301 trial.20 time to first sre was a secondary end point in the affirm trial of mdv3100 ( nct00974311 ) , but this result has not yet been reported.17 radiopharmaceuticals : there is insufficient evidence to demonstrate that sr or sm prevent or delay sres.11 antiresorptive agents : zoledronic acid delays sres compared with placebo , and denosumab is superior to zoledronic acid in delaying sres in men with mcrpc . in comparison to ar - directed therapies patients treated with abiraterone acetate have demonstrated side effects related to mineralocorticoid excess due to the drug s mechanism of action , such as fluid retention , hypertension , and hypokalemia.14 the most common side effect overall and grade 3 or 4 side effect observed with mdv3100 was fatigue , with patients rarely experiencing seizures ( 0.6% ; all grade 3).17 antiresorptive agents are likely to be used in combination with ra , as in the phase iii trial , and side effects generally appear to be nonoverlapping . in comparison to -emitters , which are renally excreted , however , there is extensive experience with ra in patients with ankylosing spondylitis.42 an increased risk for later cancers has been observed , but only in individuals treated with ra as children and , importantly , not in those treated as adults.67 in comparison , there have been reports of acute leukemia with sr but not sm.68 a large and significant improvement in os was observed when compared with placebo in men with mcrpc and symptomatic bony metastases . radiopharmaceuticals : none of the currently fda - approved radiopharmaceuticals ( eg , sr , sm ) have demonstrated an os benefit in a randomized phase iii study in men with mcrpc.11 antiresorptive agents : none of the currently fda - approved osteoclast inhibitors ( eg , zoledronic acid , denosumab ) have demonstrated os benefits over placebo . skeletal - related events ( table 4 ) : cytotoxic chemotherapy : there is insufficient evidence to demonstrate that docetaxel and cabazitaxel prevent or delay sres , although they are known to result in pain palliation and to prolong time to pain progression , disease progression , and overall survival . ar - directed therapies : abiraterone has demonstrated delay in time to first sre ( 25th percentile , 301 vs 150 days ; p < 0.0001 ) in the cou - aa-301 trial.20 time to first sre was a secondary end point in the affirm trial of mdv3100 ( nct00974311 ) , but this result has not yet been reported.17 radiopharmaceuticals : there is insufficient evidence to demonstrate that sr or sm prevent or delay sres.11 antiresorptive agents : zoledronic acid delays sres compared with placebo , and denosumab is superior to zoledronic acid in delaying sres in men with mcrpc . patients treated with abiraterone acetate have demonstrated side effects related to mineralocorticoid excess due to the drug s mechanism of action , such as fluid retention , hypertension , and hypokalemia.14 the most common side effect overall and grade 3 or 4 side effect observed with mdv3100 was fatigue , with patients rarely experiencing seizures ( 0.6% ; all grade 3).17 antiresorptive agents are likely to be used in combination with ra , as in the phase iii trial , and side effects generally appear to be nonoverlapping . in comparison to -emitters , which are renally excreted , however , there is extensive experience with ra in patients with ankylosing spondylitis.42 an increased risk for later cancers has been observed , but only in individuals treated with ra as children and , importantly , not in those treated as adults.67 in comparison , there have been reports of acute leukemia with sr but not sm.68 emerging therapies aimed at preventing skeletal morbidity in men with prostate cancer have recently been reviewed in detail.69 notable bone - targeted therapies in clinical development for mcrpc include src - targeted therapies ( dasatinib ) , and met - targeted therapies . finally , the multitargeted tyrosine kinase inhibitor xl-184 ( cabozantinib ) , which inhibits met , vefgr2 , ret , and kit , has shown impressive results in a phase ii study71 and will enter phase iii testing soon.72,73 beta emitters have been combined with chemotherapy in several studies , including two randomized studies , demonstrating the possible feasibility and safety of this approach.3134 for example , one recent phase i study by morris et al demonstrated that docetaxel and sm could be administered at full doses over repeated cycles.31 given the improved toxicity profile with ra over -emitters , it seems likely that ra could be combined with chemotherapy as well . this concept is currently being investigated in a phase i / ii trial of ra with docetaxel chemotherapy in men with bone metastasis from crpc ( nct01106352).74 as a synergistic interaction between androgen - deprivation therapy and radiotherapy that increases apoptosis is known to exist in prostate cancer,75 it is conceivable that this synergy could exist between ra and the newer ar - directed therapies ( eg , abiraterone , mdv3100 ) as well . the available data demonstrate that ra has activity in men with symptomatic bone - metastatic crpc who are either docetaxel - unfit or docetaxel - pretreated .

idiopathic parkinson 's disease ( ipd ) is the second most common neurodegenerative disorder , affecting 12% of the population over the age of 65 , with the incidence increasing steeply with age ( van den eeden et al . , 2003 ) . progression is variable and difficult to predict , but ipd is often associated with significant disability . alterations in neurovascular status ( nvs ) including measures of cerebral hemodynamic function as well as more conventional clinical and radiological measures of cerebrovascular disease ( cvd ) might be expected in ipd for two principal reasons . firstly , neurodegeneration is considered to comprise multiple interacting pathological pathways ( collins et al . , 2012 ) . recently there has been considerable interest in the disturbance of neurovascular unit ( nvu ) function and the neurovascular model of neurodegeneration ( grammas et al . , 2011 ; zlokovic , 2008 ) . the nvu is a complex , metabolically active system of endothelial cells and glial cells in close proximity to a neuron . whether altered nvu function is primary or secondary to neurodegeneration , or even attributable to the effect of pharmacotherapy , remains unclear . secondly , as ipd is strongly associated with ageing , an increased burden of comorbid cvd might be expected , but the evidence is somewhat conflicted probably on account of varying study designs and endpoints ( morley and duda , 2012 ) . there is also substantial heterogeneity within ipd with respect to clinical phenotype , including motor and non - motor features and it is possible that differences in nvs might be influential in these differing phenotypes ( lee et al . , 2009 ) . tremor dominant ( td ) and postural instability and gait disorder ( pigd ) phenotypes are recognised , based on the predominant motor features ( jankovic et al . , 1990 ) . non - motor features ( in particular neuropsychiatric and cognitive dysfunction ) have been reported in a significant proportion of ipd patients ; with cognitive decline being associated with worse motor and non - motor features ( aarsland et al . , 1999 ; hu et al . , 2014 ) . studies suggest distinct clinical courses and even variable involvement of the dopaminergic system and other pathways between phenotypes ( eggers et al . , 2012 ; mito et al . , 2006 ) magnetic resonance imaging ( mri ) can provide valuable measures of nvs , such as white matter lesion ( wml ) burden and cerebral blood flow ( cbf ) . arterial spin labelling ( asl ) employs magnetically labelled endogenous arterial blood water to quantify cerebral perfusion . asl can also measure arterial arrival time ( aat ) , the time taken for blood to travel from the labelling slab to the tissue of interest ( wang et al . aat is longest in distal branches , especially in border zone ( or watershed ) areas ( hendrikse et al . , 2008 ; alterations in resting state aat are considered likely to reflect chronic arteriolar vasodilation or collateral flow ( derdeyn et al . , 2002 ; cerebrovascular reactivity ( cvr ) can be measured by combining asl with a hypercapnic challenge . cvr reflects the capacity of the blood vessels to dilate in response to a hypercapnic challenge and can be used as a measure of brain vascular reserve ( hajjar et al . , 2010 ) . this was tested by comparing mri measurements of nvs between a group of people with ipd and age and cardiovascular risk matched controls . in addition mri images were correlated against cognitive and neuropsychiatric scores to determine any association between nvs measurements and such non - motor features of ipd . relevant approvals were obtained including ethics ( north west preston research ethics committee ) , research governance and local university approvals . eligibility criteria for ipd participants were a clinical diagnosis of ipd fulfilling the uk parkinson 's disease society ( ukpds ) brain bank ( bb ) criteria ( http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/getpdf.cgi?id=phd000042 ) without known clinical cvd ( no history of transient ischaemic attack or stroke ) or dementia ( emre et al . , 2007 ) or radiological evidence of large vessel cortical / subcortical infarct > 1.5 cm . control participants ( without ipd or above exclusion criteria ) were matched for age and cardiovascular risk factors . all participants were required to provide written informed consent and had the capacity to do so . all underwent a scan protocol on a 3 t philips achieva mri system using an 8 channel head coil at salford royal hospital . ipd phenotype was assessed using the unified parkinson 's disease rating scale ( updrs ) ( http://www.etas.ee/wp-content/uploads/2013/10/updrs.pdf ) during the scan visit . participants were further classified into three subtypes ( td , pigd , and intermediate ) by jankovic 's method ( jankovic et al . , 1990 ) . disease severity was measured using the hoehn and yahr rating scale ( hoehn and yahr , 1967 ) . routine clinical baseline data were also recorded and the levodopa equivalent doses ( ledd ) calculated ( tomlinson et al . , 2010 ) . a battery of clinical scales was administered , including the montreal cognitive assessment ( moca ) ( http://www.mocatest.org ) , the o'sullivan brief cognitive assessment for participants with cerebral small vessel disease ( o'sullivan et al . , 2005 ) , the lille apathy rating scale ( lars ) ( sockeel et al . , 2006 ) , the hamilton depression scale ( ham - d ) ( muller and dragicevic , 2003 ) and the neuropsychiatric inventory ( npi ) psychosis subscale ( fernandez et al . , 2008 ) . demographics and clinical data were compared between ipd and control participants using the unpaired student t - test with p - value set at < 0.05 . a t2-weighted flair image was acquired with the following parameters : tr 11 s , ti 2.8 s , te 120 ms , in - plane resolution of 0.45 mm , 30 axial slices of 4 mm thickness with 1 mm gap covering the whole brain . a look - locker asl sequence was used ( gunther et al . , 2001 ) , with star labelling ( edelman et al . , 1994 ) and 4 readout times of 800 , 1400 , 2000 , and 2600 ms , tr : 3500 ms ; te 22 ms ; flip angle 40 ; 3.5 3.5 6 mm voxels with a 1 mm gap between slices ; 15 slices covering the cerebrum but not the cerebellum with bipolar vascular crusher gradients added to dephase fast flowing spins and so remove large vessel signal . the labelling slab was 15 cm with a 10 mm gap between the labelling and imaging regions . 112 pairs of labelled and control images were collected , with scan duration approximately 13 min . to allow quantification of cbf an additional scan was acquired with tr = 10 s and 15 read - out times ( from 800 to 9200 ms ) in order to estimate the equilibrium magnetisation of the brain . an additional echo planar image ( epi ) was collected with the same slice positioning and the same voxel dimensions but with te = 35 ms to give typical fmri contrast for registration and normalisation purposes . a 3d t1-weighted image with 1 mm isotropic resolution was also collected . during the asl acquisition a co2 ( hypercapnic ) after 5 min of breathing room air ( from which the baseline perfusion images were extracted ) there followed 6 min of hypercapnia , administered using a non - rebreathing circuit using the fenn and craig technique ( fenn and craig , 1963 ) and a final 2 min of return to room air . this method involves a thin stream of gas ( 79% co2 balanced with 21% o2 ) being delivered through larger tubing allowing it to mix with room air . this mixture then passes through a 3-way valve which directs it to a filter and a mouth piece ( vidyasagar et al . , 2013 ) end - tidal co2 ( etco2 ) and o2 were continuously monitored using powerlab ( labchart7 v7.2.1 , 2011 ) and the co2 flow - rate was altered to ensure all participants reached an increased end tidal level approximately 1% above their baseline etco2 . prior to each scanning session the gas analysers were calibrated using a canister of gas with known concentrations of 5.03% co2 and 21.0% o2 , each participant also had a trial session of inhaled gas to allow them to become accustomed to the apparatus , this also provided an opportunity to assess an appropriate flow rate required to induce a 1% change in etco2 . wml burden was assessed semi - quantitatively using visual rating scales ( fazekas et al . , 1993 ; wahlund et al . , 2001 ) . asl data were analysed using in - house matlab ( mathworks , ma , usa ) routines using a single blood compartment model , adapted for look - locker readout ( parkes l.m . , 2012 ) . baseline cbf and aat maps were calculated using the first 5 min of asl data , during breathing of air . cvr maps were calculated using subtraction images of cbf and aat between periods of air ( 5 min ) and hypercapnia ( last 5 min , omitting the first minute of hypercapnia to allow equilibrium to be reached ) and dividing these by the value of etco2 on an individual basis . whole brain values for cbf , aat , cvrcbf ( % cbf change/etco2 ) and cvraat ( aat/etco2 ) were calculated using a simple threshold mask based on the asl control images on an individual basis . differences between ipd and control participants for cbf , aat and all cvr measures were tested using student 's t - tests in excel . differences between ipd and control participants for wml burden as measured by the visual rating scales were tested using fisher 's exact test in spss , taking into account the non - parametric nature of the scales and small sample sizes . linear regression was used to assess whether wml burden could predict cbf and aat using wml burden as a categorical variable in regression . voxel - wise analysis was also performed using the spm8 pet toolbox ( http://www.fil.ion.ucl.ac.uk/spm/ ) to compare cbf , aat and cvr maps between ipd and control participants ( phenotype specific differences were not analysed due to the small sample sizes ) . image pre - processing in spm included ( 1 ) motion correction , ( 2 ) registration and normalisation of the epi image to the epi template within spm , and application of this procedure to the perfusion maps , and ( 3 ) spatial smoothing of the normalised images using a 12 mm full - width - half - maximum kernel . ( 2005 ) according to the proportion of gray matter and white matter in each voxel , obtained from the segmented t1-weighted image . voxel - wise comparisons of cbf , aat , cvrcbf , and cvraat between the ipd and control groups were carried out using a two - sample unpaired t - test ( unequal variances ) . regions were considered significant at a p value of < 0.001 uncorrected , with a minimum cluster size of 100 voxels ( at the re - sampled voxel size of 2 mm isotropic ) , which was felt sufficient to avoid type ii errors in view of the exploratory nature of the study ( lieberman and cunningham , 2009 ) . further analysis using family - wise error ( fwe ) correction for multiple comparisons at the cluster level was performed ( using gaussian radom field theory as employed in spm8 ) . in addition , voxel wise regression of cbf and aat against moca and ham - d scores was performed within both the ipd and control groups separately . fourteen ipd participants ( mean sd age 65.1 5.9 years ) and 14 control participants ( mean sd age 64.6 4.2 years ) were enrolled into the study . six ipd participants were assigned to the pigd subgroup and 6 to the td subgroup and 2 were intermediate ( table 1 and 2 ) . thirteen ipd and all 14 control participants completed the full scanning protocol ( asl data were lacking for 1 ipd participant due to difficulties in tolerating the set - up of the gas apparatus in the scanner ) . all participants showed the expected increase in cbf and reduction in aat with the hypercapnic challenge due to induced cerebrovasodilation ( hajjar et al . , 2010 ) . two further ipd and 2 control participants were excluded from cvr analysis as the etco2 gas response was outside the expected limits ( 412 mm hg ) . twelve ipd ( 6 pigd , 5 td ) and 13 control participants completed the clinical scales . whole brain baseline cbf did not differ between the ipd and control groups ( table 3a ) . the voxel - wise analysis did reveal one region of lower cbf in patients compared to controls ( at p < 0.001 minimum cluster size 100 voxels ) in the right parietal lobe supramarginal gyrus near the angular gyrus ( talairach coordinates [ 16 80 34 ] , cluster size 231 voxels , peak t - value 3.8 , peak p - value 0.0004 ) . whole brain baseline aat was significantly prolonged in ipd participants compared to controls ( table 3a ) . voxel - wise analysis revealed widespread regions of significantly increased baseline aat in the ipd group compared to controls , particularly in the frontal and temporal regions at p < 0.001 minimum cluster threshold 100 voxels ( fig . there were no regions in the brain where aat was significantly shorter in the ipd group than in the control group . a mean difference map of aat between patients and controls was created to further shed light on the distribution of the prolonged aat ; this revealed bilateral , diffuse increases in aat in the ipd group compared to controls ( fig . whole brain measures of cvrcbf or cvraat did not differ between ipd and control participants . voxel - wise analysis did not reveal any regions of statistically significant differences in these measurements between the groups . to test for differences in wml score fisher s exact test was used , combining scores into categories ( 01 ) and 2 where appropriate since the majority of patients scored 1 or 2 . the results showed no association between any of the wml scores and whether a participant has ipd , with p - values as given in table 3b . there were no statistically significant correlations between the wml burden ( wahlund scale ) and cbf or aat . the ipd group ( mean sd , score 8.5 2.5 ) scored significantly lower than the control group ( mean sd score 10.8 2.7 , p = 0.03 ) in the digit symbol substitution test ( dsst ) . six ipd participants ( 4 pigd , 2 td ) but only one control participant met criteria for mild cognitive impairment as reflected by a score of 25 on the moca tool . three ipd participants ( 2 pigd , 1 intermediate ) met the cut - off for mild depression as measured by the ham - d scale , whereas all control participants fell within the not depressed range on this scale . all participants completed the trail making test part b ( tmt - b ) except for one ipd ( pigd group ) and one control participant . neither group exhibited significant apathy or any features of psychosis as per the clinical scales . voxel - wise analysis using linear regression was performed to identify potential regional correlations between moca and ham - d scores , with both cbf and aat . reduced cbf mainly in the parietal regions was found to correlate with reduced moca score ( table 6 , fig . 2 ) in the ipd group only . to help verify the statistical threshold used , moca scores of the ipd participants were shuffled randomly and the voxel - wise regression analysis was repeated . no regions of significant correlation were found , confirming the validity of our findings . there were no regions of significant correlation of moca score and aat in either of the two study groups . in addition ham - d scores did not correlate with either cbf or aat in either the ipd or control group . we hypothesised that nvs is altered in ipd , which may be a reflection of the neurodegenerative process or due to comorbid cvd . we revealed diffuse aat prolongation in the ipd group compared to healthy control participants and significant regional correlations between moca scores and cbf in the ipd group only . to our knowledge , the prolonged aat in the ipd group compared to controls has not been previously reported . although the differences appear predominantly in the right hemisphere the mean difference maps suggest prolonged aat is more diffuse ( fig . 1 ) . it is possible to attribute prolonged aat to any factor which increases path length or decreases the velocity of flow i.e. diameter and resistance of vessels and characteristics of the circulating blood ( liu et al . , 2012 ) . several other studies have reported on aat in non - pd populations with various reasons proposed . for example , prolonged aat has been noted in studies of ageing ( liu et al . , 2012 ) , presumably related to age driven structural cerebrovascular changes such as increased vessel tortuosity , increased rarefaction and arteriolar wall damage ( chen et al . , 2012 ; wolk and detre , 2012 ) . aat prolongation in multiple sclerosis has been attributed to widespread inflammation or chronic vasodilation of the resistance vessels ( paling et al . , 2014 ) . a study in ad revealed prolonged aat and hypoperfusion in the left inferior frontal and middle cingulate gyri ( mak et al . , 2012 ) . lastly , stroke and tia studies have attributed aat prolongation to the recruitment of collateral pathways in large artery stenosis with , in some cases , preserved perfusion ( chalela et al . , 2000 ; we did not find differences in cbf between the ipd group and controls at the p < 0.001 , minimum cluster size 100 voxels , yet at the more lenient threshold of p < 0.005 , regions of hypoperfusion in the left cuneus in the ipd group were revealed . this perfusion deficit is in keeping with both asl and positron emission tomography ( pet ) studies in ipd which have found a similar pattern of hypoperfusion and hypometabolism in ipd patients compared to healthy controls with significant correlation between pet and asl perfusion patterns ( borghammer et al . , 2010 ; these studies consistently revealed bilateral hypoperfusion in the occipital lobe ( including the cuneus ) as well as the posterior parietal regions , with variable patterns in the frontal lobe . hypoperfusion in neurodegenerative states has previously been attributed to direct tissue loss or the result of loss of functional connectivity ( borghammer et al . , 2010 ; yoshiura et al . , 2009 we feel that the small sample size may mean important differences between the 2 groups ( such as hypoperfusion ) which may be under - represented in this study . our results show that aat is a more sensitive marker of changes in nvs in ipd than cbf , and may be important to consider in future studies using asl . despite diffuse baseline aat prolongation in the ipd group , the global response of aat and cbf to the hypercapnic challenge ( cvraat and cvrcbf ) did not differ significantly between the groups , suggesting vascular responsiveness is generally preserved . if the prolonged baseline aat is indicative of chronic vasodilation then the aat and cbf response in the ipd group would be expected to be reduced ( reduced capacity for further dilation ) . however , the variability of both cvraat and cvrcbf is large , so greater participant numbers are probably required to reliably test for these differences . this study also revealed a positive correlation between cbf predominantly in the posterior regions and total moca scores in the ipd group . similarly cognitive scores including the mini mental state examination ( mmse ) scores and moca have been noted to correlate with hypoperfusion in ipd and other diseases affecting cognition , with specific patterns of hypoperfusion dependent on the disease state ( chao et al . , 2010 ; we recognise that the relatively small sample size may potentially account for differences in other non - motor features not reaching statistical significance ( table 5 ) and further exploration of their pathophysiological underpinnings is warranted . this study did not reveal differences in wml burden between the ipd group and controls ; larger sample sizes would be needed to consider phenotype - specific differences . , we used only 4 post - labelling time - points for the asl acquisition , which was necessary in order to cover the cerebrum with vascular crushing gradients enabled . more time points may have increased the precision of the aat measurements . due to the exploratory nature of the study , uncorrected p - values have been displayed alongside fwe values , so a larger data set with fwe correction is required to verify the results . it is possible that differences in nvs might be influential in differing clinical phenotypes of ipd ( lee et al . , 2009 ) , however the small sample size in this study meant that phenotype - specific differences could not be explored . led scores differed between participants , which may confound the results as previous studies have revealed regional blood flow increases with levodopa ( hershey et al . , 2003 ; kobari et al . , 1995 this exploratory work used mri perfusion and structural measures to investigate nvs in ipd . in ipd we have identified prolonged baseline aat , as well as regional posterior hypoperfusion with correlations with impaired cognition . alterations in neurovascular parameters in ipd and their associations with clinical features and treatment , as well as the potential identification of targets for intervention , warrant further study . asl data were analysed using a single blood compartment model , adapted for look locker readout ( parkes l.m . , 2012 ) . this model assumes that labelled water remains in the blood and that no labelled water leaves the voxel , an approach that has been shown to be reasonably accurate ( parkes and tofts , 2002 ) . with these assumptions , the signal in the difference image ( control label ) , m , can be described by:(1)dm(t)dt=r1m(t)+fma(t ) where r1 is the apparent r1 of blood during the look locker readout and f is cbf . for star labelling the magnetisation of arterial blood , ma , is given by:(2)ma(t)=2ma0exp(-tr1b)for t > taand t < and ma = 0 at all other times . here ma is the equilibrium magnetisation of arterial blood , is the inversion efficiency ( assumed to equal 1 ) and r1b is the true r1 of blood . according to gunther et al . ( 2001 ) r1=r1b ln(cos)/ti2 where is the flip angle and ti2 the spacing of the look locker readout . the solution for m is:(3)m(t)=2fma0rexp(-tr1)[exp(tr)-exp(tar ) ] for t > taand t < ta+ m(t)=2fma0rexp(-tr1)exp(tar)[exp(r)-1 ] for t > the mean difference signal m was fit on a voxel - wise basis to eq . 3 , extracting values for the 2 free parameters f ( cbf ) and ta ( aat ) , with fixed values for = 1000 ms ( macintosh et al . , 2010a ) , t1b = 1600 ms ( lu et al . , 2004 ) ( note r1b = 1/t1b ) , and m. the calibration images were fit on a voxel - wise basis to a saturation recovery curve , producing maps of t1 and m0 . ma was estimated from a whole brain estimate of m0 divided by the blood brain partition coefficient = 0.9 ( roberts et al . , 1996 ) .

idiopathic parkinson 's disease ( ipd ) is the second most common neurodegenerative disease , yet effective disease modifying treatments are still lacking . neurodegeneration involves multiple interacting pathological pathways . the extent to which neurovascular mechanisms are involved is not well defined in ipd . we aimed to determine whether novel magnetic resonance imaging ( mri ) techniques , including arterial spin labelling ( asl ) quantification of cerebral perfusion , can reveal altered neurovascular status ( nvs ) in ipd.fourteen participants with ipd ( mean sd age 65.1 5.9 years ) and 14 age and cardiovascular risk factor matched control participants ( mean sd age 64.6 4.2 years ) underwent a 3 t mri scan protocol . asl images were collected before , during and after a 6 minute hypercapnic challenge . flair images were used to determine white matter lesion score . quantitative images of cerebral blood flow ( cbf ) and arterial arrival time ( aat ) were calculated from the asl data both at rest and during hypercapnia . cerebrovascular reactivity ( cvr ) images were calculated , depicting the change in cbf and aat relative to the change in end - tidal co2.a significant ( p = 0.005 ) increase in whole brain averaged baseline aat was observed in ipd participants ( mean sd age 1532 138 ms ) compared to controls ( mean sd age 1335 165 ms ) . voxel - wise analysis revealed this to be widespread across the brain . however , there were no statistically significant differences in white matter lesion score , cbf , or cvr between patients and controls . regional cbf , but not aat , in the ipd group was found to correlate positively with montreal cognitive assessment ( moca ) scores . these findings provide further evidence of alterations in nvs in ipd .

Introduction Methods Results Discussion Conclusion Appendix

idiopathic parkinson 's disease ( ipd ) is the second most common neurodegenerative disorder , affecting 12% of the population over the age of 65 , with the incidence increasing steeply with age ( van den eeden et al . alterations in neurovascular status ( nvs ) including measures of cerebral hemodynamic function as well as more conventional clinical and radiological measures of cerebrovascular disease ( cvd ) might be expected in ipd for two principal reasons . firstly , neurodegeneration is considered to comprise multiple interacting pathological pathways ( collins et al . whether altered nvu function is primary or secondary to neurodegeneration , or even attributable to the effect of pharmacotherapy , remains unclear . there is also substantial heterogeneity within ipd with respect to clinical phenotype , including motor and non - motor features and it is possible that differences in nvs might be influential in these differing phenotypes ( lee et al . tremor dominant ( td ) and postural instability and gait disorder ( pigd ) phenotypes are recognised , based on the predominant motor features ( jankovic et al . non - motor features ( in particular neuropsychiatric and cognitive dysfunction ) have been reported in a significant proportion of ipd patients ; with cognitive decline being associated with worse motor and non - motor features ( aarsland et al . , 2006 ) magnetic resonance imaging ( mri ) can provide valuable measures of nvs , such as white matter lesion ( wml ) burden and cerebral blood flow ( cbf ) . arterial spin labelling ( asl ) employs magnetically labelled endogenous arterial blood water to quantify cerebral perfusion . asl can also measure arterial arrival time ( aat ) , the time taken for blood to travel from the labelling slab to the tissue of interest ( wang et al . , 2008 ; alterations in resting state aat are considered likely to reflect chronic arteriolar vasodilation or collateral flow ( derdeyn et al . , 2002 ; cerebrovascular reactivity ( cvr ) can be measured by combining asl with a hypercapnic challenge . cvr reflects the capacity of the blood vessels to dilate in response to a hypercapnic challenge and can be used as a measure of brain vascular reserve ( hajjar et al . this was tested by comparing mri measurements of nvs between a group of people with ipd and age and cardiovascular risk matched controls . in addition mri images were correlated against cognitive and neuropsychiatric scores to determine any association between nvs measurements and such non - motor features of ipd . eligibility criteria for ipd participants were a clinical diagnosis of ipd fulfilling the uk parkinson 's disease society ( ukpds ) brain bank ( bb ) criteria ( http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/getpdf.cgi?id=phd000042 ) without known clinical cvd ( no history of transient ischaemic attack or stroke ) or dementia ( emre et al . , 2007 ) or radiological evidence of large vessel cortical / subcortical infarct > 1.5 cm . control participants ( without ipd or above exclusion criteria ) were matched for age and cardiovascular risk factors . all underwent a scan protocol on a 3 t philips achieva mri system using an 8 channel head coil at salford royal hospital . ipd phenotype was assessed using the unified parkinson 's disease rating scale ( updrs ) ( http://www.etas.ee/wp-content/uploads/2013/10/updrs.pdf ) during the scan visit . a battery of clinical scales was administered , including the montreal cognitive assessment ( moca ) ( http://www.mocatest.org ) , the o'sullivan brief cognitive assessment for participants with cerebral small vessel disease ( o'sullivan et al . , 2006 ) , the hamilton depression scale ( ham - d ) ( muller and dragicevic , 2003 ) and the neuropsychiatric inventory ( npi ) psychosis subscale ( fernandez et al . demographics and clinical data were compared between ipd and control participants using the unpaired student t - test with p - value set at < 0.05 . a t2-weighted flair image was acquired with the following parameters : tr 11 s , ti 2.8 s , te 120 ms , in - plane resolution of 0.45 mm , 30 axial slices of 4 mm thickness with 1 mm gap covering the whole brain . , 1994 ) and 4 readout times of 800 , 1400 , 2000 , and 2600 ms , tr : 3500 ms ; te 22 ms ; flip angle 40 ; 3.5 3.5 6 mm voxels with a 1 mm gap between slices ; 15 slices covering the cerebrum but not the cerebellum with bipolar vascular crusher gradients added to dephase fast flowing spins and so remove large vessel signal . 112 pairs of labelled and control images were collected , with scan duration approximately 13 min . to allow quantification of cbf an additional scan was acquired with tr = 10 s and 15 read - out times ( from 800 to 9200 ms ) in order to estimate the equilibrium magnetisation of the brain . during the asl acquisition a co2 ( hypercapnic ) after 5 min of breathing room air ( from which the baseline perfusion images were extracted ) there followed 6 min of hypercapnia , administered using a non - rebreathing circuit using the fenn and craig technique ( fenn and craig , 1963 ) and a final 2 min of return to room air . , 2013 ) end - tidal co2 ( etco2 ) and o2 were continuously monitored using powerlab ( labchart7 v7.2.1 , 2011 ) and the co2 flow - rate was altered to ensure all participants reached an increased end tidal level approximately 1% above their baseline etco2 . prior to each scanning session the gas analysers were calibrated using a canister of gas with known concentrations of 5.03% co2 and 21.0% o2 , each participant also had a trial session of inhaled gas to allow them to become accustomed to the apparatus , this also provided an opportunity to assess an appropriate flow rate required to induce a 1% change in etco2 . baseline cbf and aat maps were calculated using the first 5 min of asl data , during breathing of air . cvr maps were calculated using subtraction images of cbf and aat between periods of air ( 5 min ) and hypercapnia ( last 5 min , omitting the first minute of hypercapnia to allow equilibrium to be reached ) and dividing these by the value of etco2 on an individual basis . whole brain values for cbf , aat , cvrcbf ( % cbf change/etco2 ) and cvraat ( aat/etco2 ) were calculated using a simple threshold mask based on the asl control images on an individual basis . differences between ipd and control participants for cbf , aat and all cvr measures were tested using student 's t - tests in excel . differences between ipd and control participants for wml burden as measured by the visual rating scales were tested using fisher 's exact test in spss , taking into account the non - parametric nature of the scales and small sample sizes . linear regression was used to assess whether wml burden could predict cbf and aat using wml burden as a categorical variable in regression . voxel - wise analysis was also performed using the spm8 pet toolbox ( http://www.fil.ion.ucl.ac.uk/spm/ ) to compare cbf , aat and cvr maps between ipd and control participants ( phenotype specific differences were not analysed due to the small sample sizes ) . image pre - processing in spm included ( 1 ) motion correction , ( 2 ) registration and normalisation of the epi image to the epi template within spm , and application of this procedure to the perfusion maps , and ( 3 ) spatial smoothing of the normalised images using a 12 mm full - width - half - maximum kernel . ( 2005 ) according to the proportion of gray matter and white matter in each voxel , obtained from the segmented t1-weighted image . voxel - wise comparisons of cbf , aat , cvrcbf , and cvraat between the ipd and control groups were carried out using a two - sample unpaired t - test ( unequal variances ) . further analysis using family - wise error ( fwe ) correction for multiple comparisons at the cluster level was performed ( using gaussian radom field theory as employed in spm8 ) . in addition , voxel wise regression of cbf and aat against moca and ham - d scores was performed within both the ipd and control groups separately . fourteen ipd participants ( mean sd age 65.1 5.9 years ) and 14 control participants ( mean sd age 64.6 4.2 years ) were enrolled into the study . six ipd participants were assigned to the pigd subgroup and 6 to the td subgroup and 2 were intermediate ( table 1 and 2 ) . thirteen ipd and all 14 control participants completed the full scanning protocol ( asl data were lacking for 1 ipd participant due to difficulties in tolerating the set - up of the gas apparatus in the scanner ) . all participants showed the expected increase in cbf and reduction in aat with the hypercapnic challenge due to induced cerebrovasodilation ( hajjar et al . two further ipd and 2 control participants were excluded from cvr analysis as the etco2 gas response was outside the expected limits ( 412 mm hg ) . twelve ipd ( 6 pigd , 5 td ) and 13 control participants completed the clinical scales . whole brain baseline cbf did not differ between the ipd and control groups ( table 3a ) . the voxel - wise analysis did reveal one region of lower cbf in patients compared to controls ( at p < 0.001 minimum cluster size 100 voxels ) in the right parietal lobe supramarginal gyrus near the angular gyrus ( talairach coordinates [ 16 80 34 ] , cluster size 231 voxels , peak t - value 3.8 , peak p - value 0.0004 ) . whole brain baseline aat was significantly prolonged in ipd participants compared to controls ( table 3a ) . voxel - wise analysis revealed widespread regions of significantly increased baseline aat in the ipd group compared to controls , particularly in the frontal and temporal regions at p < 0.001 minimum cluster threshold 100 voxels ( fig . there were no regions in the brain where aat was significantly shorter in the ipd group than in the control group . a mean difference map of aat between patients and controls was created to further shed light on the distribution of the prolonged aat ; this revealed bilateral , diffuse increases in aat in the ipd group compared to controls ( fig . whole brain measures of cvrcbf or cvraat did not differ between ipd and control participants . voxel - wise analysis did not reveal any regions of statistically significant differences in these measurements between the groups . to test for differences in wml score fisher s exact test was used , combining scores into categories ( 01 ) and 2 where appropriate since the majority of patients scored 1 or 2 . there were no statistically significant correlations between the wml burden ( wahlund scale ) and cbf or aat . the ipd group ( mean sd , score 8.5 2.5 ) scored significantly lower than the control group ( mean sd score 10.8 2.7 , p = 0.03 ) in the digit symbol substitution test ( dsst ) . six ipd participants ( 4 pigd , 2 td ) but only one control participant met criteria for mild cognitive impairment as reflected by a score of 25 on the moca tool . three ipd participants ( 2 pigd , 1 intermediate ) met the cut - off for mild depression as measured by the ham - d scale , whereas all control participants fell within the not depressed range on this scale . all participants completed the trail making test part b ( tmt - b ) except for one ipd ( pigd group ) and one control participant . voxel - wise analysis using linear regression was performed to identify potential regional correlations between moca and ham - d scores , with both cbf and aat . reduced cbf mainly in the parietal regions was found to correlate with reduced moca score ( table 6 , fig . 2 ) in the ipd group only . to help verify the statistical threshold used , moca scores of the ipd participants were shuffled randomly and the voxel - wise regression analysis was repeated . there were no regions of significant correlation of moca score and aat in either of the two study groups . we hypothesised that nvs is altered in ipd , which may be a reflection of the neurodegenerative process or due to comorbid cvd . we revealed diffuse aat prolongation in the ipd group compared to healthy control participants and significant regional correlations between moca scores and cbf in the ipd group only . to our knowledge , the prolonged aat in the ipd group compared to controls has not been previously reported . although the differences appear predominantly in the right hemisphere the mean difference maps suggest prolonged aat is more diffuse ( fig . a study in ad revealed prolonged aat and hypoperfusion in the left inferior frontal and middle cingulate gyri ( mak et al . lastly , stroke and tia studies have attributed aat prolongation to the recruitment of collateral pathways in large artery stenosis with , in some cases , preserved perfusion ( chalela et al . , 2000 ; we did not find differences in cbf between the ipd group and controls at the p < 0.001 , minimum cluster size 100 voxels , yet at the more lenient threshold of p < 0.005 , regions of hypoperfusion in the left cuneus in the ipd group were revealed . this perfusion deficit is in keeping with both asl and positron emission tomography ( pet ) studies in ipd which have found a similar pattern of hypoperfusion and hypometabolism in ipd patients compared to healthy controls with significant correlation between pet and asl perfusion patterns ( borghammer et al . , 2010 ; these studies consistently revealed bilateral hypoperfusion in the occipital lobe ( including the cuneus ) as well as the posterior parietal regions , with variable patterns in the frontal lobe . our results show that aat is a more sensitive marker of changes in nvs in ipd than cbf , and may be important to consider in future studies using asl . despite diffuse baseline aat prolongation in the ipd group , the global response of aat and cbf to the hypercapnic challenge ( cvraat and cvrcbf ) did not differ significantly between the groups , suggesting vascular responsiveness is generally preserved . if the prolonged baseline aat is indicative of chronic vasodilation then the aat and cbf response in the ipd group would be expected to be reduced ( reduced capacity for further dilation ) . this study also revealed a positive correlation between cbf predominantly in the posterior regions and total moca scores in the ipd group . similarly cognitive scores including the mini mental state examination ( mmse ) scores and moca have been noted to correlate with hypoperfusion in ipd and other diseases affecting cognition , with specific patterns of hypoperfusion dependent on the disease state ( chao et al . , 2010 ; we recognise that the relatively small sample size may potentially account for differences in other non - motor features not reaching statistical significance ( table 5 ) and further exploration of their pathophysiological underpinnings is warranted . this study did not reveal differences in wml burden between the ipd group and controls ; larger sample sizes would be needed to consider phenotype - specific differences . , we used only 4 post - labelling time - points for the asl acquisition , which was necessary in order to cover the cerebrum with vascular crushing gradients enabled . due to the exploratory nature of the study , uncorrected p - values have been displayed alongside fwe values , so a larger data set with fwe correction is required to verify the results . it is possible that differences in nvs might be influential in differing clinical phenotypes of ipd ( lee et al . led scores differed between participants , which may confound the results as previous studies have revealed regional blood flow increases with levodopa ( hershey et al . , 1995 this exploratory work used mri perfusion and structural measures to investigate nvs in ipd . in ipd we have identified prolonged baseline aat , as well as regional posterior hypoperfusion with correlations with impaired cognition . alterations in neurovascular parameters in ipd and their associations with clinical features and treatment , as well as the potential identification of targets for intervention , warrant further study . asl data were analysed using a single blood compartment model , adapted for look locker readout ( parkes l.m . this model assumes that labelled water remains in the blood and that no labelled water leaves the voxel , an approach that has been shown to be reasonably accurate ( parkes and tofts , 2002 ) . with these assumptions , the signal in the difference image ( control label ) , m , can be described by:(1)dm(t)dt=r1m(t)+fma(t ) where r1 is the apparent r1 of blood during the look locker readout and f is cbf . for star labelling the magnetisation of arterial blood , ma , is given by:(2)ma(t)=2ma0exp(-tr1b)for t > taand t < and ma = 0 at all other times . here ma is the equilibrium magnetisation of arterial blood , is the inversion efficiency ( assumed to equal 1 ) and r1b is the true r1 of blood . ( 2001 ) r1=r1b ln(cos)/ti2 where is the flip angle and ti2 the spacing of the look locker readout . the solution for m is:(3)m(t)=2fma0rexp(-tr1)[exp(tr)-exp(tar ) ] for t > taand t < ta+ m(t)=2fma0rexp(-tr1)exp(tar)[exp(r)-1 ] for t > the mean difference signal m was fit on a voxel - wise basis to eq . 3 , extracting values for the 2 free parameters f ( cbf ) and ta ( aat ) , with fixed values for = 1000 ms ( macintosh et al . , 2004 ) ( note r1b = 1/t1b ) , and m. the calibration images were fit on a voxel - wise basis to a saturation recovery curve , producing maps of t1 and m0 . ma was estimated from a whole brain estimate of m0 divided by the blood brain partition coefficient = 0.9 ( roberts et al .

3.11 in japan refocussed attention on the environmental and health effects of wide - spread use of nuclear power . rapid and accurate evaluation of exposure dose plays an important role in early triage , diagnosis and medical treatment of the victims during emergency response efforts in nuclear accidents . in view of some of the serious shortcomings and limits presented by physical and chemical methods used in evaluation of radiation exposure dose , biodosimetry has emerged as one of the best techniques for use in individual exposure dose evaluation in many situations such as medical rescue of radiation accident victims , bio - effects research of radiation and radiation protection . because gene expression is sensitive to environmental factors , the analysis of gene expression profiling of peripheral blood cells , particularly lymphocytes , has been used to assess the presence of certain diseases [ 28 ] . accordingly , many radiobiologists have focussed on gene expression profiling analysis to find biomarkers that are suitable for assessment of individual exposure doses under different exposure conditions [ 913 ] . considerable work has been done on radiation - induced gene alterations using cultured cell lines and peripheral blood cells irradiated in vitro or ex vivo[1419 ] . several genes , including gadd45 , cdkn1a and bbc3 , have been identified as radiation response expression genes at different exposure doses and post - irradiation times in human peripheral blood lymphocytes and tissue cells [ 2022 ] . recent reports have shown that gene expression signatures induced by ir are specific , durable and accurate in prediction of exposure doses in both mice and humans ] 2325 ] . since there is a highly relevant among exposure doses , dna damage degree and dna repair - related gene expression level , in this study 11 dna damage response genes related to dna repair ( atm , brca1 and rad50 ) , cell cycle regulation ( cdkn1a , gadd45a ) , and apoptosis ( bbc3 ) ( table 1 ) were selected as targets to screen potential bio - markers for indicating exposure dose . the radiation - induced transcription level of 11 genes was investigated in white blood cells of tbi - treated balb / c mice with a rt - pcr method . male balb / c mice , 68 weeks old , provided by the animal center of the second military medical university were divided into control ( non - irradiated ) and 4 exposure groups ( 5 mice in each group ) . the mice in the exposure groups were exposed to either 2 , 4 , 6 or 8 gy doses of co -rays with tbi at 0.80 gy / min dose rate and room temperature ( 232c ) . the co -rays source ( shanghai institute of measurement and testing technology , simt china ) provided a 98% uniform exposure field in 1513 cm sides , which was measured with a graphite cavity ionization chamber . they were housed in a pathogen - free barrier facility ( 12-hour light / dark cycle ) and were fed autoclaved standard rodent chow pre- and post - irradiation . all protocols were approved by the university committee on animal research , and all experiments were carried out in accordance with related guidelines . all samples were run 3 times and a total of 375 mice including control group were used . for each mouse approximately 0.50.7 ml of heparin - anticoagulated blood was collected from the orbital venous vein using polypropylene tubes at each post - irradiation time point . total rna was extracted from whole blood using the axygen blood total rna miniprep kit , no . ap - mn - bl - rna-250 ( axygen scientific , hangzhou , china ) following the manufacturer s recommendations . isolated white blood cells ( wbcs ) were lysed , and genomic dna and protein were removed via precipitation . the rna was quantified using a nanodrop-1000 spectrophotometer ( thermo scientific , waltham , ma ) , and the quality of the isolated rna was assayed with agarose gel electrophoresis by analyzing 18s and 28s rna . using the sybr prime script rt reagent kit , no . drr037a ( takara , dalian , china ) , 1 g of total rna was reversely transcribed to cdna according to the manufacturer s instructions . the rt - pcr reactions were performed with the rotor - gene 6000 rt - pcr system using the sybr premix ex taq , no . drr041a ( takara , dalian , china ) following the manufacturer s recommendations . the rt - pcr cycling conditions were as follows : initial denaturation at 95c for 10 min , followed by 35 cycles of denaturation at 95c for 10 s , annealing at 60c for 30 s and primer extension at 72c for 30 s. the final extension occurred at 72c for 10 min . relative fold inductions were calculated by the ct method with averaged relative levels of beta - actin , which was used for normalization . statistical analysis was performed with the graphpad prism 5.0 software package ( graphpad software , usa ) . the statistical significance of differences between groups was analyzed by one - way analysis of variance with a post - hoc tukey test . male balb / c mice , 68 weeks old , provided by the animal center of the second military medical university were divided into control ( non - irradiated ) and 4 exposure groups ( 5 mice in each group ) . the mice in the exposure groups were exposed to either 2 , 4 , 6 or 8 gy doses of co -rays with tbi at 0.80 gy / min dose rate and room temperature ( 232c ) . the co -rays source ( shanghai institute of measurement and testing technology , simt china ) provided a 98% uniform exposure field in 1513 cm sides , which was measured with a graphite cavity ionization chamber . they were housed in a pathogen - free barrier facility ( 12-hour light / dark cycle ) and were fed autoclaved standard rodent chow pre- and post - irradiation . all protocols were approved by the university committee on animal research , and all experiments were carried out in accordance with related guidelines . all samples were run 3 times and a total of 375 mice including control group were used . for each mouse approximately 0.50.7 ml of heparin - anticoagulated blood was collected from the orbital venous vein using polypropylene tubes at each post - irradiation time point . total rna was extracted from whole blood using the axygen blood total rna miniprep kit , no . ap - mn - bl - rna-250 ( axygen scientific , hangzhou , china ) following the manufacturer s recommendations . isolated white blood cells ( wbcs ) were lysed , and genomic dna and protein were removed via precipitation . the rna was quantified using a nanodrop-1000 spectrophotometer ( thermo scientific , waltham , ma ) , and the quality of the isolated rna was assayed with agarose gel electrophoresis by analyzing 18s and 28s rna . drr037a ( takara , dalian , china ) , 1 g of total rna was reversely transcribed to cdna according to the manufacturer s instructions . the rt - pcr reactions were performed with the rotor - gene 6000 rt - pcr system using the sybr premix ex taq , no . the rt - pcr cycling conditions were as follows : initial denaturation at 95c for 10 min , followed by 35 cycles of denaturation at 95c for 10 s , annealing at 60c for 30 s and primer extension at 72c for 30 s. the final extension occurred at 72c for 10 min . relative fold inductions were calculated by the ct method with averaged relative levels of beta - actin , which was used for normalization . statistical analysis was performed with the graphpad prism 5.0 software package ( graphpad software , usa ) . the statistical significance of differences between groups was analyzed by one - way analysis of variance with a post - hoc tukey test . cdkn1a , which has a regulatory role in s phase dna replication and is known to be activated by the tumor protein p53 ( tp 53 ) , had the strongest upregulation in expression level out of the 11 genes investigated in this study . as shown in figure 1a , compared with the non - irradiated control , cdkn1a gene expression gradually increased to a maximum value of approximately 88-fold for 2 gy ( p<0.0001 ) and 130-fold for 4 gy ( p<0.0001 ) at 12 h post - irradiation , and approximately 116-fold for 6 gy ( p<0.0001 ) and 100-fold for 8 gy ( p<0.0001 ) at 8 h post - exposure . then , cdkn1a gene expression gradually decreased but remained greater than 30-fold ( p<0.05 ) higher than that of the control group at 48 h post - irradiation for all exposure doses . figure 2a and b show the significant differences in cdkn1a gene expression induced by the different exposure doses . significant differences can be seen by comparing the exposure groups of 2 and 6 gy ( p<0.05 ) , 4 and 6 gy ( p<0.0001 ) , and 4 and 8 gy ( p<0.001 ) at 8 h post - exposure ( figure 2a ) . at the 12 h post - irradiation time point , atm is a predominantly nuclear protein that is involved in the dna damage repair signal transduction process . in this study , atm gene expression decreased linearly with the exposure dose , with the exception of the 12 h post - irradiation time - point after a 2 gy exposure ( figure 1b ) . compared with non - irradiated controls , it could be determined from the time course of atm gene expression ( figure 1b ) that the gene was strongly downregulated in all exposure dose ranges at 448 h post - irradiation . a significant decrease was found for the 4 gy ( p<0.001 ) and 8 gy ( p<0.05 ) irradiation groups at 4 h post - irradiation ( figure 2c ) , and highly significant changes across the dose range of 28 gy ( p<0.0001 ) were found at 24 h post - irradiation ( figure 2d ) . significant differences can also be seen by comparing the exposure groups of 2 and 4 gy ( p<0.001 ) , 2 and 6 gy ( p<0.0001 ) , 2 and 8 gy ( p<0.0001 ) , 4 and 6 gy ( p<0.0001 ) , and 4 and 8 gy ( p<0.001 ) at 24 h post - exposure ( figure 2d ) . the genes bbc3 , xpc , plk3 , rad50 , ddb2 , fdxr , gadd45a , brca1 and ier5 are largely involved in dna repair or cell cycle regulation associated with dna repair . the results show that genes brca1 , xpc and fdxr had no significant change in expression level post - irradiation across all observed dose ranges and post - irradiation time - points . the time- and dose - dependent responses of 6 other genes ( bbc3 , plk3 , ddb2 , gadd45a , rad50 and ier5 ) are shown in figure 3 . the changes in expression levels of these genes were upregulated and downregulated in an oscillating pattern across the observed time - points within certain dose ranges . to determine which genes could potentially characterize exposure doses at different post - irradiation time - points , the results are charted in figure 4 . because the change in cdkn1a expression is substantial , it is not included in figure 4 . the summary of relative expression levels of genes , which are significantly different from the control group , is shown in table 3 . cdkn1a , which has a regulatory role in s phase dna replication and is known to be activated by the tumor protein p53 ( tp 53 ) , had the strongest upregulation in expression level out of the 11 genes investigated in this study . as shown in figure 1a , compared with the non - irradiated control , cdkn1a gene expression gradually increased to a maximum value of approximately 88-fold for 2 gy ( p<0.0001 ) and 130-fold for 4 gy ( p<0.0001 ) at 12 h post - irradiation , and approximately 116-fold for 6 gy ( p<0.0001 ) and 100-fold for 8 gy ( p<0.0001 ) at 8 h post - exposure . then , cdkn1a gene expression gradually decreased but remained greater than 30-fold ( p<0.05 ) higher than that of the control group at 48 h post - irradiation for all exposure doses . figure 2a and b show the significant differences in cdkn1a gene expression induced by the different exposure doses . significant differences can be seen by comparing the exposure groups of 2 and 6 gy ( p<0.05 ) , 4 and 6 gy ( p<0.0001 ) , and 4 and 8 gy ( p<0.001 ) at 8 h post - exposure ( figure 2a ) . at the 12 h post - irradiation time point , atm is a predominantly nuclear protein that is involved in the dna damage repair signal transduction process . in this study , atm gene expression decreased linearly with the exposure dose , with the exception of the 12 h post - irradiation time - point after a 2 gy exposure ( figure 1b ) . compared with non - irradiated controls , it could be determined from the time course of atm gene expression ( figure 1b ) that the gene was strongly downregulated in all exposure dose ranges at 448 h post - irradiation . a significant decrease was found for the 4 gy ( p<0.001 ) and 8 gy ( p<0.05 ) irradiation groups at 4 h post - irradiation ( figure 2c ) , and highly significant changes across the dose range of 28 gy ( p<0.0001 ) were found at 24 h post - irradiation ( figure 2d ) . significant differences can also be seen by comparing the exposure groups of 2 and 4 gy ( p<0.001 ) , 2 and 6 gy ( p<0.0001 ) , 2 and 8 gy ( p<0.0001 ) , 4 and 6 gy ( p<0.0001 ) , and 4 and 8 gy ( p<0.001 ) at 24 h post - exposure ( figure 2d ) . the genes bbc3 , xpc , plk3 , rad50 , ddb2 , fdxr , gadd45a , brca1 and ier5 are largely involved in dna repair or cell cycle regulation associated with dna repair . the results show that genes brca1 , xpc and fdxr had no significant change in expression level post - irradiation across all observed dose ranges and post - irradiation time - points . the time- and dose - dependent responses of 6 other genes ( bbc3 , plk3 , ddb2 , gadd45a , rad50 and ier5 ) are shown in figure 3 . the changes in expression levels of these genes were upregulated and downregulated in an oscillating pattern across the observed time - points within certain dose ranges . to determine which genes could potentially characterize exposure doses at different post - irradiation time - points , the results are charted in figure 4 . because the change in cdkn1a expression is substantial the summary of relative expression levels of genes , which are significantly different from the control group , is shown in table 3 . gene expression analysis following ionizing radiation as a biomarker for individual exposure dose estimation and radiation effect prediction is becoming increasing attention [ 2628 ] . as one of the most radiosensitive targets in mammalian cells , dna damage degree induced by ir was largely dependent on type of radiation ( let ) , dose deposit in the cell , and dose rate . an instinctive protection mechanism in living organisms can repair dna damage induced by ir within certain exposure dose ranges through initiating repair - related gene expression and protein activity . in this process the quantity of repair - related gene expression and protein activating should be proportional to dna damage degree and then be logically linked with exposure dose . therefore the information associated with dna damage repair - related gene expression and protein activity may be seen as a huge data mine from which to derive biological markers for exposure dose evaluation with biodosimetry . in this study significant and meaningful exposure doses and observed post - radiation time ranges in diagnosis and treatment of acute radiation sickness were selected to obtain more complete gene expression profiles following different radiation doses and different post - irradiation times . to enhance accuracy and reliability of the results , radiosensitive mouse species and an accurate irradiation source with well - distributed exposure dose field were selected . most of the target genes observed here were proved prior to this study as radiation response genes , and some of them were shown to have a well - defined dose - effect pattern in the changes of expression level when analysis was done with separated or cultured cells irradiated in vitro or ex vivo . atm , as an important protein in the cellular response to ir , plays a critical role in initiating the repair signaling pathway of dna damage . ir could induce phosphorylation of serine 1981 of atm protein , and phosphorylated atm protein would pass the activating signal to downstream repair - related proteins . it has been reported that atm protein level did not change during the radiation - induced repair process of dna dsbs , but protein kinase activity of atm might be changed following exposure doses when checking was done by a fluorescent foci observation [ 3134 ] . in the present study , the change in transcriptional level of the atm gene was first found to be downregulated in a dose - and post - irradiation time - dependent pattern , with the exception of the 2 gy exposure dose at 12 h post - exposure , the reason for which requires further investigation . this finding implies that the information coming from atm gene expression after radiation can not be neglected as a biomarker in exposure dose indicating . strongly upregulated expression of the cdkn1a gene shown in this study slightly different from previous findings that showed only a several- or 10-fold increase in cdkn1a gene expression after radiation ( from 4-fold to 10- or 50-fold or greater increase ) [ 11,16,3538 ] . it is supposed that the difference of testing mode , primary design of cdkn1a gene composition and application of the primaries might be responsible for the diversity of the results . for example , mitsuhashi et al . reported that multiple primary applications in cdkn1a gene composition showed higher induction of cdkn1a mrna than single primary and control groups when leukocytes from multiple primary breast cancer patients were irradiated . the difference of mouse strain in experiment , exposure and treatment methods of the sample are also factors resulting in diversity of cdkn1a mrna results . the results of the present study show that peaked expression of cdkn1a gene appeared at 12 h post - irradiation for doses of 2 and 4 gy and at 8 h post - irradiation for doses of 6 and 8 gy , which is well in accord with the cell cycle arrest necessity for dna repair after radiation . as an inhibitor of cell cycle process , radiation - induced dna repair needs more cdkn1a protein expression . the heavier dna damage caused by high dose the more cdkn1a protein needed to satisfy the demand of dna repair , which accompanyed with a peak cdkn1a gene expression in the earlier phase of dna repair . combining our results with those of dressman who found only cdkn1a was in common between a pb signature of human tbi and the pb signatures of partial body irradiation exposure , suggesting that the information from cdkn1a gene expression can be a reliable and representative biomarker in estimating exposure dose within certain dose ranges . ddb2 , a damage - specific dna - binding protein 2 that can be activated by uv and x - ray irradiation , had its gene expression level reduced to approximately 20% of the initial expression level with the radiation dose increases in this study . this result is not consistent with previous reports that showed the ddb2 gene was upregulated in both in vivo and in vitro experiments . for example , some results come from just 1 tbi - treated patient and 6 hours to 4 days of observation time , and the study was performed by fractionated irradiation with a low dose and dose rate . in this study exposure dose was delivered once and dose rate was nearly 8-fold higher than mentioned above . because sublethal damage repair and somewhat adaptive response exist during fractionated irradiation by low dose and dose rate , the ddb2 expression level increase was within the range of what one would expect . recently , stoyanova reported that ddb2 could indirectly participate in nucleotide excision repair ( ner ) by regulating the cellular levels of p21 ( cdkn1a ) , and ddb2-deficient cells exhibited a significantly higher accumulation of p21 ( cdkn1a ) . they believed that ddb2 plays a critical role in attenuating the level of p21 to allow apoptosis in response to dna - damaging agents . given their conclusions , our result that cdkn1a was highly upregulated and ddb2 was downregulated show a reasonable but unclear link when radiation response of 2 genes was evaluated . gadd45a has previously been proposed as a potential dosimeter based on the linear dose response relationship observed in human cells that were irradiated in vitro , but the stress - response regulation of this gene was known to be complex . decreasing expression of this gene was observed using wbc and in many p53 wild - type cell lines when cells were irradiated in vitro ; however , other reports have also shown that ionizing radiation did not induce gadd45a expression change in a subset of p53 wild - type cell lines . in our study , gadd45a expression was upregulated approximately 23-fold only at the 48 h post - irradiation time - point within all exposure dose ranges , and no significant changes were found at other time - points . for example , the gene expression and regulation patterns in tbi mice might be more complex compared to the cultured or isolated cells irradiated in vitro or ex vivo . the sensitivity of analytic methods could also result in differences in quantification of gene expression . in hunting for biomarkers capable of indicating individual exposure dose , linear relationship with exposure dose and sensitivity of these markers it is possible that selecting and gathering non - linear change genes together , which induced by different exposure doses and up- or downregulated , at different post - exposure times , form many biomarker groups which cantian many significantly up- or downregulated genes to distinguish and evaluate different threshold doses for medical triage and diagnosis purpose . for example , in table 3 , at 4 h post - irradiation , the gene profiles of atm , xpc and cdkn1a , with statistically significant difference compared with unirradiated control group , can characterize a 4 gy exposure dose , and the information coming from ddb2 , xpc and plk3 gene expression difference can characterize a 6 gy exposure dose , and in the same way the gene expression difference information from atm , bbc3 , xpc and cdkn1a can characterize a 8 gy exposure dose . in the situation where the gene expression profiles are the same in 2 different dose scales , such as 4 and 6gy exposure groups at 8 h post - exposure in table 3 , further gene expression information screening is necessary until some genes are finally found with expression differences between the 2 different doses . then this information on different genes can be selected and grouped based on the time elapsed after radiation exposure to identify and distinguish different exposure doses . additionally , given that atm and cdkn1a gene expression levels were significantly changed across the dose range and time course ( 048 h ) , it may be possible to use these 2 genes as biomarkers to distinguish people exposed to radiation from those who were not exposed , to reassure the worried well and to assign those with significant exposure to the appropriate medical care . on the whole , although gene expression analysis is promising as a biomarker to indicate exposure dose , many problems remain to be solved before the method can be used to evaluate exposure in real - life settings .

summarybackgroundthe aim of this study was to screen molecular biomarkers for biodosimetry from dna repair - related gene expression profiles.material/methodsmice were subjected to whole - body exposure with 60co rays with a dose range of 08 gy at a dose rate of 0.80 gy / min . rna was extracted from the peripheral blood of irradiated mice at 4 , 8 , 12 , 24 and 48hrs post - irradiation . the mrna transcriptional changes of 11 genes related to dna damage and repair were detected using real - time quantitative polymerase chain reaction ( rt - pcr).resultsof the 11 genes examined , cdkn1a ( cyclin - dependent kinase inhibitor 1a or p21 , cip1 ) and atm ( ataxia telangiectasia mutated ) expression levels were found to be heavily up- and down - regulated , respectively , with exposure dose increasing at different post - irradiation times . rad50 ( rad50 homolog ) , plk3 ( polo - like kinase 3 ) , gadd45a ( growth arrest and dna damage - inducible , alpha ) , ddb2 ( damage - specific dna - binding protein 2 ) , bbc3 ( bcl2-binding component 3 ) and ier5 ( immediate early response 5 ) gene expression levels were found to undergo significant oscillating changes over a broad dose range of 28 gy at post - exposure time points observed . three of the genes were found not to change within the observed exposure dose and post - radiation time ranges.conclusionsthe results of this study add to the biodosimetry with biomarker data pool and will be helpful for constructing appropriate gene expression biomarker systems to evaluate radiation exposure doses .

Background Material and Methods Animals and exposure Blood sample collection and total RNA isolation Quantitative RT-PCR Statistical analysis Results Radiation responses of CDKN1A and ATM genes Radiation responses of the other tested genes Discussion Conclusions

rapid and accurate evaluation of exposure dose plays an important role in early triage , diagnosis and medical treatment of the victims during emergency response efforts in nuclear accidents . in view of some of the serious shortcomings and limits presented by physical and chemical methods used in evaluation of radiation exposure dose , biodosimetry has emerged as one of the best techniques for use in individual exposure dose evaluation in many situations such as medical rescue of radiation accident victims , bio - effects research of radiation and radiation protection . because gene expression is sensitive to environmental factors , the analysis of gene expression profiling of peripheral blood cells , particularly lymphocytes , has been used to assess the presence of certain diseases [ 28 ] . accordingly , many radiobiologists have focussed on gene expression profiling analysis to find biomarkers that are suitable for assessment of individual exposure doses under different exposure conditions [ 913 ] . several genes , including gadd45 , cdkn1a and bbc3 , have been identified as radiation response expression genes at different exposure doses and post - irradiation times in human peripheral blood lymphocytes and tissue cells [ 2022 ] . recent reports have shown that gene expression signatures induced by ir are specific , durable and accurate in prediction of exposure doses in both mice and humans ] 2325 ] . since there is a highly relevant among exposure doses , dna damage degree and dna repair - related gene expression level , in this study 11 dna damage response genes related to dna repair ( atm , brca1 and rad50 ) , cell cycle regulation ( cdkn1a , gadd45a ) , and apoptosis ( bbc3 ) ( table 1 ) were selected as targets to screen potential bio - markers for indicating exposure dose . the radiation - induced transcription level of 11 genes was investigated in white blood cells of tbi - treated balb / c mice with a rt - pcr method . male balb / c mice , 68 weeks old , provided by the animal center of the second military medical university were divided into control ( non - irradiated ) and 4 exposure groups ( 5 mice in each group ) . the mice in the exposure groups were exposed to either 2 , 4 , 6 or 8 gy doses of co -rays with tbi at 0.80 gy / min dose rate and room temperature ( 232c ) . they were housed in a pathogen - free barrier facility ( 12-hour light / dark cycle ) and were fed autoclaved standard rodent chow pre- and post - irradiation . for each mouse approximately 0.50.7 ml of heparin - anticoagulated blood was collected from the orbital venous vein using polypropylene tubes at each post - irradiation time point . total rna was extracted from whole blood using the axygen blood total rna miniprep kit , no . the rna was quantified using a nanodrop-1000 spectrophotometer ( thermo scientific , waltham , ma ) , and the quality of the isolated rna was assayed with agarose gel electrophoresis by analyzing 18s and 28s rna . drr037a ( takara , dalian , china ) , 1 g of total rna was reversely transcribed to cdna according to the manufacturer s instructions . the statistical significance of differences between groups was analyzed by one - way analysis of variance with a post - hoc tukey test . male balb / c mice , 68 weeks old , provided by the animal center of the second military medical university were divided into control ( non - irradiated ) and 4 exposure groups ( 5 mice in each group ) . the mice in the exposure groups were exposed to either 2 , 4 , 6 or 8 gy doses of co -rays with tbi at 0.80 gy / min dose rate and room temperature ( 232c ) . they were housed in a pathogen - free barrier facility ( 12-hour light / dark cycle ) and were fed autoclaved standard rodent chow pre- and post - irradiation . for each mouse approximately 0.50.7 ml of heparin - anticoagulated blood was collected from the orbital venous vein using polypropylene tubes at each post - irradiation time point . total rna was extracted from whole blood using the axygen blood total rna miniprep kit , no . the rna was quantified using a nanodrop-1000 spectrophotometer ( thermo scientific , waltham , ma ) , and the quality of the isolated rna was assayed with agarose gel electrophoresis by analyzing 18s and 28s rna . drr037a ( takara , dalian , china ) , 1 g of total rna was reversely transcribed to cdna according to the manufacturer s instructions . the rt - pcr cycling conditions were as follows : initial denaturation at 95c for 10 min , followed by 35 cycles of denaturation at 95c for 10 s , annealing at 60c for 30 s and primer extension at 72c for 30 s. the final extension occurred at 72c for 10 min . the statistical significance of differences between groups was analyzed by one - way analysis of variance with a post - hoc tukey test . cdkn1a , which has a regulatory role in s phase dna replication and is known to be activated by the tumor protein p53 ( tp 53 ) , had the strongest upregulation in expression level out of the 11 genes investigated in this study . as shown in figure 1a , compared with the non - irradiated control , cdkn1a gene expression gradually increased to a maximum value of approximately 88-fold for 2 gy ( p<0.0001 ) and 130-fold for 4 gy ( p<0.0001 ) at 12 h post - irradiation , and approximately 116-fold for 6 gy ( p<0.0001 ) and 100-fold for 8 gy ( p<0.0001 ) at 8 h post - exposure . then , cdkn1a gene expression gradually decreased but remained greater than 30-fold ( p<0.05 ) higher than that of the control group at 48 h post - irradiation for all exposure doses . figure 2a and b show the significant differences in cdkn1a gene expression induced by the different exposure doses . significant differences can be seen by comparing the exposure groups of 2 and 6 gy ( p<0.05 ) , 4 and 6 gy ( p<0.0001 ) , and 4 and 8 gy ( p<0.001 ) at 8 h post - exposure ( figure 2a ) . at the 12 h post - irradiation time point , atm is a predominantly nuclear protein that is involved in the dna damage repair signal transduction process . in this study , atm gene expression decreased linearly with the exposure dose , with the exception of the 12 h post - irradiation time - point after a 2 gy exposure ( figure 1b ) . compared with non - irradiated controls , it could be determined from the time course of atm gene expression ( figure 1b ) that the gene was strongly downregulated in all exposure dose ranges at 448 h post - irradiation . a significant decrease was found for the 4 gy ( p<0.001 ) and 8 gy ( p<0.05 ) irradiation groups at 4 h post - irradiation ( figure 2c ) , and highly significant changes across the dose range of 28 gy ( p<0.0001 ) were found at 24 h post - irradiation ( figure 2d ) . significant differences can also be seen by comparing the exposure groups of 2 and 4 gy ( p<0.001 ) , 2 and 6 gy ( p<0.0001 ) , 2 and 8 gy ( p<0.0001 ) , 4 and 6 gy ( p<0.0001 ) , and 4 and 8 gy ( p<0.001 ) at 24 h post - exposure ( figure 2d ) . the genes bbc3 , xpc , plk3 , rad50 , ddb2 , fdxr , gadd45a , brca1 and ier5 are largely involved in dna repair or cell cycle regulation associated with dna repair . the results show that genes brca1 , xpc and fdxr had no significant change in expression level post - irradiation across all observed dose ranges and post - irradiation time - points . the time- and dose - dependent responses of 6 other genes ( bbc3 , plk3 , ddb2 , gadd45a , rad50 and ier5 ) are shown in figure 3 . the changes in expression levels of these genes were upregulated and downregulated in an oscillating pattern across the observed time - points within certain dose ranges . to determine which genes could potentially characterize exposure doses at different post - irradiation time - points , the results are charted in figure 4 . the summary of relative expression levels of genes , which are significantly different from the control group , is shown in table 3 . cdkn1a , which has a regulatory role in s phase dna replication and is known to be activated by the tumor protein p53 ( tp 53 ) , had the strongest upregulation in expression level out of the 11 genes investigated in this study . as shown in figure 1a , compared with the non - irradiated control , cdkn1a gene expression gradually increased to a maximum value of approximately 88-fold for 2 gy ( p<0.0001 ) and 130-fold for 4 gy ( p<0.0001 ) at 12 h post - irradiation , and approximately 116-fold for 6 gy ( p<0.0001 ) and 100-fold for 8 gy ( p<0.0001 ) at 8 h post - exposure . then , cdkn1a gene expression gradually decreased but remained greater than 30-fold ( p<0.05 ) higher than that of the control group at 48 h post - irradiation for all exposure doses . figure 2a and b show the significant differences in cdkn1a gene expression induced by the different exposure doses . significant differences can be seen by comparing the exposure groups of 2 and 6 gy ( p<0.05 ) , 4 and 6 gy ( p<0.0001 ) , and 4 and 8 gy ( p<0.001 ) at 8 h post - exposure ( figure 2a ) . at the 12 h post - irradiation time point , atm is a predominantly nuclear protein that is involved in the dna damage repair signal transduction process . in this study , atm gene expression decreased linearly with the exposure dose , with the exception of the 12 h post - irradiation time - point after a 2 gy exposure ( figure 1b ) . compared with non - irradiated controls , it could be determined from the time course of atm gene expression ( figure 1b ) that the gene was strongly downregulated in all exposure dose ranges at 448 h post - irradiation . a significant decrease was found for the 4 gy ( p<0.001 ) and 8 gy ( p<0.05 ) irradiation groups at 4 h post - irradiation ( figure 2c ) , and highly significant changes across the dose range of 28 gy ( p<0.0001 ) were found at 24 h post - irradiation ( figure 2d ) . significant differences can also be seen by comparing the exposure groups of 2 and 4 gy ( p<0.001 ) , 2 and 6 gy ( p<0.0001 ) , 2 and 8 gy ( p<0.0001 ) , 4 and 6 gy ( p<0.0001 ) , and 4 and 8 gy ( p<0.001 ) at 24 h post - exposure ( figure 2d ) . the genes bbc3 , xpc , plk3 , rad50 , ddb2 , fdxr , gadd45a , brca1 and ier5 are largely involved in dna repair or cell cycle regulation associated with dna repair . the results show that genes brca1 , xpc and fdxr had no significant change in expression level post - irradiation across all observed dose ranges and post - irradiation time - points . the time- and dose - dependent responses of 6 other genes ( bbc3 , plk3 , ddb2 , gadd45a , rad50 and ier5 ) are shown in figure 3 . the changes in expression levels of these genes were upregulated and downregulated in an oscillating pattern across the observed time - points within certain dose ranges . to determine which genes could potentially characterize exposure doses at different post - irradiation time - points , the results are charted in figure 4 . because the change in cdkn1a expression is substantial the summary of relative expression levels of genes , which are significantly different from the control group , is shown in table 3 . gene expression analysis following ionizing radiation as a biomarker for individual exposure dose estimation and radiation effect prediction is becoming increasing attention [ 2628 ] . as one of the most radiosensitive targets in mammalian cells , dna damage degree induced by ir was largely dependent on type of radiation ( let ) , dose deposit in the cell , and dose rate . an instinctive protection mechanism in living organisms can repair dna damage induced by ir within certain exposure dose ranges through initiating repair - related gene expression and protein activity . in this process the quantity of repair - related gene expression and protein activating should be proportional to dna damage degree and then be logically linked with exposure dose . therefore the information associated with dna damage repair - related gene expression and protein activity may be seen as a huge data mine from which to derive biological markers for exposure dose evaluation with biodosimetry . in this study significant and meaningful exposure doses and observed post - radiation time ranges in diagnosis and treatment of acute radiation sickness were selected to obtain more complete gene expression profiles following different radiation doses and different post - irradiation times . to enhance accuracy and reliability of the results , radiosensitive mouse species and an accurate irradiation source with well - distributed exposure dose field were selected . most of the target genes observed here were proved prior to this study as radiation response genes , and some of them were shown to have a well - defined dose - effect pattern in the changes of expression level when analysis was done with separated or cultured cells irradiated in vitro or ex vivo . ir could induce phosphorylation of serine 1981 of atm protein , and phosphorylated atm protein would pass the activating signal to downstream repair - related proteins . it has been reported that atm protein level did not change during the radiation - induced repair process of dna dsbs , but protein kinase activity of atm might be changed following exposure doses when checking was done by a fluorescent foci observation [ 3134 ] . in the present study , the change in transcriptional level of the atm gene was first found to be downregulated in a dose - and post - irradiation time - dependent pattern , with the exception of the 2 gy exposure dose at 12 h post - exposure , the reason for which requires further investigation . this finding implies that the information coming from atm gene expression after radiation can not be neglected as a biomarker in exposure dose indicating . strongly upregulated expression of the cdkn1a gene shown in this study slightly different from previous findings that showed only a several- or 10-fold increase in cdkn1a gene expression after radiation ( from 4-fold to 10- or 50-fold or greater increase ) [ 11,16,3538 ] . the difference of mouse strain in experiment , exposure and treatment methods of the sample are also factors resulting in diversity of cdkn1a mrna results . the results of the present study show that peaked expression of cdkn1a gene appeared at 12 h post - irradiation for doses of 2 and 4 gy and at 8 h post - irradiation for doses of 6 and 8 gy , which is well in accord with the cell cycle arrest necessity for dna repair after radiation . the heavier dna damage caused by high dose the more cdkn1a protein needed to satisfy the demand of dna repair , which accompanyed with a peak cdkn1a gene expression in the earlier phase of dna repair . combining our results with those of dressman who found only cdkn1a was in common between a pb signature of human tbi and the pb signatures of partial body irradiation exposure , suggesting that the information from cdkn1a gene expression can be a reliable and representative biomarker in estimating exposure dose within certain dose ranges . ddb2 , a damage - specific dna - binding protein 2 that can be activated by uv and x - ray irradiation , had its gene expression level reduced to approximately 20% of the initial expression level with the radiation dose increases in this study . for example , some results come from just 1 tbi - treated patient and 6 hours to 4 days of observation time , and the study was performed by fractionated irradiation with a low dose and dose rate . in this study exposure dose was delivered once and dose rate was nearly 8-fold higher than mentioned above . because sublethal damage repair and somewhat adaptive response exist during fractionated irradiation by low dose and dose rate , the ddb2 expression level increase was within the range of what one would expect . they believed that ddb2 plays a critical role in attenuating the level of p21 to allow apoptosis in response to dna - damaging agents . gadd45a has previously been proposed as a potential dosimeter based on the linear dose response relationship observed in human cells that were irradiated in vitro , but the stress - response regulation of this gene was known to be complex . in our study , gadd45a expression was upregulated approximately 23-fold only at the 48 h post - irradiation time - point within all exposure dose ranges , and no significant changes were found at other time - points . for example , the gene expression and regulation patterns in tbi mice might be more complex compared to the cultured or isolated cells irradiated in vitro or ex vivo . the sensitivity of analytic methods could also result in differences in quantification of gene expression . in hunting for biomarkers capable of indicating individual exposure dose , linear relationship with exposure dose and sensitivity of these markers it is possible that selecting and gathering non - linear change genes together , which induced by different exposure doses and up- or downregulated , at different post - exposure times , form many biomarker groups which cantian many significantly up- or downregulated genes to distinguish and evaluate different threshold doses for medical triage and diagnosis purpose . for example , in table 3 , at 4 h post - irradiation , the gene profiles of atm , xpc and cdkn1a , with statistically significant difference compared with unirradiated control group , can characterize a 4 gy exposure dose , and the information coming from ddb2 , xpc and plk3 gene expression difference can characterize a 6 gy exposure dose , and in the same way the gene expression difference information from atm , bbc3 , xpc and cdkn1a can characterize a 8 gy exposure dose . in the situation where the gene expression profiles are the same in 2 different dose scales , such as 4 and 6gy exposure groups at 8 h post - exposure in table 3 , further gene expression information screening is necessary until some genes are finally found with expression differences between the 2 different doses . then this information on different genes can be selected and grouped based on the time elapsed after radiation exposure to identify and distinguish different exposure doses . additionally , given that atm and cdkn1a gene expression levels were significantly changed across the dose range and time course ( 048 h ) , it may be possible to use these 2 genes as biomarkers to distinguish people exposed to radiation from those who were not exposed , to reassure the worried well and to assign those with significant exposure to the appropriate medical care . on the whole , although gene expression analysis is promising as a biomarker to indicate exposure dose , many problems remain to be solved before the method can be used to evaluate exposure in real - life settings .

graphene and other two - dimensional ( 2d ) materials have been touted as promising ultrathin passivation coatings due to their extremely low permeability to gases and their ultimate thinness , offering the prospect of preserving the physical properties of surfaces with only a single atomic layer separating them from their surroundings . standard exfoliation and transfer - based techniques for producing 2d materials are not well - suited to depositing such passivating layers , where a complete , conformal coating is typically desired to prevent ingress of oxidizing species . on the other hand , catalytic growth techniques such as chemical vapor deposition ( cvd ) offer direct routes for reducing a catalyst surface and forming a uniform , conformal 2d material layer that passivates the surface enabling simplified integration into devices . indeed , cvd of graphene onto ni electrodes has been shown to prevent their oxidation during week - long exposures to atmosphere , with functional tunneling spin valve devices successfully fabricated using these graphene - passivated electrodes . however , other studies investigating graphene grown on cu foils , as well as graphene transferred onto si surfaces , have highlighted that the expected passivation is not necessarily achieved and may not be maintained over the long term . despite the huge progress in improving the quality and uniformity of cvd graphene , atomic defects still remain , both within graphene domains and at the boundaries where they join , which provide pathways for the permeation of oxidizing species . for single - layer graphene ( slg ) on cu , previous observations have revealed that , on atmospheric air exposure , oxidizing species are thereby able to intercalate between the slg and cu and thus access the whole cu surface . under these atmospheric conditions , where humidity is sufficient for condensed water vapor to serve as an electrolyte on the surface of the graphene therefore , while the reduced permeation of oxidizing species through the slg slows these processes over the short term , cu oxidation still proceeds over time and is even observed to be electrochemically enhanced in the long term due to the galvanic couple formed by the slg cu . recent progress has been made in reducing the graphene permeability by stacking multiple layers or selectively blocking defects with post - treatments , yet these approaches yield barriers significantly thicker than slg and the reported permeabilities are not sufficiently low to prevent surface oxidation over the long term ( i.e. , years ) , casting doubt on the level of passivation that can be achieved with atomically thin 2d materials . here we form continuous slg coatings on various polycrystalline transition metal catalysts ( ni , co , fe , pt ) by cvd , and using in situ , depth - resolved x - ray photoelectron spectroscopy ( xps ) we investigate the extent of oxidation following exposures to atmospheric ( moist ) air at room temperature for time frames ranging from several minutes up to many months . we thereby demonstrate that slg can effectively passivate polycrystalline ni surfaces and maintain a fully reduced ni surface even following exposure to atmospheric conditions for more than 18 months . our data reveal that of key importance to achieving this long - term passivation is a strong graphene substrate interaction , which prevents the lateral diffusion of oxidizing species along the graphene substrate interface . this enables the long - term passivation of the covered regions of a substrate that interacts strongly with graphene ( e.g. , ni , co ) , even in cases where the graphene does not form a complete , conformal coating . this is in stark contrast to metals that exhibit a weak interaction ( e.g. , cu , pt ) , where even short air exposures are sufficient to decouple the graphene from the catalyst by the intercalation of oxidizing species at edges or defects , opening a pathway by which oxidizing species can access the whole catalyst surface . we further show that the ability of the substrate to locally form a passivating oxide at defective or damaged regions in the graphene overlayer is critical in eliminating an alternative route by which oxidation of the substrate can proceed over the longer term . for catalysts that do not form such a passivating oxide ( e.g. , fe in moist air ) , while a strong graphene substrate interaction can suppress surface oxidation in the short term , for long - term exposures to atmospheric air , oxidation can proceed through the oxide layers that form close to graphene defects until eventually the metal becomes oxidized throughout . these observations reveal that the interaction between a 2d material and the underlying substrate plays a significant role in determining its performance as a passivation layer and highlight that it is the combination of 2d material and substrate that is key to preventing oxidation . we thereby rationalize apparent inconsistencies in the literature regarding the extent to which graphene passivates different substrates and highlight the key advantages offered by direct integration techniques , such as cvd , that can inherently establish a strong graphene substrate interaction . graphene is formed by cvd on polycrystalline transition metal foils ( co , fe , ni , pt ) and a ni(111 ) single crystal or by vacuum annealing of a pt(111 ) single crystal ( see methods ) , based on our extensive previous calibrations . samples are then exposed to atmospheric ( moist ) air at room temperature from times ranging from several minutes to more than 18 months , where we note that condensed water vapor may serve as an electrolyte to facilitate wet corrosion . figure 1 compares the depth - resolved xp ni 2p3/2 core level spectra of polycrystalline ni foils with different extents of graphene coverage and lengths of exposure to atmospheric conditions . depth resolution is achieved by varying the incident photon energy , ephoton , which in turn varies the kinetic energy of the photoelectrons and thus their mean escape depth , escape . the spectra for a bare ni ( 25 m ) foil that has been annealed [ 600 c , h2 ( 10 mbar ) for 15 min ] and kept under vacuum conditions ( figure 1a ) correspond to metallic ni with a dominant peak at 852.6 ev ( nim ) , confirming that the foil is fully reduced across the depths probed . following exposure to atmosphere for just 5 min ( figure 1b ) , strong ni oxide and hydroxide peaks ( niox ) appear in the most surface - sensitive spectrum ( escape 7 ) , indicating that the ni surface has become heavily oxidized . probing deeper into the sample ( escape 911 ) reveals a lower extent of oxidation , as seen from the decrease in the intensities of the niox peaks relative to the nim peaks , consistent with the rapid formation of an oxide layer on atmospheric exposure , that passivates the surface and limits oxidation from proceeding throughout the ni bulk . conversely , for a ni ( 25 m ) foil covered with a complete slg film ( figure 1c ) , the xp ni 2p3/2 core level spectra remain very similar to those measured for the fully reduced ni foil ( figure 1a ) , even following 18 months in air . this confirms the slg - covered ni surface remains reduced during extended exposure to atmospheric air and , therefore , that long - term passivation can be achieved with slg on ni surfaces . figure 1d shows that , even for a ni ( 250 m ) foil covered with noncontinuous slg islands , the surface remains largely reduced with only small niox contributions visible following 18 months of air exposure . the extent of the oxidation correlates with the area of uncovered ni , as determined by scanning electron microscopy ( figure s1 ) , indicating that only these regions are oxidized while the areas beneath the slg remain reduced . it should be noted that the absolute intensity of the ni 2p3/2 signals is weakened by graphene coverage , as apparent from the increased signal - to - noise ratio in figure 1c , d . depth - resolved xp ni 2p3/2 core level spectra for polycrystalline ni ( 25 m ) in situ immediately following annealing [ 600 c , h2 ( 10 mbar ) for 15 min ] ( a ) and after subsequent exposure to atmosphere for < 5 min ( b ) , as well as for ni ( 25 m ) covered with a complete slg layer ( c ) , and ni ( 250 m ) covered with noncontinuous slg islands ( d ) following exposure to atmosphere for > 18 months . the slg was grown by cvd [ 600 c , c6h6 ( 10 mbar ) for 15 min ] . spectra are collected at photon energies , ephoton , of 1010 , 1150 , 1300 , and 1450 ev [ from upper to lower spectra , respectively , escape 7 , 9 , 10 , and 11 ] and are normalized to have the same maximum intensity . fitted components for metallic ni ( nim ) and ni oxide / hydroxide ( niox ) are shaded blue and red , respectively . this long - term passivation behavior of graphene on ni is in contrast to that reported for graphene on cu , where long - term passivation under atmospheric conditions is not achieved , and the presence of graphene is even found to electrochemically enhance the oxidation of cu by the formation of a galvanic couple . it has been proposed that such disparities in graphene passivation behavior may relate to differences in the defect densities of the graphene formed on these different metals . however , comparison of slg grown on ni using the conditions herein and slg grown on cu that subsequently oxidizes in air reveals very similar defect densities as determined by raman spectroscopy ( following transfer to sio2 ( 300 nm)/si substrates ) . a significant difference between these two systems , however , does lie in the strength of the metal graphene interaction . we therefore draw a distinction between strongly interacting metals , such as ni , co , fe , ru , rh , and pd , where the hybridization between the graphene and metal d valence band states destroys the characteristic linear band dispersion of graphene at the k point , and weakly interacting metals , such as cu , ag , ir , pt , and au , where this linear dispersion is preserved but charge transfer between the metal and graphene ( i.e. , doping ) typically shifts the fermi level position of the graphene . dahal and batzill quantify this distinction in terms of the energy of the metal d band center with respect to the fermi level , with the transition between weakly and strongly interacting metal suggested to occur at 2 ev . we note that this refers to graphene on idealized low - index , single - crystal surfaces and that the situation for polycrystalline surfaces is more complex , with possible variations in the graphene figure 2 compares the effect of atmospheric exposures on slg grown on prototypical strongly interacting ni(111 ) and more weakly interacting pt(111 ) . the spectra are consistently fitted ( see methods ) based on our previous xps investigations of graphene on ni and other catalysts . the spectral resolution of 0.3 ev allows the relatively small shifts in binding energy associated with changes in graphene the cvd condition used on ni(111 ) [ 400 c , c2h4 ( 10 mbar ) for 2 h ] results in epitaxial slg formation by the transformation of an intermediate ni2c surface carbide.figure 2a shows the resulting xp c 1s spectrum following growth , which has a majority component at 284.8 ev corresponding to epitaxial slg , whose higher binding energy compared to isolated graphene ( 284.4 ev ) relates to the alteration of the band structure by the strong interaction with ni(111 ) . very minor contributions are also observed at 284.4 and 283.2 ev , which correspond to small amounts of rotated slg and residual ni2c , respectively . we note that , under such low - temperature growth conditions , numerous defects are included within the graphene lattice as previously confirmed by scanning tunneling microscopy.figure 2a also shows the c 1s spectrum for epitaxial slg on ni(111 ) following exposure to atmosphere for 5 days . significantly , the dominant component remains at 284.8 ev , with the absence of a shift in the peak position during air exposure confirming that the strong slg ni(111 ) coupling is maintained , in spite of the presence of defects in the slg . a shift toward the binding energy of isolated graphene ( 284.4 ev ) would be expected if the graphene catalyst interaction is weakened , as seen for rotated slg , additional graphene layers , or for epitaxial slg on ni(111 ) intercalated with au . following air exposure there is some broadening of the fitted components , consistent with the accumulation of atmospheric contaminants ( e.g. , hydrocarbons , oxygen ) . the ca component also disappears , indicating that residual ni2c is unstable under atmospheric conditions and likely oxidizes . furthermore , given that no bulk carbidic phases are found to be stabilized under these growth conditions on ni , the passivation we observe is attributed to the presence of slg rather than any surface or bulk carbidic phase . ( a ) xp c 1s core level lines of ni(111 ) covered with slg grown by cvd [ 400 c , c2h4 ( 10 mbar ) for 2 h ] in situ immediately following growth ( lower ) and after exposure to atmosphere for 5 days ( upper ) . ( b ) xp c 1s core level lines of pt(111 ) covered with slg grown by vacuum annealing [ 10 mbar , 1000 c , for 2 h ] in situ immediately following growth ( lower ) and after exposure to atmosphere for 5 min ( middle ) and 2 days ( upper ) . all spectra are collected at photon energy , ephoton , of 425 ev ( escape 7 ) and are normalized to have the same maximum intensity . ( c ) sem micrographs of slg island on polycrystalline pt ( 25 m ) after exposure to atmosphere for 5 min ( lower ) and 1 day ( upper ) , with inset schematic indicating the coupled ( purple ) and decoupled ( blue ) regions . figure 2b shows xp c 1s spectra measured for slg grown on a pt(111 ) surface by the diffusion of adventitious carbon from within the bulk of the crystal during vacuum annealing [ 10 mbar , 1000 c , for 2 h ] . the dominant component immediately following growth is at 284.0 ev , which corresponds to slg coupled to the pt surface , with the peak position shifted to lower binding energy than isolated graphene due to the p - type charge transfer doping by the higher work function pt(111 ) . on exposure to atmosphere for only short times ( 5 min ) , additional peaks begin to appear with the strongest at 284.4 ev , which grows in intensity at the expense of the 284.0 ev peak for longer exposures ( 2 days ) . this shift in peak position toward that of free - standing , undoped graphene is attributed to the weakening of the slg pt coupling by the gradual intercalation of oxidizing species at the slg pt interface , leading to the decoupling of the slg and loss of the charge transfer , i.e. , loss of p - type doping . the other more minor peaks are tentatively attributed to carbidic species ( 283.6 ev ) and reconstruction of platinum regions beneath the slg ( 285 ev ) . this decoupling behavior is also apparent in scanning electron microscopy ( sem ) images ( secondary electron , in - lens detector ) of an isolated slg island on polycrystalline pt ( figure 2c ) , where the brighter decoupled region near the perimeter ( lower image , 5 min air exposure ) proceeds inward with continuing air exposure until the whole slg island is decoupled ( upper image , 1 day air exposure ) . the reduction in secondary electron yield for the coupled slg region is attributed to the higher work function of the slg when it is p - doped . the shorter time scale for complete decoupling of this isolated slg island is ascribed to the shorter lateral distances over which intercalants must diffuse in comparison to the continuous slg film of figure 2b . we note that a similar decoupling behavior on air exposure is observed for graphene on other weakly interacting catalysts such as cu , albeit occurring over even shorter time scales , and with coupled regions showing brighter secondary electron yield than decoupled regions . this increased secondary electron yield can again be explained by alteration of the coupled graphene s work function , which in this case is expected to be lowered due to n - type charge transfer doping by cu . having established that with ni a strong graphene metal interaction protects against rapid surface oxidation by preventing intercalation of oxidizing species at the graphene metal interface , we now consider the performance of graphene in protecting other transition metals that interact strongly with graphene , namely , co ( figure 3 ) and fe ( figure 4 ) . figure 3a shows co 2p3/2 core level spectra for bare co ( 25 m ) after annealing [ h2 ( 1 mbar ) at 600 c for 15 min ] , which indicate that the co is fully reduced with a dominant metallic peak at 778.2 ev ( com ) , for both of the depths probed ( escape 8 and 11 ) . following air exposure of the bare co surface for 5 min ( figure 3b ) , peaks related to co oxides / hydroxides with binding energies above 780 ev ( coox ) dominate the most surface - sensitive co 2p3/2 spectrum ( escape 8 ) . they are also readily apparent in the more bulk - sensitive spectra ( escape 11 ) , but the com peak remains the most intense component , indicating a lower extent of oxidation . this closely parallels the oxide formation on ni and is again consistent with previous results showing the rapid formation of an oxide layer on exposure to atmospheric air that passivates the surface and slows further oxidation of the metal bulk.figure 3c reveals that , for slg - covered co ( 25 m ) exposed to air for > 6 months , the co 2p3/2 spectra are very similar to those of reduced co ( figure 3a ) . importantly , the absence of any significant coox peaks confirms that oxidation of the co surface is very limited and that the slg - covered co is maintained in a reduced state . we note that no significant carbidic phases are apparent in the co 2p3/2 spectra or corresponding c 1s spectra ( not shown ) and the passivation is thus attributed to the presence of slg . this behavior observed for co is qualitatively very similar to that for ni , with almost no oxidation of the slg - covered co evident after > 6 months , while the bare co forms a passivating oxide layer across its surface . depth - resolved xp co 2p3/2 core level spectra for polycrystalline co ( 25 m ) in situ immediately following annealing [ 600 c , h2 ( 10 mbar ) for 15 min ] ( a ) and after subsequent exposure to atmosphere for < 5 min ( b ) ; and for co ( 25 m ) covered with a complete slg layer grown by cvd [ 700 c , c2h2 ( 10 mbar ) for 15 min followed by c2h2 ( 10 mbar ) for 5 min ] following exposure to atmosphere for > 6 months . spectra are collected at photon energies , ephoton , of 1020 ( upper ) and 1400 ev ( lower ) [ respectively , escape 8 and 11 ] and are normalized to have the same maximum intensity . depth - resolved xp fe 2p3/2 core level spectra for polycrystalline fe ( 100 m ) in situ immediately following annealing [ 1000 c , h2 ( 10 mbar ) for 15 min ] ( a ) and after subsequent exposure to atmosphere for 1 h ( b ) ; and for fe ( 100 m ) covered with a complete slg layer grown by cvd [ 650 c , c2h2 ( 10 mbar ) for 30 min ] following exposure to atmosphere for 1 week ( c ) and > 6 months ( d ) . spectra are collected at photon energies , ephoton , of 920 ( upper ) and 1150 ev ( lower ) [ respectively , escape 8 and 10 ] and are normalized to have the same maximum intensity . parts a and b of figure 4 show fe 2p3/2 spectra for an annealed [ h2 ( 1 mbar ) at 900 c for 15 min ] fe foil , before and after exposure to atmosphere for 1 h , respectively . initially ( figure 4a ) a dominant peak at 706.7 ev ( fem ) is apparent for both excitation energies used ( corresponding to escape 8 and 10 ) , consistent with the fe being fully reduced . following the short atmospheric exposures , components related to fe oxides / oxyhydroxides appear around 711 ev ( feox ) , which are dominant across the depths probed , with only a very weak fem component remaining . this significant oxidation of the fe that is observed even in the more depth - sensitive spectrum is in contrast to the behavior of the bare co and ni films where the initial rapid formation of a thin oxide layer passivates the surface , limiting further oxidation of the metal bulk . this is , however , consistent with the well - established and often experienced behavior of fe in moist air where a hydrated oxide forms as a loose deposit that provides little or no passivation of the fe surface and allows oxidation to proceed throughout the bulk , in contrast to cases of purely dry or purely wet fe oxidation . figure 4c shows that , for slg - covered fe exposed to air for 5 days , while some weak feox contributions are detectable in the most surface - sensitive spectrum ( escape 8 ) , the spectra remain largely similar to those of the reduced fe ( figure 4a ) , with fem remaining by far the dominant component . however , following a longer , > 6 month air exposure ( figure 4d ) , the fe 2p3/2 spectra now more closely resemble the air - exposed bare fe foil ( figure 4b ) , showing only feox components with no detectable fem contribution . this highlights that , while the slg coverage can protect the fe from oxidation for air exposures of a few weeks , for longer - term exposure the underlying fe foil gradually oxidizes and eventually no reduced fe remains close to the surface . this same general behavior is confirmed for fe covered with thicker few - layer graphene ( flg ) films ; however , a small fem peak is still detectible in the xp spectra even after > 18 months ( see supporting information , figure s3 ) . this reveals that the fe oxidation occurs more slowly , with some fe still preserved in a metallic state , presumably as a result of the reduced permeation of oxidizing species through the thicker flg film . no significant carbidic phases are observed in the fe 2p3/2 spectra or corresponding c 1s spectra ( not shown ) of the samples measured , and thus their involvement in the observed oxidation behavior can be largely excluded . figure 5 illustrates the general model for 2d material passivation that is developed herein by investigating graphene passivation on different substrates during atmospheric air exposures . first the presence of the 2d material provides a low - permeability barrier that limits the access of oxidizing species to the substrate below , with thicker layers having even lower permeability . however , this low permeabilty alone does not typically afford long - term passivation , as intrinsic defects such as grain boundaries and atomic vacancies in the graphene still allow oxidizing species to reach the substrate close to these defects . for weakly interacting metals ( e.g. , cu , pt ) , the graphene is rapidly decoupled from the surface on air exposure by the intercalation of oxidizing species , allowing ready access to the whole metal surface and thus its rapid oxidation . for strongly interacting metals ( e.g. , ni , co , fe ) however , the graphene remains coupled on air exposure and the oxidizing species are thus only able to access the metal surface close to graphene defects , suppressing oxidation of the surface over the short term . for metals that form a passivating oxide ( e.g. , ni , co ) , these exposed regions near to defects are quickly plugged by oxide formation , and the majority of the metal surface is thereby protected from oxidation over the long term . in the case of metals whose oxide is not passivating ( e.g. , fe in moist air ) , while the strong graphene metal interaction provides short - term protection of the surface , over the longer term , oxidation can proceed through the oxide layers initially formed close to graphene defects until eventually the metal becomes oxidized throughout . graphene is easily decoupled from the surface of weakly interacting metals ( e.g. , cu , pt ) on air exposure , providing a pathway for the intercalation of oxidizing species at the graphene catalyst interface and ready access for these oxidizing species to the whole metal surface . for strongly interacting metals ( e.g. , ni , co , fe ) , graphene is not decoupled on air exposure , and the oxidizing species are thus only able to access the metal surface close to graphene defects . for metals that form a passivating oxide ( e.g. , ni , co ) , these exposed regions near to defects are quickly plugged by oxide formation , protecting the substrate from oxidation over the long term . for metals whose oxide is not passivating ( e.g. , fe ) , oxidation is initially slowed by the already formed oxide , and thus the graphene coverage provides short - term passivation . however , oxidation can proceed through the already formed oxide , eventually allowing the metal to become oxidized throughout for long - term air exposures . we emphasize that , although a passivating oxide is key to the observed long - term passivation of graphene - covered strongly interacting metals , a passivating oxide alone does not afford equivalent protection . the surfaces of bare ni and co are heavily oxidized within minutes of atmospheric exposure , whereas with graphene present negligible oxidation of the surface occurs for atmospheric exposures of several months or even years , time scales of > 5 orders of magnitude longer . this suppression of surface oxidation is important for various applications where even limited surface oxidation of metals can severely undermine performance , including ferromagnetic spin injectors , bipolar plates for polymer electrolyte membrane fuel cells , and non - noble plasmonic materials . our model is consistent with various reports in literature of the effective passivation of metals that strongly interact with graphene such as ni and ru in atmospheric air and fe in dry o2 . more weakly interacting catalysts covered with graphene , most typically cu but also ir , are reported to show oxygen intercalation even for relatively modest air exposures , and in the case of cu , surface oxidation is observed within hours of exposure to atmosphere . more direct comparisons of the gaseous oxidation of strongly and weakly interacting metals covered with 2d materials formed by cvd are scarcely reported in the literature ; however , the behavior observed during electrochemical corrosion studies of different graphene - covered metals in aqueous solutions appears consistent with the model developed herein . anodic reactions are found to be strongly suppressed for graphene - covered ni , which has a strong graphene in contrast only a very minor reduction in anodic reaction rate is observed for graphene - covered cu , whose weak graphene while our focus herein has been on passivation with 2d materials at room temperature in atmospheric air , we note that , under the more extreme conditions of full immersion in a liquid environment , the formation of a stable passivating oxide is expected to be particularly important in suppressing wet ( i.e. , electrochemical ) corrosion . under more aggressive chemical environments or at elevated temperatures , further factors may need to be taken into account when applying the graphene passivation framework that we have developed . for example , the oxidizing species may be more readily able to intercalate beneath 2d materials even when a strong interaction with the substrate exists and/or passivating oxides formed at room temperature may no longer be stable . furthermore , depending on the nature of the substrate , it can serve as a catalyst to accelerate the breakdown of the 2d material layer under such conditions . indeed the etching of h - bn on cu in the presence of oxygen has been observed at temperatures well below those at which isolated h - bn starts to degrade . therefore , careful consideration of the compatibility of the 2d material and substrate under the specific operating conditions is required , and further experimental verification of the passivation that can be achieved under such conditions may be needed . a number of previous publications have compared the passivation performance of graphene grown directly on a substrate with that of graphene transferred onto a substrate , with the latter typically giving much poorer results . this can be understood in the context of the model developed herein , given that a strong interaction with the substrate is not expected to result from typical 2d material - transfer techniques . this also applies to passivation barriers based on percolation of liquid - phase - exfoliated platelets , and thus in both of these cases any potential strong interaction with the substrate that could improve the barrier s performance may need to be activated by , for example , postdeposition thermal annealing . this highlights a key advantage of catalytic deposition techniques such as cvd , in that the establishment of an interaction between the catalyst and 2d material is integral to the growth process , allowing the direct integration of 2d materials into device structures to provide long - term passivation . we further note that , while the permeability of 2d material passivation layers can be improved by decorating defects using atomic layer deposition ( ald ) , this does not strengthen the graphene substrate interaction and thus does not benefit from the synergy between the substrate and the passivation layer . we have so far only considered graphene - covered elemental substrates , but we note that , for strongly interacting metals that do not form the required passivating oxide , alloying of the metal may offer a route to achieving long - term passivation . for example , by exchanging fe for stainless steel , a strong graphene metal interaction is still expected while the alloy can form a stable passivating oxide that can plug defects in the graphene , allowing improved corrosion resistance . similarly , where long - term passivation of a substrate with a 2d material is desired but no strong interaction exists , it may be possible to intercalate a thin layer of strongly interacting atoms at the interface to provide the required protection , although this will of course further alter the surface properties . the ability for metal atoms to intercalate beneath graphene indicates that defects are typically present in as - grown graphene or are readily formed on annealing . nevertheless , our results highlight that the protection against oxidation achieved on metals that interact strongly with graphene and form a passivating oxide is maintained even if the graphene coating is relatively defective ( figure 2a ) or not completely continuous ( figure 1d ) . this makes such protection promising even for applications where some in - service damage or wear might be expected . this raises the question of what quality of 2d material is actually required to achieve passivation of the substrate surface . the strong graphenemetal interaction that is key to preventing oxidation arises from the hybridization between the graphene and metal d valence band states . thus , if this hybridization is not maintained due to significant changes in the electronic structure of the graphene , the protection against surface oxidation is also expected to be lost . the formation of a passivating oxide is also critical in preventing oxidation from proceeding through the metal bulk over the longer term . therefore , as the distance between defects approaches the thickness of the metal s passivating oxide , the oxidation behavior will approach that of the bare metal surface . thus , for ni and co substrates , with typical passivating oxide thicknesses of the order of nanometers , reasonable passivation can still be expected from relatively defective and even nanocrystalline graphene , but this is likely to be lost on moving further along the amorphization trajectory toward tetrahedral amorphous carbon . a similar line of argument can be applied to understanding whether the suppression of intercalation on certain metals arises due to the anchoring of the graphene at edges / defects or the interaction between the graphene basal plane and the metal surface . while differences in the anchoring of graphene layers on different metals might be expected , the thicknesses of the passivating oxides on ni and co , i.e. , the distance over which oxidizing species can penetrate , would easily bypass the anchoring of the atom - thick graphene , and thus the strong interaction between the graphene basal plane and the metal surface is chiefly implicated . in summary , we have shown that slg can effectively protect ni and co surfaces from oxidation over extended periods , even when the slg does not form a continuous film . we find that crucial to achieving this long - term passivation is a strong interaction between the 2d material and the underlying substrate , preventing the intercalation of oxidizing species along their interface , which otherwise allows the rapid oxidation of the whole substrate surface . this reveals a route to the long - term protection of metal surfaces , based on the synergy between the substrate and the passivation layer , rather than just the passivation layer s standalone permeability . furthermore , we highlight that the ability of the substrate to form a passivating oxide is critical in preventing oxidation from instead proceeding through the substrate bulk , fed through defects or damaged regions in the 2d material overlayer . we are thus able to provide a consistent explanation for apparent disparities in literature regarding the ability for graphene to provide long - term passivation , which , as we highlight , depends critically on the properties of the underlying substrate . these insights are highly relevant to the application of 2d materials as effective passivation barriers , where they offer the prospect of preserving the physical properties of surfaces over the long term . we investigate commercially available polycrystalline foils of ni ( 25 or 250 m thick ) , co ( 25 m thick ) , fe ( 100 m thick ) , and pt ( 25 m thick ) , as well as 1 mm thick ni(111 ) and pt(111 ) single crystals . the polycrystalline foils and ni(111 ) are annealed [ 600900 c , h2 ( 1 mbar ) , 15 min , heated at 100 c min ] , exposed to hydrocarbons [ 400700 c , c2h4 ( 1010 mbar ) ] , and then cooled [ under vacuum ( 10 mbar ) at 100 c / min ] in custom - built cold - wall reactors unless otherwise stated . for growth on pt(111 ) , the sample is first annealed [ 1000 c , o2 ( 10 mbar ) , 30 min ] to leave a carbon - free surface ( as confirmed by in situ xp c 1s spectra ) and , following removal of o2 , graphene growth proceeds during vacuum annealing [ 1000 c , 10 mbar ] , presumably supplied by carbon dissolved with the sample s bulk . samples are exposed to atmospheric ( moist ) air at room temperature for between 5 min and 18 months and are stored in polystyrene sample boxes during this time to minimize the buildup of dust that may otherwise alter their oxidation behavior . in situ xps measurements were performed at the bessy ii synchrotron at the isiss end station of the fhi - mpg . the high - pressure setup consists of a reaction cell ( base pressure 10 mbar ) attached to a set of three differentially pumped electrostatic lenses and a differentially pumped analyzer ( phoibos 150 , specs gmbh ) , as described elsewhere . all spectra are collected in normal emission geometry , with a spot size of 80 m 150 m and a spectral resolution of 0.3 ev . ephoton is varied to achieve depth resolution , by changing the kinetic energy of the emitted photoelectrons and thus their inelastic mean free paths , escape . all spectra are background - corrected ( shirley ) and analyzed by performing a nonlinear mean square fit of the data , using doniach - nji functions convoluted with gaussian profiles . all binding energies are referenced to contemperaneously measured fermi edges . the extent of s-/flg growth is confirmed ex situ on as - grown samples using scanning electron microscopy ( sem , zeiss sigmavp , 12 kv , in - lens detector ) or after transfer of the s-/flg films to sio2 ( 300 nm)/si substrates using optical microscopy and raman spectroscopy ( renishaw raman invia microscope , 532 nm excitation ) .

the long - term ( > 18 months ) protection of ni surfaces against oxidation under atmospheric conditions is demonstrated by coverage with single - layer graphene , formed by chemical vapor deposition . in situ , depth - resolved x - ray photoelectron spectroscopy of various graphene - coated transition metals reveals that a strong graphene metal interaction is of key importance in achieving this long - term protection . this strong interaction prevents the rapid intercalation of oxidizing species at the graphene metal interface and thus suppresses oxidation of the substrate surface . furthermore , the ability of the substrate to locally form a passivating oxide close to defects or damaged regions in the graphene overlayer is critical in plugging these defects and preventing oxidation from proceeding through the bulk of the substrate . we thus provide a clear rationale for understanding the extent to which two - dimensional materials can protect different substrates and highlight the key implications for applications of these materials as barrier layers to prevent oxidation .

Introduction Results and Discussion Conclusions Methods

standard exfoliation and transfer - based techniques for producing 2d materials are not well - suited to depositing such passivating layers , where a complete , conformal coating is typically desired to prevent ingress of oxidizing species . despite the huge progress in improving the quality and uniformity of cvd graphene , atomic defects still remain , both within graphene domains and at the boundaries where they join , which provide pathways for the permeation of oxidizing species . for single - layer graphene ( slg ) on cu , previous observations have revealed that , on atmospheric air exposure , oxidizing species are thereby able to intercalate between the slg and cu and thus access the whole cu surface . under these atmospheric conditions , where humidity is sufficient for condensed water vapor to serve as an electrolyte on the surface of the graphene therefore , while the reduced permeation of oxidizing species through the slg slows these processes over the short term , cu oxidation still proceeds over time and is even observed to be electrochemically enhanced in the long term due to the galvanic couple formed by the slg cu . recent progress has been made in reducing the graphene permeability by stacking multiple layers or selectively blocking defects with post - treatments , yet these approaches yield barriers significantly thicker than slg and the reported permeabilities are not sufficiently low to prevent surface oxidation over the long term ( i.e. here we form continuous slg coatings on various polycrystalline transition metal catalysts ( ni , co , fe , pt ) by cvd , and using in situ , depth - resolved x - ray photoelectron spectroscopy ( xps ) we investigate the extent of oxidation following exposures to atmospheric ( moist ) air at room temperature for time frames ranging from several minutes up to many months . we thereby demonstrate that slg can effectively passivate polycrystalline ni surfaces and maintain a fully reduced ni surface even following exposure to atmospheric conditions for more than 18 months . our data reveal that of key importance to achieving this long - term passivation is a strong graphene substrate interaction , which prevents the lateral diffusion of oxidizing species along the graphene substrate interface . this enables the long - term passivation of the covered regions of a substrate that interacts strongly with graphene ( e.g. , cu , pt ) , where even short air exposures are sufficient to decouple the graphene from the catalyst by the intercalation of oxidizing species at edges or defects , opening a pathway by which oxidizing species can access the whole catalyst surface . we further show that the ability of the substrate to locally form a passivating oxide at defective or damaged regions in the graphene overlayer is critical in eliminating an alternative route by which oxidation of the substrate can proceed over the longer term . , fe in moist air ) , while a strong graphene substrate interaction can suppress surface oxidation in the short term , for long - term exposures to atmospheric air , oxidation can proceed through the oxide layers that form close to graphene defects until eventually the metal becomes oxidized throughout . we thereby rationalize apparent inconsistencies in the literature regarding the extent to which graphene passivates different substrates and highlight the key advantages offered by direct integration techniques , such as cvd , that can inherently establish a strong graphene substrate interaction . figure 1 compares the depth - resolved xp ni 2p3/2 core level spectra of polycrystalline ni foils with different extents of graphene coverage and lengths of exposure to atmospheric conditions . probing deeper into the sample ( escape 911 ) reveals a lower extent of oxidation , as seen from the decrease in the intensities of the niox peaks relative to the nim peaks , consistent with the rapid formation of an oxide layer on atmospheric exposure , that passivates the surface and limits oxidation from proceeding throughout the ni bulk . this confirms the slg - covered ni surface remains reduced during extended exposure to atmospheric air and , therefore , that long - term passivation can be achieved with slg on ni surfaces . depth - resolved xp ni 2p3/2 core level spectra for polycrystalline ni ( 25 m ) in situ immediately following annealing [ 600 c , h2 ( 10 mbar ) for 15 min ] ( a ) and after subsequent exposure to atmosphere for < 5 min ( b ) , as well as for ni ( 25 m ) covered with a complete slg layer ( c ) , and ni ( 250 m ) covered with noncontinuous slg islands ( d ) following exposure to atmosphere for > 18 months . this long - term passivation behavior of graphene on ni is in contrast to that reported for graphene on cu , where long - term passivation under atmospheric conditions is not achieved , and the presence of graphene is even found to electrochemically enhance the oxidation of cu by the formation of a galvanic couple . it has been proposed that such disparities in graphene passivation behavior may relate to differences in the defect densities of the graphene formed on these different metals . we note that this refers to graphene on idealized low - index , single - crystal surfaces and that the situation for polycrystalline surfaces is more complex , with possible variations in the graphene figure 2 compares the effect of atmospheric exposures on slg grown on prototypical strongly interacting ni(111 ) and more weakly interacting pt(111 ) . significantly , the dominant component remains at 284.8 ev , with the absence of a shift in the peak position during air exposure confirming that the strong slg ni(111 ) coupling is maintained , in spite of the presence of defects in the slg . figure 2b shows xp c 1s spectra measured for slg grown on a pt(111 ) surface by the diffusion of adventitious carbon from within the bulk of the crystal during vacuum annealing [ 10 mbar , 1000 c , for 2 h ] . this shift in peak position toward that of free - standing , undoped graphene is attributed to the weakening of the slg pt coupling by the gradual intercalation of oxidizing species at the slg pt interface , leading to the decoupling of the slg and loss of the charge transfer , i.e. having established that with ni a strong graphene metal interaction protects against rapid surface oxidation by preventing intercalation of oxidizing species at the graphene metal interface , we now consider the performance of graphene in protecting other transition metals that interact strongly with graphene , namely , co ( figure 3 ) and fe ( figure 4 ) . this closely parallels the oxide formation on ni and is again consistent with previous results showing the rapid formation of an oxide layer on exposure to atmospheric air that passivates the surface and slows further oxidation of the metal bulk.figure 3c reveals that , for slg - covered co ( 25 m ) exposed to air for > 6 months , the co 2p3/2 spectra are very similar to those of reduced co ( figure 3a ) . importantly , the absence of any significant coox peaks confirms that oxidation of the co surface is very limited and that the slg - covered co is maintained in a reduced state . this behavior observed for co is qualitatively very similar to that for ni , with almost no oxidation of the slg - covered co evident after > 6 months , while the bare co forms a passivating oxide layer across its surface . depth - resolved xp co 2p3/2 core level spectra for polycrystalline co ( 25 m ) in situ immediately following annealing [ 600 c , h2 ( 10 mbar ) for 15 min ] ( a ) and after subsequent exposure to atmosphere for < 5 min ( b ) ; and for co ( 25 m ) covered with a complete slg layer grown by cvd [ 700 c , c2h2 ( 10 mbar ) for 15 min followed by c2h2 ( 10 mbar ) for 5 min ] following exposure to atmosphere for > 6 months . depth - resolved xp fe 2p3/2 core level spectra for polycrystalline fe ( 100 m ) in situ immediately following annealing [ 1000 c , h2 ( 10 mbar ) for 15 min ] ( a ) and after subsequent exposure to atmosphere for 1 h ( b ) ; and for fe ( 100 m ) covered with a complete slg layer grown by cvd [ 650 c , c2h2 ( 10 mbar ) for 30 min ] following exposure to atmosphere for 1 week ( c ) and > 6 months ( d ) . this significant oxidation of the fe that is observed even in the more depth - sensitive spectrum is in contrast to the behavior of the bare co and ni films where the initial rapid formation of a thin oxide layer passivates the surface , limiting further oxidation of the metal bulk . figure 4c shows that , for slg - covered fe exposed to air for 5 days , while some weak feox contributions are detectable in the most surface - sensitive spectrum ( escape 8 ) , the spectra remain largely similar to those of the reduced fe ( figure 4a ) , with fem remaining by far the dominant component . this highlights that , while the slg coverage can protect the fe from oxidation for air exposures of a few weeks , for longer - term exposure the underlying fe foil gradually oxidizes and eventually no reduced fe remains close to the surface . this same general behavior is confirmed for fe covered with thicker few - layer graphene ( flg ) films ; however , a small fem peak is still detectible in the xp spectra even after > 18 months ( see supporting information , figure s3 ) . this reveals that the fe oxidation occurs more slowly , with some fe still preserved in a metallic state , presumably as a result of the reduced permeation of oxidizing species through the thicker flg film . no significant carbidic phases are observed in the fe 2p3/2 spectra or corresponding c 1s spectra ( not shown ) of the samples measured , and thus their involvement in the observed oxidation behavior can be largely excluded . first the presence of the 2d material provides a low - permeability barrier that limits the access of oxidizing species to the substrate below , with thicker layers having even lower permeability . however , this low permeabilty alone does not typically afford long - term passivation , as intrinsic defects such as grain boundaries and atomic vacancies in the graphene still allow oxidizing species to reach the substrate close to these defects . , cu , pt ) , the graphene is rapidly decoupled from the surface on air exposure by the intercalation of oxidizing species , allowing ready access to the whole metal surface and thus its rapid oxidation . , ni , co , fe ) however , the graphene remains coupled on air exposure and the oxidizing species are thus only able to access the metal surface close to graphene defects , suppressing oxidation of the surface over the short term . for metals that form a passivating oxide ( e.g. , ni , co ) , these exposed regions near to defects are quickly plugged by oxide formation , and the majority of the metal surface is thereby protected from oxidation over the long term . , fe in moist air ) , while the strong graphene metal interaction provides short - term protection of the surface , over the longer term , oxidation can proceed through the oxide layers initially formed close to graphene defects until eventually the metal becomes oxidized throughout . , cu , pt ) on air exposure , providing a pathway for the intercalation of oxidizing species at the graphene catalyst interface and ready access for these oxidizing species to the whole metal surface . for metals that form a passivating oxide ( e.g. , ni , co ) , these exposed regions near to defects are quickly plugged by oxide formation , protecting the substrate from oxidation over the long term . , fe ) , oxidation is initially slowed by the already formed oxide , and thus the graphene coverage provides short - term passivation . however , oxidation can proceed through the already formed oxide , eventually allowing the metal to become oxidized throughout for long - term air exposures . we emphasize that , although a passivating oxide is key to the observed long - term passivation of graphene - covered strongly interacting metals , a passivating oxide alone does not afford equivalent protection . more direct comparisons of the gaseous oxidation of strongly and weakly interacting metals covered with 2d materials formed by cvd are scarcely reported in the literature ; however , the behavior observed during electrochemical corrosion studies of different graphene - covered metals in aqueous solutions appears consistent with the model developed herein . anodic reactions are found to be strongly suppressed for graphene - covered ni , which has a strong graphene in contrast only a very minor reduction in anodic reaction rate is observed for graphene - covered cu , whose weak graphene while our focus herein has been on passivation with 2d materials at room temperature in atmospheric air , we note that , under the more extreme conditions of full immersion in a liquid environment , the formation of a stable passivating oxide is expected to be particularly important in suppressing wet ( i.e. for example , the oxidizing species may be more readily able to intercalate beneath 2d materials even when a strong interaction with the substrate exists and/or passivating oxides formed at room temperature may no longer be stable . furthermore , depending on the nature of the substrate , it can serve as a catalyst to accelerate the breakdown of the 2d material layer under such conditions . this can be understood in the context of the model developed herein , given that a strong interaction with the substrate is not expected to result from typical 2d material - transfer techniques . this also applies to passivation barriers based on percolation of liquid - phase - exfoliated platelets , and thus in both of these cases any potential strong interaction with the substrate that could improve the barrier s performance may need to be activated by , for example , postdeposition thermal annealing . we further note that , while the permeability of 2d material passivation layers can be improved by decorating defects using atomic layer deposition ( ald ) , this does not strengthen the graphene substrate interaction and thus does not benefit from the synergy between the substrate and the passivation layer . we have so far only considered graphene - covered elemental substrates , but we note that , for strongly interacting metals that do not form the required passivating oxide , alloying of the metal may offer a route to achieving long - term passivation . for example , by exchanging fe for stainless steel , a strong graphene metal interaction is still expected while the alloy can form a stable passivating oxide that can plug defects in the graphene , allowing improved corrosion resistance . similarly , where long - term passivation of a substrate with a 2d material is desired but no strong interaction exists , it may be possible to intercalate a thin layer of strongly interacting atoms at the interface to provide the required protection , although this will of course further alter the surface properties . nevertheless , our results highlight that the protection against oxidation achieved on metals that interact strongly with graphene and form a passivating oxide is maintained even if the graphene coating is relatively defective ( figure 2a ) or not completely continuous ( figure 1d ) . this raises the question of what quality of 2d material is actually required to achieve passivation of the substrate surface . the strong graphenemetal interaction that is key to preventing oxidation arises from the hybridization between the graphene and metal d valence band states . thus , if this hybridization is not maintained due to significant changes in the electronic structure of the graphene , the protection against surface oxidation is also expected to be lost . the formation of a passivating oxide is also critical in preventing oxidation from proceeding through the metal bulk over the longer term . therefore , as the distance between defects approaches the thickness of the metal s passivating oxide , the oxidation behavior will approach that of the bare metal surface . thus , for ni and co substrates , with typical passivating oxide thicknesses of the order of nanometers , reasonable passivation can still be expected from relatively defective and even nanocrystalline graphene , but this is likely to be lost on moving further along the amorphization trajectory toward tetrahedral amorphous carbon . a similar line of argument can be applied to understanding whether the suppression of intercalation on certain metals arises due to the anchoring of the graphene at edges / defects or the interaction between the graphene basal plane and the metal surface . while differences in the anchoring of graphene layers on different metals might be expected , the thicknesses of the passivating oxides on ni and co , i.e. , the distance over which oxidizing species can penetrate , would easily bypass the anchoring of the atom - thick graphene , and thus the strong interaction between the graphene basal plane and the metal surface is chiefly implicated . we find that crucial to achieving this long - term passivation is a strong interaction between the 2d material and the underlying substrate , preventing the intercalation of oxidizing species along their interface , which otherwise allows the rapid oxidation of the whole substrate surface . this reveals a route to the long - term protection of metal surfaces , based on the synergy between the substrate and the passivation layer , rather than just the passivation layer s standalone permeability . furthermore , we highlight that the ability of the substrate to form a passivating oxide is critical in preventing oxidation from instead proceeding through the substrate bulk , fed through defects or damaged regions in the 2d material overlayer . we are thus able to provide a consistent explanation for apparent disparities in literature regarding the ability for graphene to provide long - term passivation , which , as we highlight , depends critically on the properties of the underlying substrate . in situ xps measurements were performed at the bessy ii synchrotron at the isiss end station of the fhi - mpg .

the workers of different industries are exposed to various chemicals with potential peril of deoxyribonucleic acid ( dna ) damage . some of these agents may even increase the risk of different cancers , both in the workers and in those who are passively exposed to them due to environmental pollution . the workers with chronic contact with toxic compounds and other chemicals must be controlled periodically in terms of dna damage and genotoxicity . although the concentration of the perilous chemicals and other toxic agents must always be strictly kept below the threshold limit values , chronic toxicity may occur in workers with a higher degree of vulnerability . it is , therefore , essential to monitor the workplace conditions as well as the workers health status . environmental monitoring has long been used to control the working conditions concerning chemicals and toxic agents . this method is rather cheap and non - invasive , but not sufficient to assess the possible dangers of chronic exposure to legally acceptable levels of the chemicals . dna damage may occur by physical or chemical agents can lead to mutations as well as increasing the risk of new genetic disorders . being costly and time consuming , these methods seem less practical today and are giving way to other simpler and more economical techniques . besides , the mentioned methods are only applicable on cells within the reproduction and multiplication phase , which imposes another limitation on the control process . comet assay is the alternative method for dna damage monitoring , which is simple , sensitive and economical enough to be used as a routine genotoxicity test . all the mentioned advantages have highlighted this technique as a standard method for dna damage assessment in the past decade . comet assay , also known as a single cell gel method and micro gel electrophoresis , was first introduced as a micro electrophoresis method for direct observation of dna damage by johanson and ostling in the 1984 . this method , however , underwent several corrections and alterations , as those made by faust et al . , to turn into one of the most convenient and sensitive techniques for dna damage assessment . the mechanism by which comet assay detects dna damage was first explained by reydberg and johanson . based on their explanation , the cells trapped in the agarose gel and lysed under the alkaline ph , find the possibility of presenting a little opening in the dna . under the effect of the electrical flow , the dna molecules then move toward anode , to form the comets . the comet formation pattern is determined by two important factors ; the size of the dna fragments and the number of broken ends . as the percent of damage increases , the free dna fragments move in the tail and in more severe cases , the head and the tail separate completely . since this method is for dna damage assessment in single cells , it is essential to conduct the test in a way that the separation of the cells would be possible . to perform the test , suspension of the separated cells dna damage should be assessed in the cells without giving them the opportunity of being exposed to new genotoxic compounds or of repairing the previously made damages . the cells are usually suspended in agarose to form a final concentration of 0.5 - 1% in 35 - 45c and then transferred on normal or serrate slides . it is important that the gel be totally solidified before continuing the rest of the assay . should a neutral lysing buffer be used , double strand damage detection will be possible . in case that single strand damage assessment is required , an alkaline lysing buffer is needed . the time and voltage necessary for the electrophoresis depends obviously on the level of the dna damage and the salt concentration within the electrophoresis buffer . since microscopic observation of dna migration is possible with even a portion of a millimeter of dna movement , the essential dna migration which leads to comet formation requires a short electrophoresis time ( 5 - 30 min ) and a low voltage ( 0.5 - 5 v / cm ) . following the electrophoresis , one of the simplest techniques used for this purpose is to rank the resulted comets visually . another method frequently used to assess the amount of damage is measuring the distance between the dna and the nucleus . computer - based analysis of the resulted comets using available software heightens strongly the sensitivity of the assay . the final conclusions are made based upon two easily calculated factors ; tail moment which is a combination of the tail length and the total available dna in it and % dna in the tail which determines the ratio of the normal fragments over the broken ones . the purpose of the present study was to assess the dna damage caused by occupational exposure to industrial chemicals in isfahan polyacryl company workers and to investigate the effect of possible risk factors believed to predispose them to genotoxicity on the amount of dna damage , using comet assay . members were selected among the workers of the production line in isfahan polyacryl company , on the basis of filled questionnaires including personal and occupational information , record of service , working section or department and health and medication record . members were selected among the non - smoker office staffs of the same company with no record of exposure to chemicals of the production line , regardless of their age and based upon the same procedure used for the target group selection . lymphocytes were isolated from whole blood samples that has been diluted and treated with an anti - coagulant agent . briefly , cell suspensions ( 1 106 cells / ml ) were mixed with of 1% low melting point agarose ( 37c ) and were placed on the precoated slides ( 1% normal melting point agarose ) . the slides were incubated with lysis solution ( ph = 10.0 ) for 40 min . afterward , the slides were rinsed with distilled water to remove excess lysis solution . in the next step , the slides were incubated with electrophoresis buffer ( ph > 13.0 ) for 40 min . samples were electrophoresed for 40 min at 25 v with an electricity current adjusted to 300 ma . after electrophoresis , the slides were rinsed with distilled water to remove excess alkaline buffer and were placed in neutralization solution ( ph = 7.5 ) for 10 min . each slide was subsequently covered with dye solution ( 20 g / ml ethidium bromide ) for 5 min and washed with distillated water . the comets were visualized under 400 magnification using fluorescence microscopy with an excitation filter of 510 - 560 nm and barrier filter of 590 nm . all stages in comet assay were performed at 4c in dark conditions and all solutions were cool . one - way analysis of variance ( anova ) was used to compare the results of comet assay , followed by tukey 's multiple comparison post hoc tests , using graphpad prism v 5.0 ( graphpad software , san diego , ca ) software . members were selected among the workers of the production line in isfahan polyacryl company , on the basis of filled questionnaires including personal and occupational information , record of service , working section or department and health and medication record . members were selected among the non - smoker office staffs of the same company with no record of exposure to chemicals of the production line , regardless of their age and based upon the same procedure used for the target group selection . members were selected among the workers of the production line in isfahan polyacryl company , on the basis of filled questionnaires including personal and occupational information , record of service , working section or department and health and medication record . members were selected among the non - smoker office staffs of the same company with no record of exposure to chemicals of the production line , regardless of their age and based upon the same procedure used for the target group selection . lymphocytes were isolated from whole blood samples that has been diluted and treated with an anti - coagulant agent . briefly , cell suspensions ( 1 106 cells / ml ) were mixed with of 1% low melting point agarose ( 37c ) and were placed on the precoated slides ( 1% normal melting point agarose ) . the slides were incubated with lysis solution ( ph = 10.0 ) for 40 min . afterward , the slides were rinsed with distilled water to remove excess lysis solution . in the next step , the slides were incubated with electrophoresis buffer ( ph > 13.0 ) for 40 min . samples were electrophoresed for 40 min at 25 v with an electricity current adjusted to 300 ma . after electrophoresis , the slides were rinsed with distilled water to remove excess alkaline buffer and were placed in neutralization solution ( ph = 7.5 ) for 10 min . each slide was subsequently covered with dye solution ( 20 g / ml ethidium bromide ) for 5 min and washed with distillated water . the comets were visualized under 400 magnification using fluorescence microscopy with an excitation filter of 510 - 560 nm and barrier filter of 590 nm . all stages in comet assay were performed at 4c in dark conditions and all solutions were cool . one - way analysis of variance ( anova ) was used to compare the results of comet assay , followed by tukey 's multiple comparison post hoc tests , using graphpad prism v 5.0 ( graphpad software , san diego , ca ) software . to assess the genetic toxicity caused by occupational exposure to the chemicals and other toxic agents in isfahan polyacryl company , the amount of dna damage in the workers lymphocytes both in the production line ( target group ) and the staffs ( control group ) was determined and compared using the comet assay . a total of 120 workers were interred in this study , 60 of them as a control group and others as experimental groups . all workers were men , the average of their age was 35.85 4.4 years and the average of exposure duration was 13 . two critical factors including the percentage of dna in the tail and tail moment ( % dna in tail tail length ) were calculated . based on these criteria , the comparison made between the workers of different sections along with staffs showed a significant difference which means that the people working in the production line suffered from greater dna damage than the office workers [ figure 1 ] . the result of the one - way analysis ( anova ) for the percentage of dna in the tail of studding groups was significant ( p < 0.0001 ) . according to the results of the tukey 's multiple comparison post hoc test , working in all studding parts of the company increased the percentage of dna in tail significantly ( p < 0.001 ) compared with the control group [ figure 1a ] . one - way analysis result of the tail moment for all groups was significant ( p < 0.0001 ) , more over the results of tukey 's multiple comparison post hoc test for all groups showed a significant increase ( p < 0.001 ) in this parameter compared with the control group [ figure 1b ] . ( a ) comparison of % deoxyribonucleic acid in tail and ( b ) tail moment of the workers in different parts of the company . each graph has been represented as mean standard error of mean for 50 workers working in two different parts of the company is one of the other predisposing factors for some cases . according to the extracted information , about 7% of the production line workers were working in two parts . the result of the one - way analysis ( anova ) for the percentage of dna in the tail was significant ( p < 0.0001 ) . according to the results of tukey 's multiple comparison post hoc test , working in one and two parts of the company increased the percentage of dna in tail significantly ( both p < 0.001 ) compared with the control group , more over a significant difference ( p < 0.05 ) was seen between one and two - job worker groups [ figure 2a ] . one - way analysis result of the tail moment was significant ( p < 0.0001 ) . the results of tukey 's multiple comparison post hoc tests for this parameter were similar with the result of the percentage of dna in tail [ figure 2b ] . ( a ) comparison of % deoxyribonucleic acid in tail and ( b ) tail moment of workers with one and two works in the company . each graph has been represented as mean standard error of mean for 50 workers smoking is one of the personal risk factors may predispose the workers to possible genotoxicity . based on the pre - filled questionnaires , 11.6% of the total studied population used to smoke cigarettes . the percentage of dna in the tail and tail moment ( % dna in tail tail length ) , were calculated for these workers and showed a significant difference in the one - way analysis ( anova ) results ( p < 0.0001 ) . moreover , the tukey 's multiple comparison post hoc test results showed significant ( p < 0.001 ) increase in the percentage of dna in the tail for both smoker and non - smoker groups in comparison with the control group while comparison of this parameter in smokers with non - smoker was significant ( p < 0.05 ) [ figure 3a ] . the one - way analysis of the tail moment data showed significant ( p < 0.0001 ) . tukey 's multiple comparison post hoc tests showed both groups had significant difference with the control group and with each other ( respectively p < 0.001 and p < 0.01 ) [ figure 3b ] . ( b ) tail moment of the smoker and non - smoker workers in all parts of the company . to assess the genetic toxicity caused by occupational exposure to the chemicals and other toxic agents in isfahan polyacryl company , the amount of dna damage in the workers lymphocytes both in the production line ( target group ) and the staffs ( control group ) was determined and compared using the comet assay . a total of 120 workers were interred in this study , 60 of them as a control group and others as experimental groups . all workers were men , the average of their age was 35.85 4.4 years and the average of exposure duration was 13 . two critical factors including the percentage of dna in the tail and tail moment ( % dna in tail tail length ) were calculated . based on these criteria , the comparison made between the workers of different sections along with staffs showed a significant difference which means that the people working in the production line suffered from greater dna damage than the office workers [ figure 1 ] . the result of the one - way analysis ( anova ) for the percentage of dna in the tail of studding groups was significant ( p < 0.0001 ) . according to the results of the tukey 's multiple comparison post hoc test , working in all studding parts of the company increased the percentage of dna in tail significantly ( p < 0.001 ) compared with the control group [ figure 1a ] . one - way analysis result of the tail moment for all groups was significant ( p < 0.0001 ) , more over the results of tukey 's multiple comparison post hoc test for all groups showed a significant increase ( p < 0.001 ) in this parameter compared with the control group [ figure 1b ] . ( a ) comparison of % deoxyribonucleic acid in tail and ( b ) tail moment of the workers in different parts of the company . working in two different parts of the company is one of the other predisposing factors for some cases . according to the extracted information , about 7% of the production line workers were working in two parts . the result of the one - way analysis ( anova ) for the percentage of dna in the tail was significant ( p < 0.0001 ) . according to the results of tukey 's multiple comparison post hoc test , working in one and two parts of the company increased the percentage of dna in tail significantly ( both p < 0.001 ) compared with the control group , more over a significant difference ( p < 0.05 ) was seen between one and two - job worker groups [ figure 2a ] . one - way analysis result of the tail moment was significant ( p < 0.0001 ) . the results of tukey 's multiple comparison post hoc tests for this parameter were similar with the result of the percentage of dna in tail [ figure 2b ] . ( a ) comparison of % deoxyribonucleic acid in tail and ( b ) tail moment of workers with one and two works in the company . smoking is one of the personal risk factors may predispose the workers to possible genotoxicity . based on the pre - filled questionnaires , 11.6% of the total studied population used to smoke cigarettes . the percentage of dna in the tail and tail moment ( % dna in tail tail length ) , were calculated for these workers and showed a significant difference in the one - way analysis ( anova ) results ( p < 0.0001 ) . moreover , the tukey 's multiple comparison post hoc test results showed significant ( p < 0.001 ) increase in the percentage of dna in the tail for both smoker and non - smoker groups in comparison with the control group while comparison of this parameter in smokers with non - smoker was significant ( p < 0.05 ) [ figure 3a ] . the one - way analysis of the tail moment data showed significant ( p < 0.0001 ) . tukey 's multiple comparison post hoc tests showed both groups had significant difference with the control group and with each other ( respectively p < 0.001 and p < 0.01 ) [ figure 3b ] . ( a ) comparison of % deoxyribonucleic acid in tail and ( b ) tail moment of the smoker and non - smoker workers in all parts of the company . a great number of workers are exposed to different toxic agents with potential genotoxic effects . since these workers have chronic contact with these compounds and are thus exposed to potential dna damage , it is essential that they be periodically assessed using reliable biomarkers such as comet assay , sister chromatic exchange , micronucleus . many of these tests , however , are relatively expensive and complicated due to the need for cell culture and therefore , are not convenient for monitoring of a big working population . nevertheless , being rather simple , reliable and relatively less costly , comet assay is one of the most convenient methods to be used for dna damage assessment in different industrial workplaces . this method also enables us to detect different types of dna damage and to observe these damages directly in each and every of the damaged cells . besides , the possibility of data collecting from the cell surface which provides us with strong statistical analysis , the need for the limited number of sample cells for the analysis ( < 10000 ) and the possibility of using all kinds of cells with nucleus both in vitro and in vivo mark it as a very convenient method for this purpose . comet assay can also enable us to clinically control the dna - damage - inducing disorders or treatments in a short span of time . one of the other advantages offered by comet assay is its high differentiating capacity which minimizes the probability of attaining unreal positive results . it has been shown that results obtained from comet assay are in most of the cases compatible with the results acquired from cytogenetic tests , which proves that not only does the method possess enough sensitivity to detect even the smallest damage in dna , but also shows negative results whenever lack of damage can not be detected by other tests . believe that the results attained from comet assay are hopeful bioassay indexes to be used for controlling the workers exposed to industrial and non - industrial genotoxic agents . these factors include workplace , work time , record of service , occupational stress , vocational pressure , type of chemicals available in the workplace , life style , diet , physical activity , smoking , personal health and even age and sex . in this research , the office workers of isfahan polyacryl company were considered as the control group , whereas the production line workers served as the target group . the data from the target group were collected from five different company workshops , including polyester , acrylic 1 and acrylic 2 production lines , maintenance department and the power house . as shown in figure 1 , the highest amount of dna damage belongs to the workers of polyester workshop and the lowest belongs to powerhouse workers for whom the two calculated parameters ( % dna in the tail and tail moment ) offer significant differences for all groups compared with the control group . this significant dna damage observed in the production line workers can be caused by chemicals such as organic solvents , toluene , chloroform , ethanol , arsenic , chrome , acrylonitril , benzene and so on , whose genotoxic effects have long been established in numerous studies . it has also been observed that people working in two different workshops demonstrate a higher amount of dna damage , compared with those working only in one single section [ figure 2 ] . based on departmental analysis , 2.9% of the workers in the polyester production line , with as much as 31.63% of dna damage , work in two different company sections . this figure increases to 25% in the acrylic 2 section , by 28.62% of dna damage came in the second rank after polyester workshop . for acrylic 1 by 28.43% of dna damage the analysis shows that the amount of dna damage in the maintenance department workers is as much as 27.77% which is probably so high since a lot of people working there work in other sections as well . the fact that 44.5% of the workers of maintenance section work in one of the other workshops signifies that working simultaneously in two different sections can be considered as a predisposing risk factor to dna damage . in this case , for instance , if the workers with two jobs are not considered , the percent of dna damage for the maintenance department will decline to 24.6% . another risk factor evaluated in this study was smoking , which has also been taken in to consideration in some of the previous bioassay studies . cigarette and its derivatives are complex mixtures of substances with potential for genotoxicity . in this research , factors such as smoking and its record were included . according to an investigation made in italy , smoking can cause a 10% increase of % dna in the tail in smokers in comparison with a non - smoker population . nevertheless , no significant relationship was found between dna damage percent and the number of cigarettes smoked per day . another study conducted among people with two different age - range averages revealed that smoking caused up to 40% increase in dna damage percentage both in the youngsters and in the elderly . on the contrary , some investigations , mostly made in working environments , suggested that smoking may have little effect on vulnerability to dna damage . the present study , however , confirms that the percentage of dna damage in smoking workers is considerably higher than that of the non - smoking working population [ figure 3 ] . this can , of course , occur since due to the rapid intra - cell dna repair , comet assay can just detect the amount of damage recently done to dna . in a study by howard et al . revealed that no significant difference in dna damage exists between those who quit smoking more than a year before ( ex - smokers ) and those who never smoked . although most of the cases of dna damage are repaired , some of them may persist over time hence that the negative outcomes appear much later and even after the end of the workers contact with hazardous chemicals . this study showed that working in two different parts of the company and smoking are two factors of increasing dna damage of workers . it is , therefore , necessary to control the workers , every once in a while , in terms of dna damage and genotoxicity using a convenient bioassay method such as comet assay . periodic review of the workers health and amount of the chemical compounds in industrial environments is necessary .

background : chemical pollutants found in industrial environments can cause chronic genotoxicity in vulnerable individuals during the long - term exposure . the primary purpose of the present study was to assess the deoxyribonucleic acid ( dna ) damage caused by occupational exposure to industrial chemicals and secondary purpose is to investigate the effect of possible risk factors of genotoxicity.materials and methods : the blood samples of the workers of isfahan polyacryl company were evaluated in terms of genotoxicity using the comet assay method . the percentage of dna in the tail and tail moment were measured and dna damage was evaluated . furthermore , the effect of age , smoking , duration of working in the company and working in two parts of the company on the degree of vulnerability to genotoxicity was assessed.results:the amount of dna damage in the target group ( the production line workers ) was significantly higher than the control group ( the staffs ) , 3.87 versus 1.52 as tail moment , ( p < 0.0001 ) . dna damage was significantly higher in smoker groups compared with non - smoker target group and control group , 4.18 versus 3.07 and 1.52 respectively as tail moment , ( p < 0.0001 ) . furthermore , it was higher in person working in two different parts of the company compared to those work in one part and control group , 4.63 versus 3.74 and 1.52 respectively as tail moment , ( p < 0.0001).conclusion : occupational exposure to polyacryl caused dna damage . smoking and working in two parts of the company may have a significant role in dna damage .

INTRODUCTION MATERIALS AND METHODS Study groups The target group The negative control group Sample preparation Alkaline comet assay Statistical analysis RESULTS Comet assay results of workers in different parts of the company Comet assay results of workers with one and two jobs in the company Comet assay results of the smoker and non-smoker workers DISCUSSION CONCLUSION

the workers of different industries are exposed to various chemicals with potential peril of deoxyribonucleic acid ( dna ) damage . some of these agents may even increase the risk of different cancers , both in the workers and in those who are passively exposed to them due to environmental pollution . the workers with chronic contact with toxic compounds and other chemicals must be controlled periodically in terms of dna damage and genotoxicity . although the concentration of the perilous chemicals and other toxic agents must always be strictly kept below the threshold limit values , chronic toxicity may occur in workers with a higher degree of vulnerability . this method is rather cheap and non - invasive , but not sufficient to assess the possible dangers of chronic exposure to legally acceptable levels of the chemicals . dna damage may occur by physical or chemical agents can lead to mutations as well as increasing the risk of new genetic disorders . besides , the mentioned methods are only applicable on cells within the reproduction and multiplication phase , which imposes another limitation on the control process . comet assay is the alternative method for dna damage monitoring , which is simple , sensitive and economical enough to be used as a routine genotoxicity test . all the mentioned advantages have highlighted this technique as a standard method for dna damage assessment in the past decade . comet assay , also known as a single cell gel method and micro gel electrophoresis , was first introduced as a micro electrophoresis method for direct observation of dna damage by johanson and ostling in the 1984 . , to turn into one of the most convenient and sensitive techniques for dna damage assessment . the mechanism by which comet assay detects dna damage was first explained by reydberg and johanson . based on their explanation , the cells trapped in the agarose gel and lysed under the alkaline ph , find the possibility of presenting a little opening in the dna . under the effect of the electrical flow , the dna molecules then move toward anode , to form the comets . the comet formation pattern is determined by two important factors ; the size of the dna fragments and the number of broken ends . as the percent of damage increases , the free dna fragments move in the tail and in more severe cases , the head and the tail separate completely . since this method is for dna damage assessment in single cells , it is essential to conduct the test in a way that the separation of the cells would be possible . to perform the test , suspension of the separated cells dna damage should be assessed in the cells without giving them the opportunity of being exposed to new genotoxic compounds or of repairing the previously made damages . the time and voltage necessary for the electrophoresis depends obviously on the level of the dna damage and the salt concentration within the electrophoresis buffer . since microscopic observation of dna migration is possible with even a portion of a millimeter of dna movement , the essential dna migration which leads to comet formation requires a short electrophoresis time ( 5 - 30 min ) and a low voltage ( 0.5 - 5 v / cm ) . following the electrophoresis , one of the simplest techniques used for this purpose is to rank the resulted comets visually . another method frequently used to assess the amount of damage is measuring the distance between the dna and the nucleus . computer - based analysis of the resulted comets using available software heightens strongly the sensitivity of the assay . the final conclusions are made based upon two easily calculated factors ; tail moment which is a combination of the tail length and the total available dna in it and % dna in the tail which determines the ratio of the normal fragments over the broken ones . the purpose of the present study was to assess the dna damage caused by occupational exposure to industrial chemicals in isfahan polyacryl company workers and to investigate the effect of possible risk factors believed to predispose them to genotoxicity on the amount of dna damage , using comet assay . members were selected among the workers of the production line in isfahan polyacryl company , on the basis of filled questionnaires including personal and occupational information , record of service , working section or department and health and medication record . members were selected among the non - smoker office staffs of the same company with no record of exposure to chemicals of the production line , regardless of their age and based upon the same procedure used for the target group selection . lymphocytes were isolated from whole blood samples that has been diluted and treated with an anti - coagulant agent . briefly , cell suspensions ( 1 106 cells / ml ) were mixed with of 1% low melting point agarose ( 37c ) and were placed on the precoated slides ( 1% normal melting point agarose ) . in the next step , the slides were incubated with electrophoresis buffer ( ph > 13.0 ) for 40 min . one - way analysis of variance ( anova ) was used to compare the results of comet assay , followed by tukey 's multiple comparison post hoc tests , using graphpad prism v 5.0 ( graphpad software , san diego , ca ) software . members were selected among the workers of the production line in isfahan polyacryl company , on the basis of filled questionnaires including personal and occupational information , record of service , working section or department and health and medication record . members were selected among the non - smoker office staffs of the same company with no record of exposure to chemicals of the production line , regardless of their age and based upon the same procedure used for the target group selection . members were selected among the workers of the production line in isfahan polyacryl company , on the basis of filled questionnaires including personal and occupational information , record of service , working section or department and health and medication record . members were selected among the non - smoker office staffs of the same company with no record of exposure to chemicals of the production line , regardless of their age and based upon the same procedure used for the target group selection . afterward , the slides were rinsed with distilled water to remove excess lysis solution . in the next step , the slides were incubated with electrophoresis buffer ( ph > 13.0 ) for 40 min . one - way analysis of variance ( anova ) was used to compare the results of comet assay , followed by tukey 's multiple comparison post hoc tests , using graphpad prism v 5.0 ( graphpad software , san diego , ca ) software . to assess the genetic toxicity caused by occupational exposure to the chemicals and other toxic agents in isfahan polyacryl company , the amount of dna damage in the workers lymphocytes both in the production line ( target group ) and the staffs ( control group ) was determined and compared using the comet assay . a total of 120 workers were interred in this study , 60 of them as a control group and others as experimental groups . all workers were men , the average of their age was 35.85 4.4 years and the average of exposure duration was 13 . two critical factors including the percentage of dna in the tail and tail moment ( % dna in tail tail length ) were calculated . based on these criteria , the comparison made between the workers of different sections along with staffs showed a significant difference which means that the people working in the production line suffered from greater dna damage than the office workers [ figure 1 ] . the result of the one - way analysis ( anova ) for the percentage of dna in the tail of studding groups was significant ( p < 0.0001 ) . according to the results of the tukey 's multiple comparison post hoc test , working in all studding parts of the company increased the percentage of dna in tail significantly ( p < 0.001 ) compared with the control group [ figure 1a ] . one - way analysis result of the tail moment for all groups was significant ( p < 0.0001 ) , more over the results of tukey 's multiple comparison post hoc test for all groups showed a significant increase ( p < 0.001 ) in this parameter compared with the control group [ figure 1b ] . ( a ) comparison of % deoxyribonucleic acid in tail and ( b ) tail moment of the workers in different parts of the company . each graph has been represented as mean standard error of mean for 50 workers working in two different parts of the company is one of the other predisposing factors for some cases . according to the extracted information , about 7% of the production line workers were working in two parts . the result of the one - way analysis ( anova ) for the percentage of dna in the tail was significant ( p < 0.0001 ) . according to the results of tukey 's multiple comparison post hoc test , working in one and two parts of the company increased the percentage of dna in tail significantly ( both p < 0.001 ) compared with the control group , more over a significant difference ( p < 0.05 ) was seen between one and two - job worker groups [ figure 2a ] . one - way analysis result of the tail moment was significant ( p < 0.0001 ) . the results of tukey 's multiple comparison post hoc tests for this parameter were similar with the result of the percentage of dna in tail [ figure 2b ] . ( a ) comparison of % deoxyribonucleic acid in tail and ( b ) tail moment of workers with one and two works in the company . each graph has been represented as mean standard error of mean for 50 workers smoking is one of the personal risk factors may predispose the workers to possible genotoxicity . based on the pre - filled questionnaires , 11.6% of the total studied population used to smoke cigarettes . the percentage of dna in the tail and tail moment ( % dna in tail tail length ) , were calculated for these workers and showed a significant difference in the one - way analysis ( anova ) results ( p < 0.0001 ) . moreover , the tukey 's multiple comparison post hoc test results showed significant ( p < 0.001 ) increase in the percentage of dna in the tail for both smoker and non - smoker groups in comparison with the control group while comparison of this parameter in smokers with non - smoker was significant ( p < 0.05 ) [ figure 3a ] . the one - way analysis of the tail moment data showed significant ( p < 0.0001 ) . tukey 's multiple comparison post hoc tests showed both groups had significant difference with the control group and with each other ( respectively p < 0.001 and p < 0.01 ) [ figure 3b ] . ( b ) tail moment of the smoker and non - smoker workers in all parts of the company . to assess the genetic toxicity caused by occupational exposure to the chemicals and other toxic agents in isfahan polyacryl company , the amount of dna damage in the workers lymphocytes both in the production line ( target group ) and the staffs ( control group ) was determined and compared using the comet assay . a total of 120 workers were interred in this study , 60 of them as a control group and others as experimental groups . two critical factors including the percentage of dna in the tail and tail moment ( % dna in tail tail length ) were calculated . based on these criteria , the comparison made between the workers of different sections along with staffs showed a significant difference which means that the people working in the production line suffered from greater dna damage than the office workers [ figure 1 ] . the result of the one - way analysis ( anova ) for the percentage of dna in the tail of studding groups was significant ( p < 0.0001 ) . according to the results of the tukey 's multiple comparison post hoc test , working in all studding parts of the company increased the percentage of dna in tail significantly ( p < 0.001 ) compared with the control group [ figure 1a ] . one - way analysis result of the tail moment for all groups was significant ( p < 0.0001 ) , more over the results of tukey 's multiple comparison post hoc test for all groups showed a significant increase ( p < 0.001 ) in this parameter compared with the control group [ figure 1b ] . ( a ) comparison of % deoxyribonucleic acid in tail and ( b ) tail moment of the workers in different parts of the company . working in two different parts of the company is one of the other predisposing factors for some cases . according to the extracted information , about 7% of the production line workers were working in two parts . the result of the one - way analysis ( anova ) for the percentage of dna in the tail was significant ( p < 0.0001 ) . according to the results of tukey 's multiple comparison post hoc test , working in one and two parts of the company increased the percentage of dna in tail significantly ( both p < 0.001 ) compared with the control group , more over a significant difference ( p < 0.05 ) was seen between one and two - job worker groups [ figure 2a ] . one - way analysis result of the tail moment was significant ( p < 0.0001 ) . the results of tukey 's multiple comparison post hoc tests for this parameter were similar with the result of the percentage of dna in tail [ figure 2b ] . ( a ) comparison of % deoxyribonucleic acid in tail and ( b ) tail moment of workers with one and two works in the company . smoking is one of the personal risk factors may predispose the workers to possible genotoxicity . based on the pre - filled questionnaires , 11.6% of the total studied population used to smoke cigarettes . the percentage of dna in the tail and tail moment ( % dna in tail tail length ) , were calculated for these workers and showed a significant difference in the one - way analysis ( anova ) results ( p < 0.0001 ) . moreover , the tukey 's multiple comparison post hoc test results showed significant ( p < 0.001 ) increase in the percentage of dna in the tail for both smoker and non - smoker groups in comparison with the control group while comparison of this parameter in smokers with non - smoker was significant ( p < 0.05 ) [ figure 3a ] . the one - way analysis of the tail moment data showed significant ( p < 0.0001 ) . tukey 's multiple comparison post hoc tests showed both groups had significant difference with the control group and with each other ( respectively p < 0.001 and p < 0.01 ) [ figure 3b ] . ( a ) comparison of % deoxyribonucleic acid in tail and ( b ) tail moment of the smoker and non - smoker workers in all parts of the company . since these workers have chronic contact with these compounds and are thus exposed to potential dna damage , it is essential that they be periodically assessed using reliable biomarkers such as comet assay , sister chromatic exchange , micronucleus . nevertheless , being rather simple , reliable and relatively less costly , comet assay is one of the most convenient methods to be used for dna damage assessment in different industrial workplaces . this method also enables us to detect different types of dna damage and to observe these damages directly in each and every of the damaged cells . one of the other advantages offered by comet assay is its high differentiating capacity which minimizes the probability of attaining unreal positive results . it has been shown that results obtained from comet assay are in most of the cases compatible with the results acquired from cytogenetic tests , which proves that not only does the method possess enough sensitivity to detect even the smallest damage in dna , but also shows negative results whenever lack of damage can not be detected by other tests . believe that the results attained from comet assay are hopeful bioassay indexes to be used for controlling the workers exposed to industrial and non - industrial genotoxic agents . these factors include workplace , work time , record of service , occupational stress , vocational pressure , type of chemicals available in the workplace , life style , diet , physical activity , smoking , personal health and even age and sex . in this research , the office workers of isfahan polyacryl company were considered as the control group , whereas the production line workers served as the target group . the data from the target group were collected from five different company workshops , including polyester , acrylic 1 and acrylic 2 production lines , maintenance department and the power house . as shown in figure 1 , the highest amount of dna damage belongs to the workers of polyester workshop and the lowest belongs to powerhouse workers for whom the two calculated parameters ( % dna in the tail and tail moment ) offer significant differences for all groups compared with the control group . this significant dna damage observed in the production line workers can be caused by chemicals such as organic solvents , toluene , chloroform , ethanol , arsenic , chrome , acrylonitril , benzene and so on , whose genotoxic effects have long been established in numerous studies . it has also been observed that people working in two different workshops demonstrate a higher amount of dna damage , compared with those working only in one single section [ figure 2 ] . based on departmental analysis , 2.9% of the workers in the polyester production line , with as much as 31.63% of dna damage , work in two different company sections . this figure increases to 25% in the acrylic 2 section , by 28.62% of dna damage came in the second rank after polyester workshop . for acrylic 1 by 28.43% of dna damage the analysis shows that the amount of dna damage in the maintenance department workers is as much as 27.77% which is probably so high since a lot of people working there work in other sections as well . the fact that 44.5% of the workers of maintenance section work in one of the other workshops signifies that working simultaneously in two different sections can be considered as a predisposing risk factor to dna damage . in this case , for instance , if the workers with two jobs are not considered , the percent of dna damage for the maintenance department will decline to 24.6% . another risk factor evaluated in this study was smoking , which has also been taken in to consideration in some of the previous bioassay studies . according to an investigation made in italy , smoking can cause a 10% increase of % dna in the tail in smokers in comparison with a non - smoker population . another study conducted among people with two different age - range averages revealed that smoking caused up to 40% increase in dna damage percentage both in the youngsters and in the elderly . on the contrary , some investigations , mostly made in working environments , suggested that smoking may have little effect on vulnerability to dna damage . the present study , however , confirms that the percentage of dna damage in smoking workers is considerably higher than that of the non - smoking working population [ figure 3 ] . this can , of course , occur since due to the rapid intra - cell dna repair , comet assay can just detect the amount of damage recently done to dna . revealed that no significant difference in dna damage exists between those who quit smoking more than a year before ( ex - smokers ) and those who never smoked . although most of the cases of dna damage are repaired , some of them may persist over time hence that the negative outcomes appear much later and even after the end of the workers contact with hazardous chemicals . this study showed that working in two different parts of the company and smoking are two factors of increasing dna damage of workers . it is , therefore , necessary to control the workers , every once in a while , in terms of dna damage and genotoxicity using a convenient bioassay method such as comet assay . periodic review of the workers health and amount of the chemical compounds in industrial environments is necessary .

luminescent semiconductor nanocrystals , or quantum dots ( qds ) , have attracted increasing interest due to their tunable photoluminescence ( pl ) , high quantum yield , photostability , and wideband excitation in comparison with organic dyes . to realize the potential of qds as photosensitizers in green energy applications and as probes for in vivo imaging , particles . however , high - quality qds are typically prepared in organic solvents and capped with hydrophobic ligands . to suspend qds in aqueous media , two main strategies are widely employed : ( a ) encapsulation within polymeric amphiphiles and ( b ) hydrophilic ligand exchange . a relatively unexplored strategy is the incorporation of as - synthesized qds into the hydrophobic leaflets of phospholipid membranes . this is an attractive approach because it provides for a viable strategy to interface living cells with inorganic nanomaterials possessing unique optical and electronic properties . accordingly , several reports show that qd encapsulation within membranes is useful in biological imaging and in directing qd uptake within the plasma membrane of living cells . to take full advantage of lipid - qd ( l - qd ) assemblies , it is important to understand the molecular interface between these two materials , which poses a significant challenge for conventional spectroscopic analysis . for example , the interaction of nanoparticles with lipid membranes remains difficult to predict , and can be dependent on a number of factors , such as nanoparticle size , hydrophobicity , and surface charge density . consequently , nanoparticles have been observed to adsorb onto the outer leaflet of membranes , incorporate within a membrane , or cause the deformation of membranes to generate nanoscale holes . in the case of hydrophobic 2.05.0 nm qds , electron and optical microscopy indicates that the nanoparticles partition into the hydrophobic region of the lipid membrane . given that the typical diameter of qds is commiserate with the typical thickness of phospholipid membranes , it is not clear how the qd distorts the organization of the lipid bilayer , and correspondingly , how the organization of the lipid membrane influences the physical and chemical properties of the semiconductor nanocrystal . importantly , current reports do not distinguish between qds embedded within lipid membranes that are in the gel phase and those that are in the fluid phase , leaving it unclear as to how the phase of the lipid membrane would impact the qd . understanding the supramolecular organization of this organic inorganic interface is important in realizing the potential of qds as probes to manipulate and study living cells . herein , we show that the properties of lipid encapsulated qds are highly dependent on the phase of the membrane ; qds within fluid - phase membranes rapidly photo - oxidize and undergo photocorrosion , in contrast to gel - phase lipids that protect qds , rendering them photostable at ambient conditions for at least 60 days . thus , the ordered gel - phase membrane provides a simple and useful means of preparing the most photostable and water - soluble non - core / shell luminescent qds reported to date . icp - ms , pl , and ligand exchange studies further confirm the role of the lipid membrane in controlling surface accessibility of qds . dynamic light scattering ( dls ) and ft - ir measurements indicate that qds do not significantly alter the ensemble properties of the lipid vesicles . however , atomistic molecular dynamics ( md ) simulations show that qd encapsulation leads to local distortion of membrane organization , as well as rearrangement of the qd surface ligands . interestingly , membrane distortion for fully saturated gel - phase lipids is more limited than that for lipid membranes in the fluid phase . on the basis of these results , we site - selectively functionalize the encapsulated qds at the side facing the outer leaflet of the membrane with water - soluble ligands such as polyethylene glycol ( peg ) and oligonucleotides . by taking advantage of the asymmetric distribution of dna ligands , we generate hybrid gold nanoparticle - qd assemblies tethered to the lipid membrane . therefore , membrane - encapsulation offers a promising approach for the next - generation of qds ( as proposed by smith and nie ) with site - selective ligand placement . non - core / shell cdse qds were used in this study because the optical properties of these particles are extraordinarily sensitive to their surface environment , thus providing a probe to monitor accessibility to the qd surface . 3.0 nm cdse qds were synthesized via modified literature methods using oleic acid ( oa ) as a coordinating surfactant ( supporting information 1 ) . the size of cdse nanoparticles was verified by tem ( supporting information figure s1 ) . three representative phospholipids with a range of melting temperatures ( tm ) were used in this study , 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc , tm = 20 c ) , 1,2-dimyristoyl - sn - glycero-3-phosphocholine ( dmpc , tm = 23 c ) , and 1,2-distearoyl - sn - glycero-3-phosphocholine ( dspc , tm = 55 c ) . therefore , at ambient laboratory conditions , dopc is in a fluid phase ( liquid disordered phase ) , while dspc is in a gel phase ( solid ordered phase ) , and dmpc lipids are near the gel to fluid transition temperature . to generate l - qd assemblies , we mixed the phospholipids and qds at a 5000:1 molar ratio in chloroform , evaporated the solvent , then rehydrated the film in di water , and sonicated the solution ( figure 1a and supporting information 2 ) . negative stain tem of dspc - qd vesicles showed the incorporation of qds within the lipid membrane , in agreement with literature precedent ( figure 1b , c ) . this is further supported by fluorescence microscopy of qds and fluorescently doped lipid vesicles that showed a high degree of colocalization ( figure 1e g , supporting information 3 and figure s3 ) . dls of l - qd vesicles showed that qd incorporation does not significantly alter the average hydrodynamic diameter of the lipid vesicles ( 170 nm ) ( figure 1d and supporting information table s1 ) . in principle , each 170 nm vesicle should contain 20 qds when doped with a 5000:1 lipid : qd molar ratio . on the basis of absorbance and tem , however , the average number of qds per vesicle is 11 . this is reasonable and likely due to nonspecific adsorption of qds to the glassware and possible qd aggregation during the transfer into the lipid vesicle and subsequent sonication . temperature - dependent ft - ir spectroscopy of the l - qd vesicles indicated that the ensemble tm of the representative lipid , dppc ( 1,2-dipalmitoyl - sn - glycero-3-phosphocholine ) , is not affected by qd incorporation under these conditions ( figure 1h and supporting information figure s4 ) . taken together , the tem , ft - ir , dls , and pl imaging studies confirmed that qds incorporated within the membrane of vesicles but did not alter their ensemble properties . ( a ) formation of l - qd vesicles via solvent evaporation , hydration and probe sonication . ( b and c ) tem image of cdse qds and negative stain tem image of dspc - cdse vesicles . ( d ) size of dopc , dopc - cdse , dmpc , dmpc - cdse , dspc , and dspc - cdse vesicles obtained by dls measurement . ( e ) and ( f ) are representative fluorescence images of cdse qds , and nbd - pc doped in dspc vesicles . ( g ) is the merged image of ( e ) and ( f ) , which shows colocalization of cdse qds and dspc vesicles indicating the incorporation of qds in the lipid membrane . colocalization of cdse qds and dopc vesicles is shown in supporting information figure s3 . ( h ) thermal phase transition of dppc and dppc - cdse vesicles obtained by plotting the intensity of the ch2 stretch ( 2851 cm ) versus temperature from temperature - dependent ft - ir spectra . to better understand the role of membrane encapsulation in modulating the properties of qds initially , the pl spectra of cdse qds in dopc , dmpc , and dspc displayed identical peak positions and similar intensities , thus indicating that the transfer efficiency into the different types of lipid vesicles was similar ( figure 2a c , black lines ) . interestingly , we found that the pl of qds in dopc , dmpc , and dspc drastically increased , and , in some cases , this was followed by rapid decrease in a phospholipid - dependent manner when stored under ambient laboratory conditions . initially , all three samples showed a large pl intensity increase and blue shift upon one day of exposure to ambient light ( figure 2a c , red curve ) . after two days , the pl of dopc - qd vesicles dropped by 40% , blue - shifted by 13 nm , and the fwhm increased from 36 to 50 nm ( figure 2 and supporting information figure s5a ) . this trend continued as a function of time , and the pl intensity was fully quenched after 1 week ( figure 2a , green curve ) . surprisingly , the pl of qds in the gel phase dspc progressively became brighter ( up to 9-fold ) and slightly blue - shifted ( < 10 nm ) , reaching a near steady state after 1 week that was maintained for up to 2 months ( figure 2c e , supporting information figure s6 ) . importantly , the fwhm of the dspc - qd vesicles remained virtually unchanged while aging in aqueous conditions under light ( supporting information figure s5c ) . dmpc - qd vesicles , whose tm = 23 c , displayed a change of pl that was intermediate between that of fluid dopc vesicles and gel - phase dspc vesicles , first becoming bright ( 8-fold ) , then slowly quenching and blue shifting over a period of 3 weeks . note that the pl spectra of dopc - qd , dspc - qd , and dmpc - qd vesicles were nearly identical and did not display significant changes in peak position , line width , and intensity over a period of 1 week when stored in the dark ( supporting information figure s7 ) . this confirms that pl spectral changes are light - driven processes . to verify the role of oxygen in driving pl shifts , dopc - encapsulated qds were stored under a n2 atmosphere , and the pl spectra were recorded over a period of 1 month ( supporting information figure s8 ) . in this case , qds displayed a 3-fold enhancement in pl intensity at t = 9 day . this is in stark contrast to the dopc - qd samples that are fully quenched at t = 7 day . nonetheless , the dopc - qd samples stored under n2 eventually displayed quenching and blue - shifting in pl by the t = 30 day time point , which may be due to the presence of residual oxygen over this long experimental duration . taken together , these results indicate that the pl intensity changes are due to light - induced and oxygen - mediated photo - oxidation and photocorrosion processes that are controlled by the organization ( phase ) of the lipid membrane . time - dependent pl spectra of cdse qds in ( a ) dopc , ( b ) dmpc , and ( c ) dspc vesicles stored under ambient laboratory conditions . ( d ) maxima pl intensity of l - qd vesicles as a function of time . ( e ) pl blue - shifts of l - qd vesicles as a function of time . the observed pl blue shift and photobrightening followed by quenching of qds in dopc is consistent with photo - oxidation of qds . in contrast , the 9-fold enhancement in pl quantum yield and extraordinary photostability ( up to 2 months ) of dspc encapsulated qds is highly unusual , and to the best of our knowledge , has never been reported for simple qds in aqueous solvents . therefore , we propose that the divergent pl properties of l - qd assemblies in dopc and dspc are likely due to two main processes . first , the partition coefficient of oxygen in fluid lipids is approximately 1 order of magnitude larger than that in gel - phase lipids , thus increasing the rate of photo - oxidation in dopc . moreover , this observation is in agreement with literature precedent showing the physical state of the lipid bilayer significantly affects its water and small molecule permeability . for example , the water permeability of most fluid bilayers ( 10 cm / s ) is more than 2 orders of magnitude greater than that of phospholipids in the gel state ( 10 cm / s ) . to confirm this , we tested the phase - dependent accessibility of reactive species to the surface of the qds by treating the samples with h2o2 , which can chemically oxidize and quench qds ( supporting information figure s9 ) . we found that qds in dopc vesicles were quenched when treated with 110 m h2o2 , in contrast to qds in dspc that maintained their emission after identical treatment to the chemical oxidant . therefore , gel - phase lipids act as a more effective barrier than fluid - phase lipids to protect qds from reactive species . this suggests an explanation for the differential rates of pl blue - shift ( figure 2e ) , but does not account for the ultrastability and brightness of cdse - dspc assemblies . second , to explain the observed time - dependent difference of qd pl and ultrastability in gel - phase vesicles , we hypothesize that qd surface oxide species have different dissolution rates as a function of the phase of the lipid membrane ( figure 3a , b ) . specifically , we propose that oxide species ( seo2 and cdo ) remain bound to qds within the dspc membrane , offering a protective shell , in contrast to qds in dopc membranes ( and other water - stabilized qds ) where oxides are continually released possibly due to the lateral motion and free volume in the fluid lipid membrane . to test the model of lipid - dependent oxide dissolution , icp - ms was used to measure the concentration of se species in the supernatant of the l - qd samples ( figure 3c ) . no significant change in se content was observed in the supernatant of samples containing dspc - qd stored at ambient conditions for seven days . in contrast , identically treated dopc - qd samples showed a 1 order of magnitude increase in dissolved [ se ] ( from 2.8 to 25.9 m ) , thus indicating that these qds are undergoing sustained photocorrosion and dissolution . therefore , fluid - phase dopc - qd vesicles may form transient core / shell qds that are continuously whittled due to dissolution of the oxides . this is supported by pl data that show broadening and blue - shifting of spectra for dopc and dmpc incorporated qds , which is significantly less pronounced for dspc - qd vesicles ( supporting information figure s5 ) . moreover , the diffusion rate of oxide species across the lipid membrane may parallel other small molecule permeability data ( vide supra ) , which shows phase - dependent behavior . taken together , the icp - ms results and the magnitude of the pl shift suggest that approximately one monolayer of oxide forms on the surface of the nanocrystal in dspc , thus increasing the pl quantum yield , and rendering these particles as the most photostable simple qd reported , to the best of our knowledge . given that dopc is chemically distinct from dspc and dmpc , and is more prone to oxidation , we designed an experiment to ensure that these differential pl shifts are due to the phase ( organization ) of the lipid membrane rather than chemical differences in phospholipid structure . we generated dmpc - qd assemblies and divided the sample into two identical aliquots , one stored at 40 c ( fluid phase ) and the second at 4 c ( gel phase ) ( supporting information 7 ) . both samples were illuminated under the same light source , and the pl spectra were collected as a function of time ( supporting information figure s10 ) . in agreement with the trends observed for dopc - qd and dspc - qd assemblies ( figure 2a and c ) , we found that the pl intensity of gel - phase dmpc qds increased by 10-fold over a period of 7 days , whereas fluid - phase dmpc - qds displayed an initial increase in pl followed by a blue - shift and rapid 5-fold quenching of emission between day one and day seven . these data further confirm the remarkable role of the lipid membrane organization ( phase ) in controlling photo - oxidation and photostability of qds as proposed in figure 3 . proposed model of lipid phase - dependent photo - oxidation behavior for cdse qds in ( a ) gel phase lipid vesicles and ( b ) fluid phase lipid vesicles . ( c ) table summarizing [ se ] in the supernatant of the l - qd vesicle solutions as a function of time . data were obtained by icp - ms . given that the qd likely changes the local microenvironment of the lipid membrane , we next set out to use atomistic md simulations to better understand how lipid structure and dynamics are perturbed by the qd . to accommodate for the 10% polydispersity of particle diameters , 2.6 and 3.4 atomistic simulations of 2.6 nm cdse qd passivated with 121 oa ligands in dopc , dmpc , and dspc vesicles were performed for up to 150 ns . snapshots are shown in figure 4a c ( see supporting information figure s11 for 3.4 nm cdse ) . simulations show that in all cases , qds incorporate within the lipid membrane , and lead to distortion of the lipid membrane organization as well as rearrangement of the oa aliphatic tails . importantly , the oa aliphatic chains rearranged to achieve a greater density within the plane of the membrane , while avoiding the aqueous bilayer interface . as evident from the snapshots , dspc lipid tails near the qd were disrupted from their ordered gel - phase structures . to quantify the fluidity of lipid tails , we plotted the time autocorrelation function associated with tail orientation in figure 4d ; a steeper decay indicates faster reorientation , and a lower final value indicates a wider range of motion ( see supporting information 8) . qd - perturbed dspc tails are more available to reorient than those that remain in a gel - phase conformation ( far from qd surface ) ; however , even for the perturbed dspc tails , reorientation is slower and more limited than for dmpc or dopc . as evident from supporting information video 1 , the oa ligands also exhibit a narrower range of motion in dspc than in dopc . dopc tail dynamics are essentially unaffected by the qd , while dmpc tails experience some slowing near the qd , but retain more fluid character than dspc tails . it is reasonable to infer from the simulated tail dynamics that the rates of any events involving lipid tail rearrangement near the qd ( e.g. , ligand exchange ) will be slower for dspc than that for dopc lipids , with dmpc intermediate . the same trends were observed for both 2.6 and 3.4 qd sizes with different numbers of oa ligands passivating the qds ( supporting information figure s12 ) . therefore , the atomistic md simulations are in support of experimental results ( figure 2 ) , and the proposed lipid - dependent photo - oxidation model is shown in figure 3 . representative snapshots of atomistic md simulation of 2.6 nm cdse nanocrystal ( 0150 ns ) in fluid phase dopc ( a ) , dmpc ( b ) , and gel phase dspc vesicles ( c ) . ( d ) time correlation function of tail orientations for lipids perturbed by an embedded 2.6 nm cdse qd ( solid line ) and lipids far from the qd ( dotted line ) , averaged over the final 50 ns of simulation trajectories . on the basis of the md simulations , the surface of qds near the water lipid interface likely presents regions of reduced density of capping ligands and increased surface accessibility , thus offering sites for selective ligand exchange . this is highly desirable , given the lack of methods to direct the location of ligand binding on the surface of nanoparticles in general . to test this idea , we first determined whether water - soluble ligands , such as sh - peg ( sh-(ch2ch2o)8ch3 ) , would displace oa on qds encapsulated in dopc and dspc . we observed ligand exchange in both types of lipids at 100 m sh - peg , as evidenced by pl quenching ( supporting information figure s13 ) . in support of the md simulations , dspc membranes were more organized near the qd and blocked ligand exchange for sh - peg concentrations up to 10 m , in contrast to dopc , which showed quenching at these conditions . to further verify ligand exchange , we performed pl microscopy colocalization using fluorescent ligand ( 10 m , sh-(ch2ch2o)82-cy5 ) and fluorescently doped lipid vesicles ( nbd - pc ) ( supporting information 9 ) . the colocalization of nbd - pc ( figure 5a ) , cdse qds ( figure 5b ) , and sh-(ch2ch2o)82-cy5 ( figure 5c ) , as highlighted in the overlay ( figure 5d ) , confirms partial ligand exchange in fluid dopc vesicles . in contrast , no colocalization of the dspc - cdse qd vesicles was observed with the cy5 emission as indicated by the overlay image ( figure 5i ) . control dopc vesicles that lacked qds also did not show colocalization between the nbd - pc emission and cy5 ( supporting information figure s14 ) , confirming that ligand binding to the vesicle is mediated by the qd . we next aimed to determine the average number of exchanged ligands per dopc membrane - encapsulated qd . this is important , because extensive exchange would likely result in the translocation of qds into the aqueous phase and complete quenching of pl . vis absorption indicated that the stoichiometry between cy5 and qds was approximately two to three ligands per particle , suggesting that only a few sites are available for ligand exchange under these conditions ( supporting information figure s15 ) . ligand exchange for cdse qds encapsulated within dopc ( a e ) and dspc ( f j ) vesicles . colocalization of fluorescence emission from nbd - pc ( a ) , qds ( b ) , and cy5 ( c ) in individual dopc vesicles indicates ligand exchange of qds within fluid lipid vesicles ( d and e ) . ( f j ) shows representative fluorescence microscopy images of qds embedded within dspc vesicles . ( f ) and ( g ) display colocalization , but ( h ) shows no localization with the qd embedded within the dspc vesicles , indicating the lack of ligand exchange ( i and j ) . the cy5 signal in ( h ) ( dotted circle ) is due to the nonspecific adsorption of the ligand on the surface of glass , which is also shown in the dopc - cdse sample in ( c ) ( dotted circles ) . having established that 23 ligands will bind to qds within fluid - phase dopc vesicles ( 10 m , t 10 h ) , we next asked whether these ligands are available for directional binding to other materials . incorporating ligands site - selectively with specific binding directionalities is a first step toward building next generation qds . to achieve this goal , we performed ligand exchange with single - stranded thiolated dna ( 10 m , 5-sh - gcc tat gaa tga gct tca gtg-3 , t 10 h ) . after washing , dna - modified gold nanoparticles ( aunps ) that presented a complementary sequence were added and allowed to hybridize for 1 h ( supporting information 10 ) . the vesicles were subsequently adsorbed to a glass slide and imaged ( figure 6 ) . the fluorescence emission of qds identified the position of dopc - cdse vesicles ( figure 6a ) , while dark field scattering microscopy indicated the position of aunps ( figure 6b ) . the overlay image shows that more than 60% of l - qd vesicles were colocalized with aunps after hybridization ( figure 6c , solid circles ) . to verify that dna - modified aunps were bound to qds through specific watson crick base pairing , we incubated the sample with di water for 1 h as a stringency to dehybridize dna duplexes . subsequent pl and darkfield microscopy imaging confirms complete disassembly of aunps from qds as indicated by the lack of colocalization between the two channels ( figure 6e h ) . ensemble pl measurements of these dna - programmed aunp - qd structures ( supporting information figure s16 ) show significant quenching of the qd emission , confirming the proximity ( 120 nm ) of aunp to the qd surface . taken together , these data confirm that membrane - encapsulated qds are site - specifically functionalized with dna ligands that direct the assembly of dna - aunp structures ( figure 6d and h ) . qd - dopc vesicles were incubated with sh - dna and hybridized with complementary dna - aunp , and then imaged using pl microscopy ( a , qd ) , darkfield scattering microscopy ( b , aunp ) , and then overlaid to show colocalization ( c ) . when this sample was treated with di water , the qd emission was no longer localized with aunp darkfield scattering ( e g ) , indicating dehybridization of double - stranded dna and disassembly of qd - dna - aunp structures . ( d ) and ( h ) show the models for assembly and disassembly of qd - dna - aunp hybrid structures . in summary , lipid vesicles offer a dynamic molecular template that places qds within a two - dimensional and confined environment . we found that the molecular organization ( phase ) of the lipid controls access to the qd surface , and drastically controls photostability of qd . we showed that qds in fluid dopc vesicles are susceptible to photocorrosion and oxide diffusion , which would likely increase toxicity in biological settings . in contrast , the qds in crystalline gel phase lipids are ultrastable , remaining bright over a period of at least 2 months in aqueous solution exposed to ambient light and oxygen . md simulations showed that the qd incorporation led to distortion of the lipid membrane organization as well as rearrangement of the surface ligand tails in all cases , which support experimental findings and indicate higher ligand accessibility ( lipid disorder and ligand reorganization ) at qd sites near the lipid water interface . we tested qd accessibility and showed ( thiolated peg and dna ) ligand binding to the qd surface , driving the assembly of hybrid semiconductor noble metal structures associated with the lipid membrane . therefore , membrane encapsulated qds offer a promising template to generate the next generation of qds with site - selective control of ligand organization . as a corollary , hybrid inorganic nanoparticles ( e.g. , cdse au ) generated using this strategy may also be useful for fluorescence - based imaging , magnetic - based targeting , delivery , cell separation , and mri applications . small unilamellar vesicles were prepared by hydration of dried lipid - cdse films followed by probe sonication . typically , chloroform solutions of lipid ( 2.54 mol ) and cdse qds ( 0.5 nmol ) were mixed in a 10 ml glass vial and dried under a slight vacuum using a r-210 rotavapor . the obtained multilamellar vesicles ( mlv ) were further sonicated for 1 min ( amplitude : 30% ) using a probe sonicator to make small unilamellar vesicles ( suv ) . tem measurements were acquired on a hitachi h-7500 transmission electron microscope at an accelerating voltage of 75 kv . the l - qd vesicles were visualized by negative staining tem with 1% methylamine tungstate ( supporting information 1 ) . dynamic light scattering ( dls ) was used to estimate the hydrodynamic diameters of lipid vesicle samples as well as l - qd samples ( supporting information 2 ) . l - qd vesicles were imaged in aqueous solution at room temperature using a nikon eclipse ti microscope driven by the elements software package ( supporting information 3 ) . equilibrium temperature - dependent ftir spectra were recorded on a varian 3100 ftir spectrometer equipped with liquid nitrogen cooled mercury cadmium telluride ( mct ) detector ( supporting information 4 ) . the concentration of se species in the supernatant of the l - qd samples was measured by a vg plasma quad iii simultaneous inductively coupled plasma - mass spectrometer ( icp - ms ) . l - qd vesicle samples were stored under ambient laboratory conditions under room light , while control samples were wrapped in aluminum foil and placed in the dark ( supporting information 5 ) . cdse nanocrystals of diameter 2.6 or 3.4 nm ( compositions cd257se205 and cd505se413 ) were generated from a wurtzite lattice . oa ligands were covalently attached to surface cd sites , and ligand - covered structures were equilibrated in a vacuum over 1 ns md trajectories . qds were then inserted into a vacant space between the leaflets of solvated , pre - equilibrated lipid bilayers that had been separated by 7 nm . the leaflets closed around the qd within 150 ps of md simulation to produce bilayer - embedded qd structures , which were then simulated for 150 ns at 300 k and 1 bar pressure . numbers of lipids , ligands , and solvent used for each simulation are listed in supporting information table s2 . interaction parameters from the literature were used for lipids , cdse , and for the oleic acid ligands ; however , negative charges on each oa oxygen site were increased to compensate for the excess cd charge , making each qd neutral . lipid tail orientational relaxation functions were obtained by first finding the unit vector i directed from carbon 4 to carbon 9 on the sn-2 lipid tail of each lipid i , then calculating the average autocorrelation function ci(t ) = i()i( + t) over the final 50 ns of the trajectory . the averages of ci(t ) were taken over lipids i classified as either bulk - like ( far from the qd ) or perturbed ( near the qd ) based on their distance in the xy plane from the qd center . the cutoff radius was defined as the distance at which the lipid bilayer recovered its unperturbed thickness . in the case of dspc , a subset of lipids inside the cutoff radius had gel - like properties ; in these , the orientational relaxation function persisted above 0.92 . these 2030% of lipids were excluded from the average plotted in figure 4 . in the ligand exchange experiment with thiolated peg ligand ( sh-(ch2ch2o)8ch3 ) , 1 , 10 , and 100 m thiolated peg ligand was incubated with a 500 nm qd suspension encapsulated in dopc and dspc vesicles overnight in the dark ( 10 h ) ( supporting information figure s13 ) . in the ligand exchange experiment with fluorescently tagged ligand , 10 m sh-(ch2ch2o)82-cy5 was added to l - qd vesicles overnight in the dark ( 10 h ) , and then excess ligand was removed via rinsing with di water five times before imaging .

lipid vesicle encapsulation is an efficient approach to transfer quantum dots ( qds ) into aqueous solutions , which is important for renewable energy applications and biological imaging . however , little is known about the molecular organization at the interface between a qd and lipid membrane . to address this issue , we investigated the properties of 3.0 nm cdse qds encapsulated within phospholipid membranes displaying a range of phase transition temperatures ( tm ) . theoretical and experimental results indicate that the qd locally alters membrane structure , and in turn , the physical state ( phase ) of the membrane controls the optical and chemical properties of the qds . using photoluminescence , icp - ms , optical microscopy , and ligand exchange studies , we found that the tm of the membrane controls optical and chemical properties of lipid vesicle - embedded qds . importantly , qds encapsulated within gel - phase membranes were ultrastable , providing the most photostable non - core / shell qds in aqueous solution reported to date . atomistic molecular dynamics simulations support these observations and indicate that membranes are locally disordered displaying greater disordered organization near the particle solution interface . using this asymmetry in membrane organization near the particle , we identify a new approach for site - selective modification of qds by specifically functionalizing the qd surface facing the outer lipid leaflet to generate gold nanoparticle qd assemblies programmed by watson crick base - pairing .

Introduction Results and Discussion Methods

luminescent semiconductor nanocrystals , or quantum dots ( qds ) , have attracted increasing interest due to their tunable photoluminescence ( pl ) , high quantum yield , photostability , and wideband excitation in comparison with organic dyes . to realize the potential of qds as photosensitizers in green energy applications and as probes for in vivo imaging , particles . to suspend qds in aqueous media , two main strategies are widely employed : ( a ) encapsulation within polymeric amphiphiles and ( b ) hydrophilic ligand exchange . to take full advantage of lipid - qd ( l - qd ) assemblies , it is important to understand the molecular interface between these two materials , which poses a significant challenge for conventional spectroscopic analysis . in the case of hydrophobic 2.05.0 nm qds , electron and optical microscopy indicates that the nanoparticles partition into the hydrophobic region of the lipid membrane . given that the typical diameter of qds is commiserate with the typical thickness of phospholipid membranes , it is not clear how the qd distorts the organization of the lipid bilayer , and correspondingly , how the organization of the lipid membrane influences the physical and chemical properties of the semiconductor nanocrystal . importantly , current reports do not distinguish between qds embedded within lipid membranes that are in the gel phase and those that are in the fluid phase , leaving it unclear as to how the phase of the lipid membrane would impact the qd . herein , we show that the properties of lipid encapsulated qds are highly dependent on the phase of the membrane ; qds within fluid - phase membranes rapidly photo - oxidize and undergo photocorrosion , in contrast to gel - phase lipids that protect qds , rendering them photostable at ambient conditions for at least 60 days . thus , the ordered gel - phase membrane provides a simple and useful means of preparing the most photostable and water - soluble non - core / shell luminescent qds reported to date . icp - ms , pl , and ligand exchange studies further confirm the role of the lipid membrane in controlling surface accessibility of qds . dynamic light scattering ( dls ) and ft - ir measurements indicate that qds do not significantly alter the ensemble properties of the lipid vesicles . however , atomistic molecular dynamics ( md ) simulations show that qd encapsulation leads to local distortion of membrane organization , as well as rearrangement of the qd surface ligands . interestingly , membrane distortion for fully saturated gel - phase lipids is more limited than that for lipid membranes in the fluid phase . on the basis of these results , we site - selectively functionalize the encapsulated qds at the side facing the outer leaflet of the membrane with water - soluble ligands such as polyethylene glycol ( peg ) and oligonucleotides . by taking advantage of the asymmetric distribution of dna ligands , we generate hybrid gold nanoparticle - qd assemblies tethered to the lipid membrane . therefore , membrane - encapsulation offers a promising approach for the next - generation of qds ( as proposed by smith and nie ) with site - selective ligand placement . non - core / shell cdse qds were used in this study because the optical properties of these particles are extraordinarily sensitive to their surface environment , thus providing a probe to monitor accessibility to the qd surface . 3.0 nm cdse qds were synthesized via modified literature methods using oleic acid ( oa ) as a coordinating surfactant ( supporting information 1 ) . three representative phospholipids with a range of melting temperatures ( tm ) were used in this study , 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc , tm = 20 c ) , 1,2-dimyristoyl - sn - glycero-3-phosphocholine ( dmpc , tm = 23 c ) , and 1,2-distearoyl - sn - glycero-3-phosphocholine ( dspc , tm = 55 c ) . therefore , at ambient laboratory conditions , dopc is in a fluid phase ( liquid disordered phase ) , while dspc is in a gel phase ( solid ordered phase ) , and dmpc lipids are near the gel to fluid transition temperature . to generate l - qd assemblies , we mixed the phospholipids and qds at a 5000:1 molar ratio in chloroform , evaporated the solvent , then rehydrated the film in di water , and sonicated the solution ( figure 1a and supporting information 2 ) . negative stain tem of dspc - qd vesicles showed the incorporation of qds within the lipid membrane , in agreement with literature precedent ( figure 1b , c ) . on the basis of absorbance and tem , however , the average number of qds per vesicle is 11 . temperature - dependent ft - ir spectroscopy of the l - qd vesicles indicated that the ensemble tm of the representative lipid , dppc ( 1,2-dipalmitoyl - sn - glycero-3-phosphocholine ) , is not affected by qd incorporation under these conditions ( figure 1h and supporting information figure s4 ) . taken together , the tem , ft - ir , dls , and pl imaging studies confirmed that qds incorporated within the membrane of vesicles but did not alter their ensemble properties . ( e ) and ( f ) are representative fluorescence images of cdse qds , and nbd - pc doped in dspc vesicles . ( g ) is the merged image of ( e ) and ( f ) , which shows colocalization of cdse qds and dspc vesicles indicating the incorporation of qds in the lipid membrane . to better understand the role of membrane encapsulation in modulating the properties of qds initially , the pl spectra of cdse qds in dopc , dmpc , and dspc displayed identical peak positions and similar intensities , thus indicating that the transfer efficiency into the different types of lipid vesicles was similar ( figure 2a c , black lines ) . interestingly , we found that the pl of qds in dopc , dmpc , and dspc drastically increased , and , in some cases , this was followed by rapid decrease in a phospholipid - dependent manner when stored under ambient laboratory conditions . surprisingly , the pl of qds in the gel phase dspc progressively became brighter ( up to 9-fold ) and slightly blue - shifted ( < 10 nm ) , reaching a near steady state after 1 week that was maintained for up to 2 months ( figure 2c e , supporting information figure s6 ) . importantly , the fwhm of the dspc - qd vesicles remained virtually unchanged while aging in aqueous conditions under light ( supporting information figure s5c ) . dmpc - qd vesicles , whose tm = 23 c , displayed a change of pl that was intermediate between that of fluid dopc vesicles and gel - phase dspc vesicles , first becoming bright ( 8-fold ) , then slowly quenching and blue shifting over a period of 3 weeks . note that the pl spectra of dopc - qd , dspc - qd , and dmpc - qd vesicles were nearly identical and did not display significant changes in peak position , line width , and intensity over a period of 1 week when stored in the dark ( supporting information figure s7 ) . taken together , these results indicate that the pl intensity changes are due to light - induced and oxygen - mediated photo - oxidation and photocorrosion processes that are controlled by the organization ( phase ) of the lipid membrane . time - dependent pl spectra of cdse qds in ( a ) dopc , ( b ) dmpc , and ( c ) dspc vesicles stored under ambient laboratory conditions . in contrast , the 9-fold enhancement in pl quantum yield and extraordinary photostability ( up to 2 months ) of dspc encapsulated qds is highly unusual , and to the best of our knowledge , has never been reported for simple qds in aqueous solvents . therefore , we propose that the divergent pl properties of l - qd assemblies in dopc and dspc are likely due to two main processes . first , the partition coefficient of oxygen in fluid lipids is approximately 1 order of magnitude larger than that in gel - phase lipids , thus increasing the rate of photo - oxidation in dopc . moreover , this observation is in agreement with literature precedent showing the physical state of the lipid bilayer significantly affects its water and small molecule permeability . to confirm this , we tested the phase - dependent accessibility of reactive species to the surface of the qds by treating the samples with h2o2 , which can chemically oxidize and quench qds ( supporting information figure s9 ) . we found that qds in dopc vesicles were quenched when treated with 110 m h2o2 , in contrast to qds in dspc that maintained their emission after identical treatment to the chemical oxidant . second , to explain the observed time - dependent difference of qd pl and ultrastability in gel - phase vesicles , we hypothesize that qd surface oxide species have different dissolution rates as a function of the phase of the lipid membrane ( figure 3a , b ) . specifically , we propose that oxide species ( seo2 and cdo ) remain bound to qds within the dspc membrane , offering a protective shell , in contrast to qds in dopc membranes ( and other water - stabilized qds ) where oxides are continually released possibly due to the lateral motion and free volume in the fluid lipid membrane . to test the model of lipid - dependent oxide dissolution , icp - ms was used to measure the concentration of se species in the supernatant of the l - qd samples ( figure 3c ) . therefore , fluid - phase dopc - qd vesicles may form transient core / shell qds that are continuously whittled due to dissolution of the oxides . moreover , the diffusion rate of oxide species across the lipid membrane may parallel other small molecule permeability data ( vide supra ) , which shows phase - dependent behavior . taken together , the icp - ms results and the magnitude of the pl shift suggest that approximately one monolayer of oxide forms on the surface of the nanocrystal in dspc , thus increasing the pl quantum yield , and rendering these particles as the most photostable simple qd reported , to the best of our knowledge . given that dopc is chemically distinct from dspc and dmpc , and is more prone to oxidation , we designed an experiment to ensure that these differential pl shifts are due to the phase ( organization ) of the lipid membrane rather than chemical differences in phospholipid structure . in agreement with the trends observed for dopc - qd and dspc - qd assemblies ( figure 2a and c ) , we found that the pl intensity of gel - phase dmpc qds increased by 10-fold over a period of 7 days , whereas fluid - phase dmpc - qds displayed an initial increase in pl followed by a blue - shift and rapid 5-fold quenching of emission between day one and day seven . these data further confirm the remarkable role of the lipid membrane organization ( phase ) in controlling photo - oxidation and photostability of qds as proposed in figure 3 . proposed model of lipid phase - dependent photo - oxidation behavior for cdse qds in ( a ) gel phase lipid vesicles and ( b ) fluid phase lipid vesicles . given that the qd likely changes the local microenvironment of the lipid membrane , we next set out to use atomistic md simulations to better understand how lipid structure and dynamics are perturbed by the qd . simulations show that in all cases , qds incorporate within the lipid membrane , and lead to distortion of the lipid membrane organization as well as rearrangement of the oa aliphatic tails . importantly , the oa aliphatic chains rearranged to achieve a greater density within the plane of the membrane , while avoiding the aqueous bilayer interface . as evident from the snapshots , dspc lipid tails near the qd were disrupted from their ordered gel - phase structures . to quantify the fluidity of lipid tails , we plotted the time autocorrelation function associated with tail orientation in figure 4d ; a steeper decay indicates faster reorientation , and a lower final value indicates a wider range of motion ( see supporting information 8) . qd - perturbed dspc tails are more available to reorient than those that remain in a gel - phase conformation ( far from qd surface ) ; however , even for the perturbed dspc tails , reorientation is slower and more limited than for dmpc or dopc . dopc tail dynamics are essentially unaffected by the qd , while dmpc tails experience some slowing near the qd , but retain more fluid character than dspc tails . it is reasonable to infer from the simulated tail dynamics that the rates of any events involving lipid tail rearrangement near the qd ( e.g. therefore , the atomistic md simulations are in support of experimental results ( figure 2 ) , and the proposed lipid - dependent photo - oxidation model is shown in figure 3 . on the basis of the md simulations , the surface of qds near the water lipid interface likely presents regions of reduced density of capping ligands and increased surface accessibility , thus offering sites for selective ligand exchange . in support of the md simulations , dspc membranes were more organized near the qd and blocked ligand exchange for sh - peg concentrations up to 10 m , in contrast to dopc , which showed quenching at these conditions . the colocalization of nbd - pc ( figure 5a ) , cdse qds ( figure 5b ) , and sh-(ch2ch2o)82-cy5 ( figure 5c ) , as highlighted in the overlay ( figure 5d ) , confirms partial ligand exchange in fluid dopc vesicles . vis absorption indicated that the stoichiometry between cy5 and qds was approximately two to three ligands per particle , suggesting that only a few sites are available for ligand exchange under these conditions ( supporting information figure s15 ) . ligand exchange for cdse qds encapsulated within dopc ( a e ) and dspc ( f j ) vesicles . colocalization of fluorescence emission from nbd - pc ( a ) , qds ( b ) , and cy5 ( c ) in individual dopc vesicles indicates ligand exchange of qds within fluid lipid vesicles ( d and e ) . the cy5 signal in ( h ) ( dotted circle ) is due to the nonspecific adsorption of the ligand on the surface of glass , which is also shown in the dopc - cdse sample in ( c ) ( dotted circles ) . to verify that dna - modified aunps were bound to qds through specific watson crick base pairing , we incubated the sample with di water for 1 h as a stringency to dehybridize dna duplexes . ensemble pl measurements of these dna - programmed aunp - qd structures ( supporting information figure s16 ) show significant quenching of the qd emission , confirming the proximity ( 120 nm ) of aunp to the qd surface . we found that the molecular organization ( phase ) of the lipid controls access to the qd surface , and drastically controls photostability of qd . in contrast , the qds in crystalline gel phase lipids are ultrastable , remaining bright over a period of at least 2 months in aqueous solution exposed to ambient light and oxygen . md simulations showed that the qd incorporation led to distortion of the lipid membrane organization as well as rearrangement of the surface ligand tails in all cases , which support experimental findings and indicate higher ligand accessibility ( lipid disorder and ligand reorganization ) at qd sites near the lipid water interface . we tested qd accessibility and showed ( thiolated peg and dna ) ligand binding to the qd surface , driving the assembly of hybrid semiconductor noble metal structures associated with the lipid membrane . therefore , membrane encapsulated qds offer a promising template to generate the next generation of qds with site - selective control of ligand organization . the concentration of se species in the supernatant of the l - qd samples was measured by a vg plasma quad iii simultaneous inductively coupled plasma - mass spectrometer ( icp - ms ) . the leaflets closed around the qd within 150 ps of md simulation to produce bilayer - embedded qd structures , which were then simulated for 150 ns at 300 k and 1 bar pressure . interaction parameters from the literature were used for lipids , cdse , and for the oleic acid ligands ; however , negative charges on each oa oxygen site were increased to compensate for the excess cd charge , making each qd neutral . in the ligand exchange experiment with thiolated peg ligand ( sh-(ch2ch2o)8ch3 ) , 1 , 10 , and 100 m thiolated peg ligand was incubated with a 500 nm qd suspension encapsulated in dopc and dspc vesicles overnight in the dark ( 10 h ) ( supporting information figure s13 ) .

bladder cancer is the seventh most common malignancy in men and 8 most common malignancy in women . in india , bladder cancer is the fifth most common cancer in men according to delhi based registry with age adjusted incidence rate of 5.8/100,000 person years . it is predominantly a disease of the male population with male to female ratio of 8.6:1 with the median age at presentation of 60 years ( ranges from 18 to 90 years ) . for non - muscle invasive bladder carcinoma ( nmibc ) , transurethral resection of bladder tumor ( turbt ) is the cornerstone of treatment . reasons for such a high recurrence rate for nmibc have been cited as incomplete resection in initial turbt , aggressive tumor biology , implantation of the tumor cells and possibly a field change to start with . the conventional turbt ( ct ) technique involves piecemeal resection of the tumor , which is contrary to the established oncological principles of removing tumor in other parts of the body . unfortunately , there has never been a certain way to provide a complete resection of the visible tumor in the bladder . similarly , it has been observed in one study that almost 81% of the tumors recurred at the site of previous resection , which indirectly suggests that the technique of resecting tumor is not adequate . in view of the high rate of recurrence , perhaps turbt should be modified to provide en - bloc resection of the specimen , based on the established oncological principle of dissecting through normal tissue . these studies on en - bloc tumor removal were attractive in their simplicity , use of existing equipment , clinical safety profile and adequacy of pathology , but they have not commented on the essential outcome , i.e. , recurrence and progression rate . this study was aimed at conceptualizing the technique of sculpting and resecting in form of et and comparing its outcomes in terms of recurrence and progression with the conventional technique of incising and scattering . after seeking an institutional ethics committee approval , a prospective non - randomized interventional study was conducted between september 2007 to june 2011 . end points of the study , i.e. , recurrence and progression rates were compared in two groups . et was carried out by a single surgeon . to minimize the bias toward deliberate attempt by a surgeon who did et to resect tumor completely , all three urologists were senior consultants with more than 10 years of experience in endourology . clinical data on the size ( measured by ultrasonography in 37 patients and contrast enhanced computerized tomogram scan on 33 patients ) and the location of the tumor were recorded . two sided z - test with pooled variance was used to calculate the sample size . with existing recurrence rate of 60% with ct , an estimate of reduction of recurrence rate by 40% gave a sample of 23 patients in each group to achieve 82% power at the significance level of 0.05 . patients presenting for the first time with single tumor on imaging and the tumor size from 2 to 4 cm were included in the study . patients with prior turbt and recurrences , pedunculated tumors , multiple tumors , biopsy specimen with absent detrusor muscles and those with tumor size of more than 4 cm were excluded . tumors less than 2 cm were excluded as the technique would not make a real difference in them . patients were followed - up after an initial turbt with check cystoscopy at 3 months interval for initial 2 years , 6 monthly for next 2 years and then yearly a total of 70 patients were included , 30 had et and 40 had ct . as patients with stage t2 and those with absence of detrusor muscle on the first turbt were excluded , for the final analyses there were 21 and 24 patients in et and ct groups respectively [ graph 1 ] . the usual tungsten loop electrode and 26 fr resectoscope ( storz , germany ) with 30 the angle of the loop electrode was changed manually from 90 to 45 for et as published before [ figure 1 ] . feasibility of en - bloc resection was initially established in tumors of less than 2 cm in six cases before initiating this study . angle of the loop electrode was changed manually from 90 to 45 for en - bloc transurethral resection of bladder tumor normal mucosa around the tumor base was scored by using coagulation current at setting of 60 - 80 w ( valley lab , usa ) . retrograde resection was started in the deep layer of detrusor muscle elevating the tumor off the base with the help of angle of the loop and beak of the resectoscope . free lying tumor which was too large to be taken out through urethra was cut into 2 - 3 pieces in a way that all the layers are there in the piece of the tumor tissue . in a partially filled bladder with minimal inflow , the tumors were divided using a cutting current with the tumor sited over the trigone for the current to pass . once the groove was created , the loop was entangled in the groove and tumor was lifted away from the trigone and then cut against the beak of the resectoscope . use of a nephroscope and grasping forceps was helpful in retrieving the tumor , the largest single piece being a 3 cm tumor in a female . six , weekly doses of intravesical bacillus calmette - guerin ( bcg ) ( oncovac , danish 1331 strain ) was instilled in patients with ta high grade and t1 disease . the compliance rate for bcg at our center is as high as 85% and was similar in both ct and et . histopathological examination was carried out using the 2004 world health organization - international society of urological pathological grading system . blood loss was calculated in terms of fall in hemoglobin levels measured before and a day after turbt . recurrences free survival ( rfs ) and progression - free survival ( pfs ) were calculated using kaplan - meier method and long rank test . two sided z - test with pooled variance was used to calculate the sample size . with existing recurrence rate of 60% with ct , an estimate of reduction of recurrence rate by 40% gave a sample of 23 patients in each group to achieve 82% power at the significance level of 0.05 . patients presenting for the first time with single tumor on imaging and the tumor size from 2 to 4 cm were included in the study . patients with prior turbt and recurrences , pedunculated tumors , multiple tumors , biopsy specimen with absent detrusor muscles and those with tumor size of more than 4 cm were excluded . tumors less than 2 cm were excluded as the technique would not make a real difference in them . patients were followed - up after an initial turbt with check cystoscopy at 3 months interval for initial 2 years , 6 monthly for next 2 years and then yearly . progression was defined as any upgradation of stage or grade . a total of 70 patients were included , 30 had et and 40 had ct . as patients with stage t2 and those with absence of detrusor muscle on the first turbt were excluded , for the final analyses there were 21 and 24 patients in et and ct groups respectively [ graph 1 ] . the usual tungsten loop electrode and 26 fr resectoscope ( storz , germany ) with 30 the angle of the loop electrode was changed manually from 90 to 45 for et as published before [ figure 1 ] . feasibility of en - bloc resection was initially established in tumors of less than 2 cm in six cases before initiating this study . angle of the loop electrode was changed manually from 90 to 45 for en - bloc transurethral resection of bladder tumor normal mucosa around the tumor base was scored by using coagulation current at setting of 60 - 80 w ( valley lab , usa ) . retrograde resection was started in the deep layer of detrusor muscle elevating the tumor off the base with the help of angle of the loop and beak of the resectoscope . free lying tumor which was too large to be taken out through urethra was cut into 2 - 3 pieces in a way that all the layers are there in the piece of the tumor tissue . in a partially filled bladder with minimal inflow , the tumors were divided using a cutting current with the tumor sited over the trigone for the current to pass . once the groove was created , the loop was entangled in the groove and tumor was lifted away from the trigone and then cut against the beak of the resectoscope . use of a nephroscope and grasping forceps was helpful in retrieving the tumor , the largest single piece being a 3 cm tumor in a female . six , weekly doses of intravesical bacillus calmette - guerin ( bcg ) ( oncovac , danish 1331 strain ) was instilled in patients with ta high grade and t1 disease . the compliance rate for bcg at our center is as high as 85% and was similar in both ct and et . histopathological examination was carried out using the 2004 world health organization - international society of urological pathological grading system . blood loss was calculated in terms of fall in hemoglobin levels measured before and a day after turbt . recurrences free survival ( rfs ) and progression - free survival ( pfs ) were calculated using kaplan - meier method and long rank test . two sided z - test with pooled variance was used to calculate the sample size . with existing recurrence rate of 60% with ct , an estimate of reduction of recurrence rate by 40% gave a sample of 23 patients in each group to achieve 82% power at the significance level of 0.05 . patients presenting for the first time with single tumor on imaging and the tumor size from 2 to 4 cm were included in the study . patients with prior turbt and recurrences , pedunculated tumors , multiple tumors , biopsy specimen with absent detrusor muscles and those with tumor size of more than 4 cm were excluded . tumors less than 2 cm were excluded as the technique would not make a real difference in them . patients were followed - up after an initial turbt with check cystoscopy at 3 months interval for initial 2 years , 6 monthly for next 2 years and then yearly . progression was defined as any upgradation of stage or grade . a total of 70 patients were included , 30 had et and 40 had ct . as patients with stage t2 and those with absence of detrusor muscle on the first turbt were excluded , for the final analyses there were 21 and 24 patients in et and ct groups respectively [ graph 1 ] . the usual tungsten loop electrode and 26 fr resectoscope ( storz , germany ) with 30 lens and monopolar cautery was used . the angle of the loop electrode was changed manually from 90 to 45 for et as published before [ figure 1 ] . feasibility of en - bloc resection was initially established in tumors of less than 2 cm in six cases before initiating this study . angle of the loop electrode was changed manually from 90 to 45 for en - bloc transurethral resection of bladder tumor normal mucosa around the tumor base was scored by using coagulation current at setting of 60 - 80 w ( valley lab , usa ) . retrograde resection was started in the deep layer of detrusor muscle elevating the tumor off the base with the help of angle of the loop and beak of the resectoscope . free lying tumor which was too large to be taken out through urethra was cut into 2 - 3 pieces in a way that all the layers are there in the piece of the tumor tissue . in a partially filled bladder with minimal inflow , the tumors were divided using a cutting current with the tumor sited over the trigone for the current to pass . once the groove was created , the loop was entangled in the groove and tumor was lifted away from the trigone and then cut against the beak of the resectoscope . use of a nephroscope and grasping forceps was helpful in retrieving the tumor , the largest single piece being a 3 cm tumor in a female . six , weekly doses of intravesical bacillus calmette - guerin ( bcg ) ( oncovac , danish 1331 strain ) was instilled in patients with ta high grade and t1 disease . the compliance rate for bcg at our center is as high as 85% and was similar in both ct and et . histopathological examination was carried out using the 2004 world health organization - international society of urological pathological grading system . blood loss was calculated in terms of fall in hemoglobin levels measured before and a day after turbt . recurrences free survival ( rfs ) and progression - free survival ( pfs ) were calculated using kaplan - meier method and long rank test . patients who had detrusor muscle invasion or had no detrusor muscle in the first turbt were excluded from the analyses . patients in whom follow - up was not present were also excluded [ graph 1 ] . at the final analysis 21 patients of et the mean age was 52.6 12.2 and 55 13.6 years in et and ct group . median follow - up period was 40 months and 30 months in et and ct groups respectively [ tables 1 and 2 ] . stage , grade and locations of tumor in two groups on initial turbt tumor characteristics and the outcome in two groups of turbt recurrence rate was 28.6% versus 62.5% ( p = 0.03 ) and progression rate was 19% versus 33.3% ( p = 0.32 ) in et versus ct group respectively [ table 2 ] . in the et group , the recurrence in non - lamina invasive nmibc tumors was 16% , where as it was 53% in ct . similarly in lamina invasive tumor , the recurrence rate was 44% and 72% in et and ct group . mean rfs was 45.1 months ( 95% ci : 19.0 - 38 months ) with et and 28.5 months ( 95% ci : 35.4 - 54.7 months ) in ct with p value of 0.018 [ figure 2 and table 2 ] . regarding the site of tumor recurrence , only 1 ( 16% ) patient had recurrence at the same site in et group while 6 ( 40% ) patients had recurrence at the same site with ct [ table 3 ] . recurrence free survival site of recurrences in two groups four patients in en - bloc and eight patients in conventional group had disease progression ( 19% vs. 33.3% , p = 0.32 ) . mean pfs in et and ct group was 48.32 months ( 95% ci : 35.5 - 53.0 months ) and 44.26 months ( 95% ci : 39.0 - 57.5 months ) with p value of 0.46 [ figure 3 and table 2 ] . in et group , three patients had progressed without prior recurrence ; one had progressed after one recurrence . in ct group , six patients had progressed on first recurrence ; one had progressed after one recurrence and one patient had progressed after two recurrences . progression free survival turbt is a procedure with a varied outcome in terms of adequacy of resection , recurrence and progression . there are a few surrogate markers to assess adequate resection such as presence of detrusor muscle in the specimen and the rate of subsequent recurrence . successful management of bladder tumors ( particularly non - muscle - invasive tumors ) , relies on adequate initial resection and accurate histological diagnosis . an ideal turbt would mean complete resection of the visible tumor , resection of the surrounding healthy looking mucosa for up to 1 cm and then the removal of detrusor muscle . herr and donat described three ways to measure the quality of a good turbt , i.e. , complete resection , presence of deep muscle in the specimen and the rate of recurrence at the site of previous turbt . they also suggested classifying tumor resection as r0 ; microscopic negative margin , r1 with microscopic positive margin and r2 that is macroscopic positive margin . this kind of assessment is not practical in ct , but could be possible in en - bloc resection , where we can have a piece of tumor tissue , which has all three layers , i.e. , urothelium , lamina propria and detrusor muscle in contiguity . the outer - most surface of the detrusor muscle in the resected specimen could then be inked to assess margin status and thereby discerning a true perspective of level of resection , i.e. , r0-r1 [ figure 4 ] . en - bloc resection provides a tissue piece of the bladder with all the layers in contiguity to assess the t stage and margin status . in inset , a 3 cm tumor removed en - bloc inadequacy of ct is not only judged by absence of detrusor muscle in an initial specimen , but also by the rate of recurrence . recurrence is seen in 50 - 70% of non - muscle invasive bladder cancer , mostly during the 1 year . these may be due to incomplete resection , cell implantation or the tumor biology itself . incomplete resection seems to be the most important reason for the recurrence . in a review of seven randomized controlled trials , after controlling established factors for recurrence such as tumor size , multiplicity , stage and grade , it was concluded that a wide range of recurrence rate , i.e. , 0 - 46% , was due to the difference in quality of resection . inadequate resection leading to higher rate of recurrence at the same site is supported by another study where 81% of recurred tumor occurred at the site of previous resection . various techniques using different kinds of loops and laser have been described to improve the quality of turbt . en - bloc resection technique is one of the ways to provide better pathological evaluation for ta and t1 tumors . in a study on et , although the authors did not describe presence or absence of detrusor muscle in the resected specimen , they concluded that invasion of lamina was better delineated with en - bloc resection . a limitation of this technique was the inability to use a flat loop for tumors located at the anterior and upper posterior wall . it may not matter much whether we do et or ct for a small size papillary tumor , but for tumors of 2 - 4 cm size , et would give better tissue configuration for assessing the exact t stage and commenting on margin status . there is paucity of literature on et for tumors of 2 - 4 cm size . we used a 45 angle loop electrode , which helped in scooping out the tumor in retrograde fashion . once the tumor was resected en - bloc , removal of 4 cm size tumor from the urethra is a challenge particularly in a male patient . we could remove 3 cm size tumor in a female , but in males we had to cut the free lying tumor into two or three pieces . the most common technique was to place a nephroscope and hold the tumor with forceps to remove the whole assembly along with the tumor . results of this prospective non - randomized study have shown that recurrence rate in et was significantly lower than ct . best results of et were seen in non - lamina invasive bladder cancer , where recurrence rate was reduced to 16% as compared with 53% with ct . therefore , en - bloc resection not only provided a tissue piece with all the layers of bladder in contiguity for better histopathological characterization , but also resulted in reduction in the recurrence rate . looking at the pattern of recurrence in our patients , it seems that et is a better way to reduce the risk of recurrence as it provides a complete resection as only 1 ( 16% ) patient had recurrence at the same site as compared to 6 ( 40% ) patients in ct group 6 ( 40% ) patients in ct group regarding the learning curve , et is rather more controlled technique of resection than ct as it gives better hemostasis and thereby a good vision , which is crucial to avoid complications . depth of the resection could also be modified with et and it does not take more than three cases to get a knack of this technique if it is started with a relatively small tumor . first , tumor retrieval from the bladder was technically difficult and it had to be cut into 2 - 3 pieces once it was lying free in the bladder . second , there could be surgeon related factors as ct was done by two different surgeons . turbt is a procedure with a varied outcome in terms of adequacy of resection , recurrence and progression . there are a few surrogate markers to assess adequate resection such as presence of detrusor muscle in the specimen and the rate of subsequent recurrence . successful management of bladder tumors ( particularly non - muscle - invasive tumors ) , relies on adequate initial resection and accurate histological diagnosis . an ideal turbt would mean complete resection of the visible tumor , resection of the surrounding healthy looking mucosa for up to 1 cm and then the removal of detrusor muscle . herr and donat described three ways to measure the quality of a good turbt , i.e. , complete resection , presence of deep muscle in the specimen and the rate of recurrence at the site of previous turbt . they also suggested classifying tumor resection as r0 ; microscopic negative margin , r1 with microscopic positive margin and r2 that is macroscopic positive margin . this kind of assessment is not practical in ct , but could be possible in en - bloc resection , where we can have a piece of tumor tissue , which has all three layers , i.e. , urothelium , lamina propria and detrusor muscle in contiguity . the outer - most surface of the detrusor muscle in the resected specimen could then be inked to assess margin status and thereby discerning a true perspective of level of resection , i.e. , r0-r1 [ figure 4 ] . en - bloc resection provides a tissue piece of the bladder with all the layers in contiguity to assess the t stage and margin status . in inset , a 3 cm tumor removed en - bloc inadequacy of ct is not only judged by absence of detrusor muscle in an initial specimen , but also by the rate of recurrence . recurrence is seen in 50 - 70% of non - muscle invasive bladder cancer , mostly during the 1 year . these may be due to incomplete resection , cell implantation or the tumor biology itself . incomplete resection seems to be the most important reason for the recurrence . in a review of seven randomized controlled trials , after controlling established factors for recurrence such as tumor size , multiplicity , stage and grade , it was concluded that a wide range of recurrence rate , i.e. , 0 - 46% , was due to the difference in quality of resection . inadequate resection leading to higher rate of recurrence at the same site is supported by another study where 81% of recurred tumor occurred at the site of previous resection . various techniques using different kinds of loops and laser have been described to improve the quality of turbt . en - bloc resection technique is one of the ways to provide better pathological evaluation for ta and t1 tumors . in a study on et , although the authors did not describe presence or absence of detrusor muscle in the resected specimen , they concluded that invasion of lamina was better delineated with en - bloc resection . a limitation of this technique was the inability to use a flat loop for tumors located at the anterior and upper posterior wall . it may not matter much whether we do et or ct for a small size papillary tumor , but for tumors of 2 - 4 cm size , et would give better tissue configuration for assessing the exact t stage and commenting on margin status . there is paucity of literature on et for tumors of 2 - 4 cm size . we used a 45 angle loop electrode , which helped in scooping out the tumor in retrograde fashion . once the tumor was resected en - bloc , removal of 4 cm size tumor from the urethra is a challenge particularly in a male patient . we could remove 3 cm size tumor in a female , but in males we had to cut the free lying tumor into two or three pieces . the most common technique was to place a nephroscope and hold the tumor with forceps to remove the whole assembly along with the tumor . results of this prospective non - randomized study have shown that recurrence rate in et was significantly lower than ct . the progression rate , though not significant , was lower with et . best results of et were seen in non - lamina invasive bladder cancer , where recurrence rate was reduced to 16% as compared with 53% with ct . therefore , en - bloc resection not only provided a tissue piece with all the layers of bladder in contiguity for better histopathological characterization , but also resulted in reduction in the recurrence rate . looking at the pattern of recurrence in our patients , it seems that et is a better way to reduce the risk of recurrence as it provides a complete resection as only 1 ( 16% ) patient had recurrence at the same site as compared to 6 ( 40% ) patients in ct group 6 ( 40% ) patients in ct group regarding the learning curve , et is rather more controlled technique of resection than ct as it gives better hemostasis and thereby a good vision , which is crucial to avoid complications . depth of the resection could also be modified with et and it does not take more than three cases to get a knack of this technique if it is started with a relatively small tumor . first , tumor retrieval from the bladder was technically difficult and it had to be cut into 2 - 3 pieces once it was lying free in the bladder . second , there could be surgeon related factors as ct was done by two different surgeons . turbt is a procedure with a varied outcome in terms of adequacy of resection , recurrence and progression . there are a few surrogate markers to assess adequate resection such as presence of detrusor muscle in the specimen and the rate of subsequent recurrence . successful management of bladder tumors ( particularly non - muscle - invasive tumors ) , relies on adequate initial resection and accurate histological diagnosis . an ideal turbt would mean complete resection of the visible tumor , resection of the surrounding healthy looking mucosa for up to 1 cm and then the removal of detrusor muscle . herr and donat described three ways to measure the quality of a good turbt , i.e. , complete resection , presence of deep muscle in the specimen and the rate of recurrence at the site of previous turbt . they also suggested classifying tumor resection as r0 ; microscopic negative margin , r1 with microscopic positive margin and r2 that is macroscopic positive margin . this kind of assessment is not practical in ct , but could be possible in en - bloc resection , where we can have a piece of tumor tissue , which has all three layers , i.e. , urothelium , lamina propria and detrusor muscle in contiguity . the outer - most surface of the detrusor muscle in the resected specimen could then be inked to assess margin status and thereby discerning a true perspective of level of resection , i.e. , r0-r1 [ figure 4 ] . en - bloc resection provides a tissue piece of the bladder with all the layers in contiguity to assess the t stage and margin status . in inset , a 3 cm tumor removed en - bloc inadequacy of ct is not only judged by absence of detrusor muscle in an initial specimen , but also by the rate of recurrence . recurrence is seen in 50 - 70% of non - muscle invasive bladder cancer , mostly during the 1 year . these may be due to incomplete resection , cell implantation or the tumor biology itself . incomplete resection seems to be the most important reason for the recurrence . in a review of seven randomized controlled trials , after controlling established factors for recurrence such as tumor size , multiplicity , stage and grade , it was concluded that a wide range of recurrence rate , i.e. , 0 - 46% , was due to the difference in quality of resection . inadequate resection leading to higher rate of recurrence at the same site is supported by another study where 81% of recurred tumor occurred at the site of previous resection . various techniques using different kinds of loops and laser have been described to improve the quality of turbt . en - bloc resection technique is one of the ways to provide better pathological evaluation for ta and t1 tumors . in a study on et , although the authors did not describe presence or absence of detrusor muscle in the resected specimen , they concluded that invasion of lamina was better delineated with en - bloc resection . a limitation of this technique was the inability to use a flat loop for tumors located at the anterior and upper posterior wall . it may not matter much whether we do et or ct for a small size papillary tumor , but for tumors of 2 - 4 cm size , et would give better tissue configuration for assessing the exact t stage and commenting on margin status . there is paucity of literature on et for tumors of 2 - 4 cm size . we used a 45 angle loop electrode , which helped in scooping out the tumor in retrograde fashion . once the tumor was resected en - bloc , removal of 4 cm size tumor from the urethra is a challenge particularly in a male patient . we could remove 3 cm size tumor in a female , but in males we had to cut the free lying tumor into two or three pieces . the most common technique was to place a nephroscope and hold the tumor with forceps to remove the whole assembly along with the tumor . results of this prospective non - randomized study have shown that recurrence rate in et was significantly lower than ct . the progression rate , though not significant , was lower with et . best results of et were seen in non - lamina invasive bladder cancer , where recurrence rate was reduced to 16% as compared with 53% with ct . therefore , en - bloc resection not only provided a tissue piece with all the layers of bladder in contiguity for better histopathological characterization , but also resulted in reduction in the recurrence rate . looking at the pattern of recurrence in our patients , it seems that et is a better way to reduce the risk of recurrence as it provides a complete resection as only 1 ( 16% ) patient had recurrence at the same site as compared to 6 ( 40% ) patients in ct group 6 ( 40% ) patients in ct group regarding the learning curve , et is rather more controlled technique of resection than ct as it gives better hemostasis and thereby a good vision , which is crucial to avoid complications . depth of the resection could also be modified with et and it does not take more than three cases to get a knack of this technique if it is started with a relatively small tumor . first , tumor retrieval from the bladder was technically difficult and it had to be cut into 2 - 3 pieces once it was lying free in the bladder . second , there could be surgeon related factors as ct was done by two different surgeons . it demands meticulous care and attention to achieve adequate cancer control and improve recurrence rates . in our study , though et did not significantly affect the progression rate , it showed a significant reduction in the recurrence rate for nmibc in comparison with ct . the concept of removing a bladder tumor by ct should be changed to a technique of en - bloc resection to provide better cancer control and long term outcome in non - muscle invasive bladder cancer .

purpose : conventional , transurethral resection of bladder tumor ( turbt ) involves piecemeal resection of the tumor and has a very high recurrence rate . we evaluated the outcome of en - bloc turbt ( et ) in comparison with conventional turbt ( ct ) in non - muscle invasive bladder carcinoma in terms of recurrencew and progression.materials and methods : from september 2007 to june 2011 , in a prospective non - randomized interventional setting , et was compared with ct in patients with solitary tumor of 2 - 4 cm size in terms of recurrence and progression . pedunculated tumors , size > 4 cm , tumors with associated hydroureteronephrosis and biopsy specimen with absent detrusor muscles were excluded . fisher 's exact test and survival analyses were used to compare the demography and the outcome.results:a total of 21 patients of et were compared with 24 patients of ct . mean tumor size was 2.8 cm in et and 3.3 cm in ct group . location of tumor , stage and grade were comparable in both groups . recurrence rate was 28.6% versus 62.5% ( p = 0.03 ) and progression rate was 19% versus 33.3% ( p = 0.32 ) in et versus ct group respectively . recurrence free survival was 45.1 ( 95% ci : 19.0 - 38 months ) and 28.5 ( 95% ci : 35.4 - 54.7 months ) in et and ct group ( p = 0.018 ) . progression free survival in et and ct was 48.32 ( 95% ci : 35.5 - 53.0 months ) and 44.26 ( 95% ci : 39.0 - 57.5 months ) , p = 0.46.conclusion:there was a significant reduction in the recurrence rate and time to recurrence with et . rate of progression was also relatively less with et , though not statistically significant .

INTRODUCTION MATERIALS AND METHODS None Sample size Inclusion criteria Exclusion criteria Treatment and follow-up Final analysis Technique of ET, sculpting and resection technique Statistical analysis RESULTS None Discussion CONCLUSION

for non - muscle invasive bladder carcinoma ( nmibc ) , transurethral resection of bladder tumor ( turbt ) is the cornerstone of treatment . reasons for such a high recurrence rate for nmibc have been cited as incomplete resection in initial turbt , aggressive tumor biology , implantation of the tumor cells and possibly a field change to start with . the conventional turbt ( ct ) technique involves piecemeal resection of the tumor , which is contrary to the established oncological principles of removing tumor in other parts of the body . unfortunately , there has never been a certain way to provide a complete resection of the visible tumor in the bladder . in view of the high rate of recurrence , perhaps turbt should be modified to provide en - bloc resection of the specimen , based on the established oncological principle of dissecting through normal tissue . this study was aimed at conceptualizing the technique of sculpting and resecting in form of et and comparing its outcomes in terms of recurrence and progression with the conventional technique of incising and scattering . after seeking an institutional ethics committee approval , a prospective non - randomized interventional study was conducted between september 2007 to june 2011 . , recurrence and progression rates were compared in two groups . clinical data on the size ( measured by ultrasonography in 37 patients and contrast enhanced computerized tomogram scan on 33 patients ) and the location of the tumor were recorded . with existing recurrence rate of 60% with ct , an estimate of reduction of recurrence rate by 40% gave a sample of 23 patients in each group to achieve 82% power at the significance level of 0.05 . patients presenting for the first time with single tumor on imaging and the tumor size from 2 to 4 cm were included in the study . patients with prior turbt and recurrences , pedunculated tumors , multiple tumors , biopsy specimen with absent detrusor muscles and those with tumor size of more than 4 cm were excluded . as patients with stage t2 and those with absence of detrusor muscle on the first turbt were excluded , for the final analyses there were 21 and 24 patients in et and ct groups respectively [ graph 1 ] . feasibility of en - bloc resection was initially established in tumors of less than 2 cm in six cases before initiating this study . angle of the loop electrode was changed manually from 90 to 45 for en - bloc transurethral resection of bladder tumor normal mucosa around the tumor base was scored by using coagulation current at setting of 60 - 80 w ( valley lab , usa ) . retrograde resection was started in the deep layer of detrusor muscle elevating the tumor off the base with the help of angle of the loop and beak of the resectoscope . free lying tumor which was too large to be taken out through urethra was cut into 2 - 3 pieces in a way that all the layers are there in the piece of the tumor tissue . use of a nephroscope and grasping forceps was helpful in retrieving the tumor , the largest single piece being a 3 cm tumor in a female . recurrences free survival ( rfs ) and progression - free survival ( pfs ) were calculated using kaplan - meier method and long rank test . with existing recurrence rate of 60% with ct , an estimate of reduction of recurrence rate by 40% gave a sample of 23 patients in each group to achieve 82% power at the significance level of 0.05 . patients presenting for the first time with single tumor on imaging and the tumor size from 2 to 4 cm were included in the study . patients with prior turbt and recurrences , pedunculated tumors , multiple tumors , biopsy specimen with absent detrusor muscles and those with tumor size of more than 4 cm were excluded . as patients with stage t2 and those with absence of detrusor muscle on the first turbt were excluded , for the final analyses there were 21 and 24 patients in et and ct groups respectively [ graph 1 ] . feasibility of en - bloc resection was initially established in tumors of less than 2 cm in six cases before initiating this study . angle of the loop electrode was changed manually from 90 to 45 for en - bloc transurethral resection of bladder tumor normal mucosa around the tumor base was scored by using coagulation current at setting of 60 - 80 w ( valley lab , usa ) . retrograde resection was started in the deep layer of detrusor muscle elevating the tumor off the base with the help of angle of the loop and beak of the resectoscope . free lying tumor which was too large to be taken out through urethra was cut into 2 - 3 pieces in a way that all the layers are there in the piece of the tumor tissue . use of a nephroscope and grasping forceps was helpful in retrieving the tumor , the largest single piece being a 3 cm tumor in a female . recurrences free survival ( rfs ) and progression - free survival ( pfs ) were calculated using kaplan - meier method and long rank test . with existing recurrence rate of 60% with ct , an estimate of reduction of recurrence rate by 40% gave a sample of 23 patients in each group to achieve 82% power at the significance level of 0.05 . patients presenting for the first time with single tumor on imaging and the tumor size from 2 to 4 cm were included in the study . patients with prior turbt and recurrences , pedunculated tumors , multiple tumors , biopsy specimen with absent detrusor muscles and those with tumor size of more than 4 cm were excluded . as patients with stage t2 and those with absence of detrusor muscle on the first turbt were excluded , for the final analyses there were 21 and 24 patients in et and ct groups respectively [ graph 1 ] . feasibility of en - bloc resection was initially established in tumors of less than 2 cm in six cases before initiating this study . angle of the loop electrode was changed manually from 90 to 45 for en - bloc transurethral resection of bladder tumor normal mucosa around the tumor base was scored by using coagulation current at setting of 60 - 80 w ( valley lab , usa ) . free lying tumor which was too large to be taken out through urethra was cut into 2 - 3 pieces in a way that all the layers are there in the piece of the tumor tissue . use of a nephroscope and grasping forceps was helpful in retrieving the tumor , the largest single piece being a 3 cm tumor in a female . recurrences free survival ( rfs ) and progression - free survival ( pfs ) were calculated using kaplan - meier method and long rank test . at the final analysis 21 patients of et the mean age was 52.6 12.2 and 55 13.6 years in et and ct group . stage , grade and locations of tumor in two groups on initial turbt tumor characteristics and the outcome in two groups of turbt recurrence rate was 28.6% versus 62.5% ( p = 0.03 ) and progression rate was 19% versus 33.3% ( p = 0.32 ) in et versus ct group respectively [ table 2 ] . in the et group , the recurrence in non - lamina invasive nmibc tumors was 16% , where as it was 53% in ct . similarly in lamina invasive tumor , the recurrence rate was 44% and 72% in et and ct group . mean rfs was 45.1 months ( 95% ci : 19.0 - 38 months ) with et and 28.5 months ( 95% ci : 35.4 - 54.7 months ) in ct with p value of 0.018 [ figure 2 and table 2 ] . regarding the site of tumor recurrence , only 1 ( 16% ) patient had recurrence at the same site in et group while 6 ( 40% ) patients had recurrence at the same site with ct [ table 3 ] . recurrence free survival site of recurrences in two groups four patients in en - bloc and eight patients in conventional group had disease progression ( 19% vs. 33.3% , p = 0.32 ) . mean pfs in et and ct group was 48.32 months ( 95% ci : 35.5 - 53.0 months ) and 44.26 months ( 95% ci : 39.0 - 57.5 months ) with p value of 0.46 [ figure 3 and table 2 ] . in ct group , six patients had progressed on first recurrence ; one had progressed after one recurrence and one patient had progressed after two recurrences . progression free survival turbt is a procedure with a varied outcome in terms of adequacy of resection , recurrence and progression . successful management of bladder tumors ( particularly non - muscle - invasive tumors ) , relies on adequate initial resection and accurate histological diagnosis . an ideal turbt would mean complete resection of the visible tumor , resection of the surrounding healthy looking mucosa for up to 1 cm and then the removal of detrusor muscle . , complete resection , presence of deep muscle in the specimen and the rate of recurrence at the site of previous turbt . this kind of assessment is not practical in ct , but could be possible in en - bloc resection , where we can have a piece of tumor tissue , which has all three layers , i.e. en - bloc resection provides a tissue piece of the bladder with all the layers in contiguity to assess the t stage and margin status . in inset , a 3 cm tumor removed en - bloc inadequacy of ct is not only judged by absence of detrusor muscle in an initial specimen , but also by the rate of recurrence . recurrence is seen in 50 - 70% of non - muscle invasive bladder cancer , mostly during the 1 year . in a review of seven randomized controlled trials , after controlling established factors for recurrence such as tumor size , multiplicity , stage and grade , it was concluded that a wide range of recurrence rate , i.e. en - bloc resection technique is one of the ways to provide better pathological evaluation for ta and t1 tumors . in a study on et , although the authors did not describe presence or absence of detrusor muscle in the resected specimen , they concluded that invasion of lamina was better delineated with en - bloc resection . it may not matter much whether we do et or ct for a small size papillary tumor , but for tumors of 2 - 4 cm size , et would give better tissue configuration for assessing the exact t stage and commenting on margin status . there is paucity of literature on et for tumors of 2 - 4 cm size . once the tumor was resected en - bloc , removal of 4 cm size tumor from the urethra is a challenge particularly in a male patient . we could remove 3 cm size tumor in a female , but in males we had to cut the free lying tumor into two or three pieces . results of this prospective non - randomized study have shown that recurrence rate in et was significantly lower than ct . best results of et were seen in non - lamina invasive bladder cancer , where recurrence rate was reduced to 16% as compared with 53% with ct . therefore , en - bloc resection not only provided a tissue piece with all the layers of bladder in contiguity for better histopathological characterization , but also resulted in reduction in the recurrence rate . looking at the pattern of recurrence in our patients , it seems that et is a better way to reduce the risk of recurrence as it provides a complete resection as only 1 ( 16% ) patient had recurrence at the same site as compared to 6 ( 40% ) patients in ct group 6 ( 40% ) patients in ct group regarding the learning curve , et is rather more controlled technique of resection than ct as it gives better hemostasis and thereby a good vision , which is crucial to avoid complications . turbt is a procedure with a varied outcome in terms of adequacy of resection , recurrence and progression . there are a few surrogate markers to assess adequate resection such as presence of detrusor muscle in the specimen and the rate of subsequent recurrence . successful management of bladder tumors ( particularly non - muscle - invasive tumors ) , relies on adequate initial resection and accurate histological diagnosis . an ideal turbt would mean complete resection of the visible tumor , resection of the surrounding healthy looking mucosa for up to 1 cm and then the removal of detrusor muscle . , complete resection , presence of deep muscle in the specimen and the rate of recurrence at the site of previous turbt . this kind of assessment is not practical in ct , but could be possible in en - bloc resection , where we can have a piece of tumor tissue , which has all three layers , i.e. en - bloc resection provides a tissue piece of the bladder with all the layers in contiguity to assess the t stage and margin status . in inset , a 3 cm tumor removed en - bloc inadequacy of ct is not only judged by absence of detrusor muscle in an initial specimen , but also by the rate of recurrence . recurrence is seen in 50 - 70% of non - muscle invasive bladder cancer , mostly during the 1 year . in a review of seven randomized controlled trials , after controlling established factors for recurrence such as tumor size , multiplicity , stage and grade , it was concluded that a wide range of recurrence rate , i.e. en - bloc resection technique is one of the ways to provide better pathological evaluation for ta and t1 tumors . in a study on et , although the authors did not describe presence or absence of detrusor muscle in the resected specimen , they concluded that invasion of lamina was better delineated with en - bloc resection . it may not matter much whether we do et or ct for a small size papillary tumor , but for tumors of 2 - 4 cm size , et would give better tissue configuration for assessing the exact t stage and commenting on margin status . there is paucity of literature on et for tumors of 2 - 4 cm size . once the tumor was resected en - bloc , removal of 4 cm size tumor from the urethra is a challenge particularly in a male patient . we could remove 3 cm size tumor in a female , but in males we had to cut the free lying tumor into two or three pieces . results of this prospective non - randomized study have shown that recurrence rate in et was significantly lower than ct . the progression rate , though not significant , was lower with et . best results of et were seen in non - lamina invasive bladder cancer , where recurrence rate was reduced to 16% as compared with 53% with ct . therefore , en - bloc resection not only provided a tissue piece with all the layers of bladder in contiguity for better histopathological characterization , but also resulted in reduction in the recurrence rate . looking at the pattern of recurrence in our patients , it seems that et is a better way to reduce the risk of recurrence as it provides a complete resection as only 1 ( 16% ) patient had recurrence at the same site as compared to 6 ( 40% ) patients in ct group 6 ( 40% ) patients in ct group regarding the learning curve , et is rather more controlled technique of resection than ct as it gives better hemostasis and thereby a good vision , which is crucial to avoid complications . depth of the resection could also be modified with et and it does not take more than three cases to get a knack of this technique if it is started with a relatively small tumor . turbt is a procedure with a varied outcome in terms of adequacy of resection , recurrence and progression . there are a few surrogate markers to assess adequate resection such as presence of detrusor muscle in the specimen and the rate of subsequent recurrence . successful management of bladder tumors ( particularly non - muscle - invasive tumors ) , relies on adequate initial resection and accurate histological diagnosis . an ideal turbt would mean complete resection of the visible tumor , resection of the surrounding healthy looking mucosa for up to 1 cm and then the removal of detrusor muscle . , complete resection , presence of deep muscle in the specimen and the rate of recurrence at the site of previous turbt . this kind of assessment is not practical in ct , but could be possible in en - bloc resection , where we can have a piece of tumor tissue , which has all three layers , i.e. en - bloc resection provides a tissue piece of the bladder with all the layers in contiguity to assess the t stage and margin status . in inset , a 3 cm tumor removed en - bloc inadequacy of ct is not only judged by absence of detrusor muscle in an initial specimen , but also by the rate of recurrence . recurrence is seen in 50 - 70% of non - muscle invasive bladder cancer , mostly during the 1 year . in a review of seven randomized controlled trials , after controlling established factors for recurrence such as tumor size , multiplicity , stage and grade , it was concluded that a wide range of recurrence rate , i.e. in a study on et , although the authors did not describe presence or absence of detrusor muscle in the resected specimen , they concluded that invasion of lamina was better delineated with en - bloc resection . it may not matter much whether we do et or ct for a small size papillary tumor , but for tumors of 2 - 4 cm size , et would give better tissue configuration for assessing the exact t stage and commenting on margin status . there is paucity of literature on et for tumors of 2 - 4 cm size . once the tumor was resected en - bloc , removal of 4 cm size tumor from the urethra is a challenge particularly in a male patient . results of this prospective non - randomized study have shown that recurrence rate in et was significantly lower than ct . the progression rate , though not significant , was lower with et . best results of et were seen in non - lamina invasive bladder cancer , where recurrence rate was reduced to 16% as compared with 53% with ct . therefore , en - bloc resection not only provided a tissue piece with all the layers of bladder in contiguity for better histopathological characterization , but also resulted in reduction in the recurrence rate . looking at the pattern of recurrence in our patients , it seems that et is a better way to reduce the risk of recurrence as it provides a complete resection as only 1 ( 16% ) patient had recurrence at the same site as compared to 6 ( 40% ) patients in ct group 6 ( 40% ) patients in ct group regarding the learning curve , et is rather more controlled technique of resection than ct as it gives better hemostasis and thereby a good vision , which is crucial to avoid complications . depth of the resection could also be modified with et and it does not take more than three cases to get a knack of this technique if it is started with a relatively small tumor . in our study , though et did not significantly affect the progression rate , it showed a significant reduction in the recurrence rate for nmibc in comparison with ct . the concept of removing a bladder tumor by ct should be changed to a technique of en - bloc resection to provide better cancer control and long term outcome in non - muscle invasive bladder cancer .

traumatic injury , often accompanied by hemorrhagic shock ( t / hs ) , continues to be the most common cause of death for young people and constitutes a significant source of morbidity and mortality for all ages [ 1 , 2 , 151 ] . western europe [ 147150 ] and a budding epidemic throughout asia and the middle east . traumatic brain injury ( tbi ) is also a major cause of disability , with survivors acquiring long - term cognitive , motor , behavioural or speech - language disabilities . the various forms of traumatic injury therefore represent a pandemic disease that affects every nation in the world without regard for economic development , racial or religious predominance , or political ideology ; this disease is acute in onset and often results in chronic , debilitating health problems affecting far beyond the individual victims . further complicating the primary damage in acute trauma is the increased susceptibility to sepsis and multiple organ dysfunction syndrome ( mods ) , a poorly understood syndrome of sequential and gradual loss of organ function . mods is the most frequent cause of late deaths post - injury , accounting for substantial morbidity and mortality [ 4 , 5 ] . mods is considered to be due , in part , to excessive or maladaptive activation of inflammatory pathways . organs such as the liver and the gut not only become damaged or dysfunctional from trauma - induced inflammation , but in turn further perpetuate this inflammatory vicious cycle [ 7 , 8 , 21 ] . furthermore , patients admitted to the intensive care unit following trauma and hemorrhage often become susceptible to infection second hit further complicating attempts at immunomodulation early in the clinical course ( fig . one hit represents the initial , massive tissue injury and shock and sirs along with remote organ injury . second hit refers to the less intense sirs that normally resolves but leaves the patient vulnerable to a secondary inflammatory hit that can reactivate the sirs and precipitate late mods . trauma acts as a trigger of a complex cascade of posttraumatic events that can be divided into a hemodynamic , metabolic , neuro - endocrine and immune responses leading to a multifocal pathophysiologic process . however , inflammation is not in itself detrimental . it is in most cases a well - coordinated communication network operating at an intermediate time scale between neural and longer - term endocrine processes . inflammation is necessary for the removal or reduction of challenges to the organism and subsequent restoration of homeostasis . however , hemorrhage and trauma , perhaps combined with failed attempts at therapy [ 13 , 14 ] , can induce a dysregulated acute inflammatory response that affects several organ systems and sets in motion a vicious cycle of inflammation damage inflammation [ 12 , 1518 ] driven by cytokines , chemokines , and products of damaged , dysfunctional , or stressed tissue ( fig . traumatic injury signals various cell types to produce cytokines , chemokines , and damps . in turn , damps re - activate and further propagate the production of inflammatory mediators , setting in motion a positive feedback loop of inflammationdamageinflammation . thus , though the inflammatory response is pivotal in clearing invading organisms and offending agents and promoting tissue repair , these same responses carried out under a set of extreme conditions can also compromise healthy tissue and further exacerbate inflammation [ 12 , 19 ] . a central question then is : how do we harness the beneficial effects of inflammation and allow proper lines of communication while simultaneously not allowing inflammation to exceed a threshold that becomes self - sustaining ? this review article will focus on the common inflammatory / immune responses to t / hs and tbi , and will aim to give an overview of both the current state of relevant translational / clinical research and several novel approaches being undertaken as trauma research moves from the bench to the bedside . the pathophysiology of t / hs and tbi is now understood to consist of different phases that form a continuum [ 7 , 107 ] . death from post - traumatic injury occurs in three phases . in the first phase , patients die immediately because of devastating trauma . in the second phase , which occurs during early resuscitation , death may be related to hypoxia or hypovolemia . in the third phase , days or weeks following injury , death may be due to general physical consequences of injury of which the dominant manifestations are adult respiratory distress syndrome ( ards ) and mods . in 1995 , two models were proposed for the exaggerated immune inflammatory response , known colloquially as the one hit model , which accounts for the initial , massive tissue injury and shock that gives rise to an intense systemic inflammatory response syndrome ( sirs ) with remote organ injury . the second hit model indicates the initial , less intense sirs that normally resolves but leaves the patient vulnerable to a secondary inflammatory hit that can reactivate the sirs and precipitate late mods ( fig . 1 ) in the case of tbi , primary brain injury consists primarily of unavoidable brain damage that occurs at the immediate moment of impact , resulting in the disruption of brain parenchyma and cerebral blood vessels . a secondary brain injury develops in the minutes to months following the original insult , progressively contributing to worsened neurological impairment . death of resident cells of the central nervous system has traditionally been thought to take place in two phases : an early necrotic and an ongoing , long - term apoptotic phase [ 154 , 155 ] . thirteen years after these two models regarding the pathophysiology of t / hs and tbi were proposed , the question arises of how the clinical community has benefited from these two theoretical models , with regard to decreasing patient mortality post - traumatic injury ; we will attempt to address this thorny question in this review . we know that the post - traumatic inflammatory process occurs at multiple scales and involves the activation of signaling pathways that mobilize inflammatory cells , and stimulate the secretion of multiple inflammatory mediators / biomarkers . the complexity of this response has stymied attempts at therapeutic modulation of trauma - induced inflammation , resulting in a dearth of therapeutic options , though , as we discuss below , novel approaches from the systems biology field may help in deciphering this complexity . cytokines are a broad class of protein hormones that mediate inflammatory and immune responses in a complex , context - sensitive manner [ 12 , 88 ] ( fig . 3 ] . not surprisingly , cytokines play a major role in the body 's response to t / hs and tbi [ 107 , 109 ] . major cytokines that participate in the response to trauma include tumor necrosis factor alpha ( tnf- ) , interleukin-1 beta ( il-1 ) , il-2 , il-6 , il-8 [ 20 , 24 , 25 ] , il-4 and recently il-18 . on the other hand , the cytokine il-10 counteracts the effects of the pro - inflammatory cytokines il-1 , il-6 and tnf- in various contexts , including severe hemorrhagic shock . unlike septic shock , where the cascade of cytokines is well defined , the role of cytokines in trauma and hemorrhagic shock is not well elucidated , the experimental and clinical data are conflicting , and the response in humans ( as opposed to animal models of t / hs ) is still poorly understood . circulating levels of cytokines have been detected in animal models and in patients with severe sepsis , and these levels have some correlation with outcome . production of the free radical nitric oxide ( no ) , which is produced in inflammatory settings by the enzyme inducible no synthase ( inos ) , was shown to be a central mediator of post - t / hs inflammation in mice . in human trauma patients , circulating no reaction products reflect the severity of injury during the first two hours after the traumatic insult , suggesting that increased no production might play a role in the very early post injury period . the spectrum of cytokines , chemokines , and damps in t / hs and tbi . the inflammatory response generated in response to t / hs or tbi can be assessed by measuring a panoply of cytokines , chemokines , damps , and ultimate markers of endorgan damage chemokines represent a class of cytokine - like immune modulators that are gaining attention as potential therapeutic targets for various inflammatory diseases [ 112 , 113 ] ( fig . chemokines are produced by a variety of immune cells ( innate and adaptive immunity ) such as macrophages , lymphocytes , neutrophils and dendritic cells that mediate various functions of these cells , including recruitment of other cells . chemokines have been the focus of intense study in relation to t / hs . the complex interaction between cytokines and chemokines may underlie the crucial role of these inflammatory modulators in the inflammatory process following t / hs and tbi and in other disease setting such as tumors , infection , and autoimmune disease . indeed , chemokines initiate recruitment of peripheral leukocytes after tbi , and evidence now exists for their intra - cerebral production [ 153 , 156 , 157 ] . among chemokines , macrophage inflammatory protein-1 alpha ( mip-1 ) appears to orchestrate both acute and chronic inflammatory host responses at the site of injury or infection , mainly by recruiting inflammatory cells [ 49 , 50 ] . additionally , mip-1 mediates an extensive repertoire of pro - inflammatory activities , including stimulating the secretion of tnf- , il-1 , and il-6 by peritoneal macrophages . studies in mice have shown that short - term manipulation of mip-1 following t / hs might be advantageous for diminishing the inflammatory response and improving vital organ dysfunction . as in most cases of therapeutic immunomodulation , inhibition of mip-1 is a two - edged sword , in this case an increased risk of late infection . monocyte chemoattractant protein ( mcp-1 ) , macrophage inflammatory protein-1 beta ( mip-1 ) , regulated on activation normal t cell expressed and secreted ( rantes ) , eotaxin , interferon - inducible protein 10 ( ip-10 ) , monokine induced by gamma interferon ( mig ) , and il-8 are chemokines that may offer novel therapeutic or diagnostic targets for t / hs . pathogen - associated molecular patterns ( pamps ) , damage - associated molecular patterns ( damp 's , also known as alarmins ) , and their receptors ( e.g. toll - like receptors [ tlr]-2 and -4 ; receptor for advanced glycation end products [ rage ] ) represent a parallel and perhaps integrative system that is turned on during infection as well as tissue injury , including t / hs and perhaps also tbi ( fig . pamps encompass a diverse set of microbial molecules that share various recognizable biochemical features that alert the organism to intruding pathogens [ 92 , 93 ] . such exogenous pamps are recognized by cells of the innate and acquired immune system , primarily through tlrs , which activate several signaling pathways among which nf-b is the most distinctive . for example , gram - negative bacterial lipopolysaccharide ( lps ) is the prototypical pamp . in an analogous fashion , damps are produced by injured tissue and stimulate or propagate inflammation through the production of cytokines ; in this way , damps play an important role in the pro - inflammatory cascade of innate immunity 9295 ( figs . 2 and 3 ) . molecules in this class of inflammatory mediators include high - mobility group box 1 ( hmgb1 ) , s100a and b , uric acid , il-1 , heat shock proteins , and a growing list of additional molecules ( fig . hmgb1 is produced in diverse settings such as infection , trauma , ischemia , t / hs , and tbi , which may contribute to the pathogenesis of severe sepsis along with other early , classical pro - inflammatory cytokines such as tnf- and il-1 . in animal studies , hmgb1 was shown to be a key mediator of inflammation in models of sterile injury , including hemorrhagic shock [ 99 , 100 ] . serum hmgb1 concentrations were significantly increased 16 - 32 h after exposure to lipopolysaccharide , and systemic administration of hmgb1 was lethal . antibodies to hmgb1 were shown to be protective even in the setting of established septic shock in mice . traumatic hemorrhage can be a consequence of direct injury to blood vessels , with massive bleeding , or as a result of diffuse bleeding secondary to coagulopathy in vessels too small and too numerous for surgical management . in the last few decades , the pathophysiology of the systemic response to t / hs has been studied extensively in an attempt to elucidate the hemodynamic mechanisms and immunological alterations associated with t / hs . however , translating these experimental findings into clinically applicable therapy has proven difficult , and investigators in this field are challenged by two sometimes mutually incompatible goals . researchers desire to minimize the animal - to - animal variability and at the same time seek to simulate clinical conditions . ideally , the experimental setup mimics the clinical situation associated with hemorrhagic shock in the trauma patient , while providing the controlled conditions that maximize reproducibility and standardization . there are three common variants of preclinical animal models , which all have their advantages and disadvantages . these experimental preparations are the uncontrolled hemorrhage model and the controlled hemorrhage model that is divided into two : the fixed pressure regimens , and the fixed volume models . although the standardization and reproducibility of this model is poor , it can be combined with organ and tissue injury , and allows for assessment of compensatory mechanisms . on the other hand , controlled hemorrhage offers a much better management of the degree of shock induced . in fixed volume model , animals are bled to a fixed amount of blood , usually based on the weight of the animal . it is not as clinically relevant as the uncontrolled hemorrhage model , but one can achieve a reasonably good management of the degree of shock induced . in a fixed pressure model , also called wiggers model , blood pressure is monitored and blood is removed or reinfused to achieve a fixed pressure . in these models , the degree and duration of hypotension can be controlled by using a variable stress ( blood loss ) to maintain a constant level of response ( blood level ) . however , the clinical comparability is poor and animals often need to be heparinized . heparin has been shown to confound results in experimental models of hemorrhagic shock like release of catecholamine 's and alter cytokine levels [ 34 , 35 ] . recent advances in computerized automation , however , raise the possibility that very precise hemorrhage can be carried out in both rats and mice . animal models of tbi include both paradigms of focal injury such as closed cortical impact , fluid percussion , or stab wound injury , as well as models that involve diffuse injury that occurs from the tissue distortion , or shear , caused by inertial forces present at the moment of injury [ 177 , 178 ] . these are most commonly separated into four main pathologies : traumatic axonal injury ( tai ) , diffuse hypoxic brain damage , diffuse brain swelling and diffuse vascular injury , which seems to be the worst of the four [ 177179 ] . in these animal models , il-1 and tnf- have been implicated as primary pro - inflammatory cytokines , while a potentially beneficial , anti - inflammatory role has been ascribed to il-10 . interleukin-1 has been characterized extensively in animal models of tbi as a promoter of neuroinflammation [ 158 , 159 ] . the neuronal damage resulting from il-1 release appears to be indirect , due to synergistic action with other pro - inflammatory cytokines such as tnf- [ 160 , 161 ] . like il-1 , tnf- has been regarded as a purely pro - inflammatory cytokine in the short history of tbi research . the time course of release of tnf- has is remarkably consistent across experimental paradigms of focal tbi in rodents ( closed cortical impact , fluid percussion , or stab wound injury ) , with detectable levels at 1 h post - injury , maximal concentration at 3 - 8 h , and a decline in release by 24 h within the brain [ 162 , 163 ] . in diffuse injury models , serum levels of tnf- rise within 24 h with an absence of expression in brain tissue , suggesting that diffuse injury induces a different immune response . similar to tnf- , il-6 has shown to play a role in neuroinflammation that is detected by 1 h post - injury in animal models , followed by a peak concentration between 2 and 8 h [ 153 , 165 , 166 ] . on the anti - inflammatory side , experimental studies have demonstrated a beneficial effect of il-10 , with exogenous administration of this cytokine aiding neurological recovery and reducing pro - inflammatory cytokine expression . translational research aims to apply scientific discoveries in basic science into the clinical level hoping to provide measures that predict outcome and to decrease the mortality rate in humans . in the setting of t / hs and tbi , initial efforts to understand the role of cytokines focused on post - traumatic blood levels and pharmacological therapy aimed at enhancing the protective cytokines and inhibiting the damaging cytokines are underway and have shown some improved survival rates in experimental animals . conclusions from these studies were that , at low concentrations , cytokines are important to the host response to trauma whereas in higher concentrations they are deleterious . the best characterized and , apparently , earliest and most fundamental cytokine in the trauma - induced pro - inflammatory cascade is tnf-. tnf- triggers the production of other cytokines , which amplify and propagate the inflammatory response where raised plasma tnf- have been found in hemorrhagic shock patients [ 97 , 98 ] . tnf- also participates in the generation of free radicals such as no [ 110 , 121 ] . clinical studies have demonstrated that levels of several inflammatory mediators , such as il-6 , il-8 and il-10 , correlate closely with severity of injury and complication rates [ 101104 ] . from the family of damps , serum hmgb1 was significantly increased in patients with sepsis , and the highest concentrations were observed in samples from patients who died . recent studies in hs patients suggested that hmgb1 may be involved in the pathogenesis of human hs outcome , though further studies are needed to determine hmgb1 role in the inflammatory response to trauma . we have demonstrated recently that mean post - t / hs hmgb1 levels samples within the first 24 h were higher in non - survivors vs. survivors , and that these levels correlated with various indices of injury severity including marshall score , creatinine , and circulating liver transferases . various intrinsic factors such as age , gender , race , body temperature , resuscitation , and hypotensive period , among others , play a role in how the body responds to acute traumatic injury . in addition , aspects of the injury itself ( assessed clinically as iss score , marshall score , lactate , and base deficit ) , as well as treatment with agents such as inotropes , are additional important variables that impact clinical outcomes . it is daunting to attempt to study this multitude of variables in the acute clinical setting , and thus they are often examined separately . for example , the effect of aging on the immune response to traumatic injury has been studied . indeed , the two processes ( inflammation and aging ) , have prompted some authors to coin the term inflamm - aging for this complex process . however , the characteristics of the aged inflammatory response vary occasionally between rodents ( the experimental animals typically used for studies of inflammation ) and humans . interestingly , inflammation in the aged is characterized by a confounding array of alterations in cytokine production rather than a clear - cut increase or decrease . several studies in vitro have reported enhanced production of il-6 , tnf- and il-1 in elderly human peripheral blood mononuclear cells compared to younger controls after inflammatory stimulation . in contrast , and illustrating the complex interplay of age and gender , spontaneous production of il-8 by elderly males is lower than that produced by elderly females and young controls . furthermore , there is a lower degree of in vitro - stimulated production of the chemokines mip-1 , rantes and il-8 by natural killer cells from elderly donors compared to younger ones . zhang et al showed elevated serum levels of cytokines , including ifn- , il12p40 and tnf- in aged compared with young mice . others have also shown that lps - induced cytokine production is increased in the serum of aged mice [ 46 , 47 ] . studies focused on gender specific differences in the response to traumatic injury in animal models suggest that this dimorphic response is , at least in part , based on the levels of estrogen , testosterone , or their derivatives [ 5254 ] . in this respect aet ( 5-androstene-3b , 7b , 17b - triol ) administered subcutaneously provided significant survival effect in a 40%-volume hemorrhage trauma model in rats . this was the first study to report the ability of aet to improve survival after traumatic shock . a clinical study provided evidence for differences in the early cytokine response between females and males after injury , with males having persistently elevated il-6 cytokine expression over time as compared to similarly injured females . an alternative hypothesis states that x - linked genetic differences between males and females , independent of hormonal status , responsible for these gender - based differential outcomes after injury in humans [ 56 , 57 , 58 ] . these studies suggest a new avenue for t / hs research and interaction with the field of endocrinology . for example , il-1 levels correlated with poor clinical outcome in either adult or pediatric population . patients with elevated cerebrospinal fluid ( csf ) levels of il-1 tended to have significantly poorer glasgow outcome scores [ 168 , 169 ] . tnf- in both serum and csf has been documented in clinical settings of patients with severe tbi . paradoxically , both neuroprotective and neurotoxic effects of tnf- have been suggested in human tbi , in terms of the inverse relationship of tnf- with the both pro - inflammatory il-18 and the anti - inflammatory il-10 [ 171 , 172 ] . measurements in a tbi population displayed maximal levels of il-6 in the csf between 3 and 6 days , with a steady decline in release thereafter . evidence for the intrathecal production of anti - inflammatory cytokines in tbi patients also exists . for example , il-10 was increased acutely within 24 h of injury , correlating with decreases in tnf-. in addition , transforming growth factor- 1 ( tgf-1 ) was elevated in both csf at day 1 and serum at 3 weeks post - injury [ 153 , 171 , 174 ] . interestingly , serum levels of il-10 were elevated in both the severely head injured , as well as those suffering polytrauma , potentially rendering this cytokine a nonspecific marker of tbi as well as pointing to common mechanisms of injury response in t / hs and tbi [ 169 , 175 , 176 ] . the acute inflammatory response is generally recognized as a complex system , both in structure and behavior . understanding and potentially manipulating the acute inflammatory response requires an extension beyond the traditional scientific paradigm of analysis via sequential reductionist experimentation . accomplishing this task requires a formal , explicit means of synthesis , heretofore an intuitive process carried out in the mind of the researcher . the emerging scientific discipline of systems biology , encompassing the search for information relating to the behavior of many biological components interacting in unison and often embodied in -omics technologies ( genomics , proteomics , metabolomics , etc . ) holds promise with regard to gaining definitive insights into biological processes [ 123132 ] . both genomic [ 81 , 133138 ] and proteomic [ 115 , 139143 ] approaches have begun to yield insights into the mechanisms of the response to t / hs . computational simulations are often used to integrate genomic and proteomic information , and have been used extensively by researchers dealing with such complex dynamic systems as studied in many fields [ 5962 ] but only recently in biology [ 6367 ] . both inflammation and associated processes ( e.g. apoptosis and organ damage / dysfunction ) have been studied at the molecular and cellular levels [ 6871 ] . given the central role of organ damage / dysfunction in acute illness , modeling at the tissue and organ level has also played an essential function , especially examining the issue of physiologic variability 73 . this type of modeling has been successful in yielding basic insights into acute inflammation [ 7578 ] including quantitative insights into the biology underlying experimental paradigms of acute inflammation in animals [ 7981 ] . a more recent concept has been that of translational systems biology [ 11 , 72 , 82 , 108 ] , which includes computational simulations of clinical trials [ 8386 ] , potential clinical diagnostics in the form of patient - specific models , streamlined usage of experimental animals , and rational device design . using these approaches , we have shed basic insights into the basic interactions of trauma with hemorrhage in mice , and have already begun to create patient - specific , predictive simulations in human t / hs and tbi . new knowledge derived from a rich set of studies in cells , animals , and humans , combined with computational methods that are rapidly coming into use , promises to revolutionize the way in which clinical studies and clinical practice in t / hs and tbi are being conducted . we are rapidly gaining a new understanding of the complex interactions between injury and the inflammatory response and vice versa , and these new insights will hopefully serve as the foundation for improving patient care worldwide . we may envision a point at which an integrated , rational , and iterative program of simulated clinical trials , in vitro screening for new drug compounds , pre - clinical studies , and human clinical trials will lead to a raft of new therapeutic options for t / hs and tbi ( fig . 4 ) . this new frontier increasingly requires training not only in clinical medicine , but also in quantitative sciences , bioinformatics , and translational science . moreover , this new approach highlights the need for inter- and multi - disciplinary teams . finally , emphasis should be placed on applying this new methodology to the difficult , complex clinical scenarios of combined t / hs and tbi , and especially integrating additional factors such as age , gender , genetics , and co - morbidities . despite the many challenges that remain , we are optimistic that a bright future lies ahead for the care of traumatic injury and critical illness . a vision for the future of drug design for t / hs and tbi . the future of rational drug design for t / hs and tbi may involve the use of in silico ( computer simulated ) that would be based on a mechanistic understanding of the inflammatory response as well as pharmacokinetic and pharmacodynamic principles and used to determine the optimal properties , dosage , timing , and inclusion / exclusion criteria for a given drug candidate 's clinical trial . key aspects of these simulations would be tested iteratively in cell culture experiments and pre - clinical animal models , streamlining the process ( and reducing the time and cost ) of clinical trial design and implementation .

traumatic injury / hemorrhagic shock ( t / hs ) elicits an acute inflammatory response that may result in death . inflammation describes a coordinated series of molecular , cellular , tissue , organ , and systemic responses that drive the pathology of various diseases including t / hs and traumatic brain injury ( tbi ) . inflammation is a finely tuned , dynamic , highly - regulated process that is not inherently detrimental , but rather required for immune surveillance , optimal post - injury tissue repair , and regeneration . the inflammatory response is driven by cytokines and chemokines and is partially propagated by damaged tissue - derived products ( damage - associated molecular patterns ; damp 's ) . damps perpetuate inflammation through the release of pro - inflammatory cytokines , but may also inhibit anti - inflammatory cytokines . various animal models of t / hs in mice , rats , pigs , dogs , and non - human primates have been utilized in an attempt to move from bench to bedside . novel approaches , including those from the field of systems biology , may yield therapeutic breakthroughs in t / hs and tbi in the near future .

Introduction Trauma and the immune response from a clinical perspective Animal models of T/HS and TBI Human studies of T/HS Systems biology approaches can shed insight into inflammation at the cellular, tissue, organ, and organism levels Conclusions and future prospects

traumatic injury , often accompanied by hemorrhagic shock ( t / hs ) , continues to be the most common cause of death for young people and constitutes a significant source of morbidity and mortality for all ages [ 1 , 2 , 151 ] . traumatic brain injury ( tbi ) is also a major cause of disability , with survivors acquiring long - term cognitive , motor , behavioural or speech - language disabilities . the various forms of traumatic injury therefore represent a pandemic disease that affects every nation in the world without regard for economic development , racial or religious predominance , or political ideology ; this disease is acute in onset and often results in chronic , debilitating health problems affecting far beyond the individual victims . mods is the most frequent cause of late deaths post - injury , accounting for substantial morbidity and mortality [ 4 , 5 ] . organs such as the liver and the gut not only become damaged or dysfunctional from trauma - induced inflammation , but in turn further perpetuate this inflammatory vicious cycle [ 7 , 8 , 21 ] . furthermore , patients admitted to the intensive care unit following trauma and hemorrhage often become susceptible to infection second hit further complicating attempts at immunomodulation early in the clinical course ( fig . however , inflammation is not in itself detrimental . inflammation is necessary for the removal or reduction of challenges to the organism and subsequent restoration of homeostasis . however , hemorrhage and trauma , perhaps combined with failed attempts at therapy [ 13 , 14 ] , can induce a dysregulated acute inflammatory response that affects several organ systems and sets in motion a vicious cycle of inflammation damage inflammation [ 12 , 1518 ] driven by cytokines , chemokines , and products of damaged , dysfunctional , or stressed tissue ( fig . traumatic injury signals various cell types to produce cytokines , chemokines , and damps . thus , though the inflammatory response is pivotal in clearing invading organisms and offending agents and promoting tissue repair , these same responses carried out under a set of extreme conditions can also compromise healthy tissue and further exacerbate inflammation [ 12 , 19 ] . this review article will focus on the common inflammatory / immune responses to t / hs and tbi , and will aim to give an overview of both the current state of relevant translational / clinical research and several novel approaches being undertaken as trauma research moves from the bench to the bedside . the pathophysiology of t / hs and tbi is now understood to consist of different phases that form a continuum [ 7 , 107 ] . death from post - traumatic injury occurs in three phases . in the first phase , patients die immediately because of devastating trauma . in the second phase , which occurs during early resuscitation , death may be related to hypoxia or hypovolemia . in 1995 , two models were proposed for the exaggerated immune inflammatory response , known colloquially as the one hit model , which accounts for the initial , massive tissue injury and shock that gives rise to an intense systemic inflammatory response syndrome ( sirs ) with remote organ injury . 1 ) in the case of tbi , primary brain injury consists primarily of unavoidable brain damage that occurs at the immediate moment of impact , resulting in the disruption of brain parenchyma and cerebral blood vessels . a secondary brain injury develops in the minutes to months following the original insult , progressively contributing to worsened neurological impairment . thirteen years after these two models regarding the pathophysiology of t / hs and tbi were proposed , the question arises of how the clinical community has benefited from these two theoretical models , with regard to decreasing patient mortality post - traumatic injury ; we will attempt to address this thorny question in this review . we know that the post - traumatic inflammatory process occurs at multiple scales and involves the activation of signaling pathways that mobilize inflammatory cells , and stimulate the secretion of multiple inflammatory mediators / biomarkers . the complexity of this response has stymied attempts at therapeutic modulation of trauma - induced inflammation , resulting in a dearth of therapeutic options , though , as we discuss below , novel approaches from the systems biology field may help in deciphering this complexity . not surprisingly , cytokines play a major role in the body 's response to t / hs and tbi [ 107 , 109 ] . major cytokines that participate in the response to trauma include tumor necrosis factor alpha ( tnf- ) , interleukin-1 beta ( il-1 ) , il-2 , il-6 , il-8 [ 20 , 24 , 25 ] , il-4 and recently il-18 . on the other hand , the cytokine il-10 counteracts the effects of the pro - inflammatory cytokines il-1 , il-6 and tnf- in various contexts , including severe hemorrhagic shock . unlike septic shock , where the cascade of cytokines is well defined , the role of cytokines in trauma and hemorrhagic shock is not well elucidated , the experimental and clinical data are conflicting , and the response in humans ( as opposed to animal models of t / hs ) is still poorly understood . circulating levels of cytokines have been detected in animal models and in patients with severe sepsis , and these levels have some correlation with outcome . production of the free radical nitric oxide ( no ) , which is produced in inflammatory settings by the enzyme inducible no synthase ( inos ) , was shown to be a central mediator of post - t / hs inflammation in mice . in human trauma patients , circulating no reaction products reflect the severity of injury during the first two hours after the traumatic insult , suggesting that increased no production might play a role in the very early post injury period . the spectrum of cytokines , chemokines , and damps in t / hs and tbi . the inflammatory response generated in response to t / hs or tbi can be assessed by measuring a panoply of cytokines , chemokines , damps , and ultimate markers of endorgan damage chemokines represent a class of cytokine - like immune modulators that are gaining attention as potential therapeutic targets for various inflammatory diseases [ 112 , 113 ] ( fig . chemokines are produced by a variety of immune cells ( innate and adaptive immunity ) such as macrophages , lymphocytes , neutrophils and dendritic cells that mediate various functions of these cells , including recruitment of other cells . chemokines have been the focus of intense study in relation to t / hs . the complex interaction between cytokines and chemokines may underlie the crucial role of these inflammatory modulators in the inflammatory process following t / hs and tbi and in other disease setting such as tumors , infection , and autoimmune disease . additionally , mip-1 mediates an extensive repertoire of pro - inflammatory activities , including stimulating the secretion of tnf- , il-1 , and il-6 by peritoneal macrophages . studies in mice have shown that short - term manipulation of mip-1 following t / hs might be advantageous for diminishing the inflammatory response and improving vital organ dysfunction . as in most cases of therapeutic immunomodulation , inhibition of mip-1 is a two - edged sword , in this case an increased risk of late infection . monocyte chemoattractant protein ( mcp-1 ) , macrophage inflammatory protein-1 beta ( mip-1 ) , regulated on activation normal t cell expressed and secreted ( rantes ) , eotaxin , interferon - inducible protein 10 ( ip-10 ) , monokine induced by gamma interferon ( mig ) , and il-8 are chemokines that may offer novel therapeutic or diagnostic targets for t / hs . pathogen - associated molecular patterns ( pamps ) , damage - associated molecular patterns ( damp 's , also known as alarmins ) , and their receptors ( e.g. toll - like receptors [ tlr]-2 and -4 ; receptor for advanced glycation end products [ rage ] ) represent a parallel and perhaps integrative system that is turned on during infection as well as tissue injury , including t / hs and perhaps also tbi ( fig . in an analogous fashion , damps are produced by injured tissue and stimulate or propagate inflammation through the production of cytokines ; in this way , damps play an important role in the pro - inflammatory cascade of innate immunity 9295 ( figs . molecules in this class of inflammatory mediators include high - mobility group box 1 ( hmgb1 ) , s100a and b , uric acid , il-1 , heat shock proteins , and a growing list of additional molecules ( fig . hmgb1 is produced in diverse settings such as infection , trauma , ischemia , t / hs , and tbi , which may contribute to the pathogenesis of severe sepsis along with other early , classical pro - inflammatory cytokines such as tnf- and il-1 . in animal studies , hmgb1 was shown to be a key mediator of inflammation in models of sterile injury , including hemorrhagic shock [ 99 , 100 ] . serum hmgb1 concentrations were significantly increased 16 - 32 h after exposure to lipopolysaccharide , and systemic administration of hmgb1 was lethal . antibodies to hmgb1 were shown to be protective even in the setting of established septic shock in mice . in the last few decades , the pathophysiology of the systemic response to t / hs has been studied extensively in an attempt to elucidate the hemodynamic mechanisms and immunological alterations associated with t / hs . however , translating these experimental findings into clinically applicable therapy has proven difficult , and investigators in this field are challenged by two sometimes mutually incompatible goals . ideally , the experimental setup mimics the clinical situation associated with hemorrhagic shock in the trauma patient , while providing the controlled conditions that maximize reproducibility and standardization . there are three common variants of preclinical animal models , which all have their advantages and disadvantages . these experimental preparations are the uncontrolled hemorrhage model and the controlled hemorrhage model that is divided into two : the fixed pressure regimens , and the fixed volume models . although the standardization and reproducibility of this model is poor , it can be combined with organ and tissue injury , and allows for assessment of compensatory mechanisms . it is not as clinically relevant as the uncontrolled hemorrhage model , but one can achieve a reasonably good management of the degree of shock induced . heparin has been shown to confound results in experimental models of hemorrhagic shock like release of catecholamine 's and alter cytokine levels [ 34 , 35 ] . animal models of tbi include both paradigms of focal injury such as closed cortical impact , fluid percussion , or stab wound injury , as well as models that involve diffuse injury that occurs from the tissue distortion , or shear , caused by inertial forces present at the moment of injury [ 177 , 178 ] . these are most commonly separated into four main pathologies : traumatic axonal injury ( tai ) , diffuse hypoxic brain damage , diffuse brain swelling and diffuse vascular injury , which seems to be the worst of the four [ 177179 ] . in these animal models , il-1 and tnf- have been implicated as primary pro - inflammatory cytokines , while a potentially beneficial , anti - inflammatory role has been ascribed to il-10 . interleukin-1 has been characterized extensively in animal models of tbi as a promoter of neuroinflammation [ 158 , 159 ] . the neuronal damage resulting from il-1 release appears to be indirect , due to synergistic action with other pro - inflammatory cytokines such as tnf- [ 160 , 161 ] . like il-1 , tnf- has been regarded as a purely pro - inflammatory cytokine in the short history of tbi research . the time course of release of tnf- has is remarkably consistent across experimental paradigms of focal tbi in rodents ( closed cortical impact , fluid percussion , or stab wound injury ) , with detectable levels at 1 h post - injury , maximal concentration at 3 - 8 h , and a decline in release by 24 h within the brain [ 162 , 163 ] . in diffuse injury models , serum levels of tnf- rise within 24 h with an absence of expression in brain tissue , suggesting that diffuse injury induces a different immune response . similar to tnf- , il-6 has shown to play a role in neuroinflammation that is detected by 1 h post - injury in animal models , followed by a peak concentration between 2 and 8 h [ 153 , 165 , 166 ] . on the anti - inflammatory side , experimental studies have demonstrated a beneficial effect of il-10 , with exogenous administration of this cytokine aiding neurological recovery and reducing pro - inflammatory cytokine expression . in the setting of t / hs and tbi , initial efforts to understand the role of cytokines focused on post - traumatic blood levels and pharmacological therapy aimed at enhancing the protective cytokines and inhibiting the damaging cytokines are underway and have shown some improved survival rates in experimental animals . the best characterized and , apparently , earliest and most fundamental cytokine in the trauma - induced pro - inflammatory cascade is tnf-. tnf- triggers the production of other cytokines , which amplify and propagate the inflammatory response where raised plasma tnf- have been found in hemorrhagic shock patients [ 97 , 98 ] . from the family of damps , serum hmgb1 was significantly increased in patients with sepsis , and the highest concentrations were observed in samples from patients who died . recent studies in hs patients suggested that hmgb1 may be involved in the pathogenesis of human hs outcome , though further studies are needed to determine hmgb1 role in the inflammatory response to trauma . we have demonstrated recently that mean post - t / hs hmgb1 levels samples within the first 24 h were higher in non - survivors vs. survivors , and that these levels correlated with various indices of injury severity including marshall score , creatinine , and circulating liver transferases . various intrinsic factors such as age , gender , race , body temperature , resuscitation , and hypotensive period , among others , play a role in how the body responds to acute traumatic injury . in addition , aspects of the injury itself ( assessed clinically as iss score , marshall score , lactate , and base deficit ) , as well as treatment with agents such as inotropes , are additional important variables that impact clinical outcomes . it is daunting to attempt to study this multitude of variables in the acute clinical setting , and thus they are often examined separately . however , the characteristics of the aged inflammatory response vary occasionally between rodents ( the experimental animals typically used for studies of inflammation ) and humans . furthermore , there is a lower degree of in vitro - stimulated production of the chemokines mip-1 , rantes and il-8 by natural killer cells from elderly donors compared to younger ones . zhang et al showed elevated serum levels of cytokines , including ifn- , il12p40 and tnf- in aged compared with young mice . others have also shown that lps - induced cytokine production is increased in the serum of aged mice [ 46 , 47 ] . studies focused on gender specific differences in the response to traumatic injury in animal models suggest that this dimorphic response is , at least in part , based on the levels of estrogen , testosterone , or their derivatives [ 5254 ] . a clinical study provided evidence for differences in the early cytokine response between females and males after injury , with males having persistently elevated il-6 cytokine expression over time as compared to similarly injured females . these studies suggest a new avenue for t / hs research and interaction with the field of endocrinology . paradoxically , both neuroprotective and neurotoxic effects of tnf- have been suggested in human tbi , in terms of the inverse relationship of tnf- with the both pro - inflammatory il-18 and the anti - inflammatory il-10 [ 171 , 172 ] . measurements in a tbi population displayed maximal levels of il-6 in the csf between 3 and 6 days , with a steady decline in release thereafter . evidence for the intrathecal production of anti - inflammatory cytokines in tbi patients also exists . in addition , transforming growth factor- 1 ( tgf-1 ) was elevated in both csf at day 1 and serum at 3 weeks post - injury [ 153 , 171 , 174 ] . interestingly , serum levels of il-10 were elevated in both the severely head injured , as well as those suffering polytrauma , potentially rendering this cytokine a nonspecific marker of tbi as well as pointing to common mechanisms of injury response in t / hs and tbi [ 169 , 175 , 176 ] . the acute inflammatory response is generally recognized as a complex system , both in structure and behavior . understanding and potentially manipulating the acute inflammatory response requires an extension beyond the traditional scientific paradigm of analysis via sequential reductionist experimentation . accomplishing this task requires a formal , explicit means of synthesis , heretofore an intuitive process carried out in the mind of the researcher . the emerging scientific discipline of systems biology , encompassing the search for information relating to the behavior of many biological components interacting in unison and often embodied in -omics technologies ( genomics , proteomics , metabolomics , etc . ) both genomic [ 81 , 133138 ] and proteomic [ 115 , 139143 ] approaches have begun to yield insights into the mechanisms of the response to t / hs . computational simulations are often used to integrate genomic and proteomic information , and have been used extensively by researchers dealing with such complex dynamic systems as studied in many fields [ 5962 ] but only recently in biology [ 6367 ] . apoptosis and organ damage / dysfunction ) have been studied at the molecular and cellular levels [ 6871 ] . a more recent concept has been that of translational systems biology [ 11 , 72 , 82 , 108 ] , which includes computational simulations of clinical trials [ 8386 ] , potential clinical diagnostics in the form of patient - specific models , streamlined usage of experimental animals , and rational device design . using these approaches , we have shed basic insights into the basic interactions of trauma with hemorrhage in mice , and have already begun to create patient - specific , predictive simulations in human t / hs and tbi . new knowledge derived from a rich set of studies in cells , animals , and humans , combined with computational methods that are rapidly coming into use , promises to revolutionize the way in which clinical studies and clinical practice in t / hs and tbi are being conducted . we are rapidly gaining a new understanding of the complex interactions between injury and the inflammatory response and vice versa , and these new insights will hopefully serve as the foundation for improving patient care worldwide . we may envision a point at which an integrated , rational , and iterative program of simulated clinical trials , in vitro screening for new drug compounds , pre - clinical studies , and human clinical trials will lead to a raft of new therapeutic options for t / hs and tbi ( fig . this new frontier increasingly requires training not only in clinical medicine , but also in quantitative sciences , bioinformatics , and translational science . finally , emphasis should be placed on applying this new methodology to the difficult , complex clinical scenarios of combined t / hs and tbi , and especially integrating additional factors such as age , gender , genetics , and co - morbidities . a vision for the future of drug design for t / hs and tbi . the future of rational drug design for t / hs and tbi may involve the use of in silico ( computer simulated ) that would be based on a mechanistic understanding of the inflammatory response as well as pharmacokinetic and pharmacodynamic principles and used to determine the optimal properties , dosage , timing , and inclusion / exclusion criteria for a given drug candidate 's clinical trial .

the interpro resource ( http://www.ebi.ac.uk/interpro ) ( 1 ) provides an integrated set of computational models ( or signatures ) for protein family classification and the prediction of structural and functional domains , sites and repeats . the predictive signatures are built by the 11 interpro member databases that , together with the interpro team at the ebi , comprise the interpro consortium . the member databases are gene3d ( 2 ) , hamap ( 3 ) , panther ( 4 ) , pfam ( 5 ) , pirsf ( 6 ) , prints ( 7 ) , prodom ( 8) , prosite ( 9 ) , smart ( 10 ) , superfamily ( 11 ) and tigrfams ( 12 ) . the interpro team at the ebi integrates the predictive signatures from these member databases into interpro entries. each entry may include one or more signatures that either identify the same feature , or classify proteins into the same family . additionally , entries are collated into two biologically principled hierarchies , one of which describes protein families , the other protein domains . interpro entries are curated by a team of experts in a variety of fields in biology . the curation process includes the creation of entries , the structuring of the entry hierarchies , the provision of detailed abstracts describing each entry and the addition of useful cross - references to other databases and ontologies . an example interpro entry , as viewed on the main interpro website , is illustrated in figure 1 . this example entry comprises two member database signatures , one from pfam and the other from superfamily . in total , this interpro entry matches 2753 uniprotkb protein sequences . figure 1.an example human - curated interpro entry , illustrating the detailed description provided for the entry and cross references to the go and the member database signatures from which the entry is composed . an example human - curated interpro entry , illustrating the detailed description provided for the entry and cross references to the go and the member database signatures from which the entry is composed . the integration described above is useful because the individual member databases have distinct but overlapping interests and use a number of different algorithms and modeling techniques . from the perspective of the biologist or bioinformatician wishing to use these predictive techniques , interpro allows consideration of all of the available signatures from a single resource , without the need to be concerned with differences or overlap between the foci of the individual member databases . as well as integrating the member database signatures , interpro calculates matches to these signatures for the whole of the uniprot knowledge base ( uniprotkb , http://www.uniprot.org ) and the uniparc sequence archive ( 13 ) . figure 2 illustrates a set of matches to a single uniprotkb protein sequence , which matches three interpro entries . the matches of interpro signatures and entries to the sequences in uniprotkb are available from the main interpro website as well as from the interpro biomart , however , at the time of writing , the matches to uniparc sequences are only available from the biomart . it is expected that uniparc matches will be included in a future version of the main interpro website . the interpro biomart is built on the technology developed by the biomart project ( http://www.biomart.org ) ( 14 , 15 ) , a collaboration between the ontario institute for cancer research ( oicr ) and the european bioinformatics institute ( ebi ) . it is also incorporated into the biomart central server at http://www.biomart.org/biomart/martview ( 16 ) . figure 2.a protein for which matches have been calculated by interpro . for this sequence , interpro provides a prediction of protein family membership , an overview of the domain organization and the details of matches to member database signatures . at the foot of the view can be seen associated go terms , based upon the calculated matches to interpro entries . a protein for which matches have been calculated by interpro . for this sequence , interpro provides a prediction of protein family membership , an overview of the domain organization and the details of matches to member database signatures . at the foot of the view can be seen associated go terms , based upon the calculated matches to interpro entries . the adoption of biomart as a mechanism to share the data in interpro has been motivated by the benefits that biomart brings : the ability to build complex filters on the data ; the facility to select specifically which data types are returned ( equivalent to the columns of a spreadsheet ) ; the capacity of biomart to handle queries that return many thousands of rows of data and the provision of a web service with an associated data federation mechanism . the interpro biomart provides three data sources : interpro entry annotation , uniprotkb protein matches and uniparc protein matches. match information can be obtained from both the interpro entry annotation data source and the uniprotkb protein matches data source . these two data sources provide a different slant on the contents of interpro , as described below . interpro entry annotation data source focuses on descriptions of the interpro entries and the hierarchical relationships between them . the user can therefore build filters using this annotation and retrieve more detailed information , such as assigned gene ontology ( 19 ) terms and cross - references to other , related databases . uniprotkb protein matches data set is focused on the uniprotkb protein entity , allowing queries to be built based on attributes of the protein sequence , including options to filter on the taxonomic group annotated on the sequence . this data set also provides the opportunity to retrieve match information with respect to member database signatures as well as summarized match information , described as supermatch is determined where one or more member database signatures that have been integrated together into the same entry have overlapping matches to the protein in the same region of the sequence . supermatch are then calculated as the most extreme bounds of the matches of all the member databases signatures comprising the entry . uniparc protein matches data set provides equivalent information to the uniprotkb protein matches data set , coordinated on sequences included in the uniparc database , a non - redundant , historical archive of protein sequences extracted from public databases . at the time of writing , the uniparc database includes 25.6 million unique sequences ; the interpro match calculation pipeline is run against all of these sequences and the results are made available from this biomart data set . this service allows matches to be returned for sequences that are present in ( for example ) model organism protein sequence databases which are not yet represented in uniprotkb . for users interested in matches for specific protein sequences , this data set supports filtering by uniparc i d or sequence checksum ( crc-64 or md5 ) , as does the uniprotkb protein matches data set . if the user wishes to query using protein accessions or identifiers from third - party sequence databases , various services are available that allow protein identifier cross - referencing , including the protein identifier cross reference service , picr ( http://www.ebi.ac.uk/tools/picr/ ) ( 20 ) and the uniprot i d mapping service ( http://www.uniprot.org/ ) . both of these services can be used to convert protein identifiers or accessions from a large number of protein sequence databases to uniparc sequence identifiers . the three interpro biomart data sources include matches to the full taxonomy range in uniprotkb or uniparc . in this respect , the interpro biomart is different in structure to the ensembl biomart ( http://www.ensembl.org/biomart/martview ) ( 17 , 18 ) which is organized into species - specific data sets . the interpro biomart provides three data sources : interpro entry annotation , uniprotkb protein matches and uniparc protein matches. match information can be obtained from both the interpro entry annotation data source and the uniprotkb protein matches data source . these two data sources provide a different slant on the contents of interpro , as described below . interpro entry annotation data source focuses on descriptions of the interpro entries and the hierarchical relationships between them . the user can therefore build filters using this annotation and retrieve more detailed information , such as assigned gene ontology ( 19 ) terms and cross - references to other , related databases . uniprotkb protein matches data set is focused on the uniprotkb protein entity , allowing queries to be built based on attributes of the protein sequence , including options to filter on the taxonomic group annotated on the sequence . this data set also provides the opportunity to retrieve match information with respect to member database signatures as well as summarized match information , described as supermatch is determined where one or more member database signatures that have been integrated together into the same entry have overlapping matches to the protein in the same region of the sequence . supermatch are then calculated as the most extreme bounds of the matches of all the member databases signatures comprising the entry . uniparc protein matches data set provides equivalent information to the uniprotkb protein matches data set , coordinated on sequences included in the uniparc database , a non - redundant , historical archive of protein sequences extracted from public databases . at the time of writing , the uniparc database includes 25.6 million unique sequences ; the interpro match calculation pipeline is run against all of these sequences and the results are made available from this biomart data set . this service allows matches to be returned for sequences that are present in ( for example ) model organism protein sequence databases which are not yet represented in uniprotkb . for users interested in matches for specific protein sequences , this data set supports filtering by uniparc i d or sequence checksum ( crc-64 or md5 ) , as does the uniprotkb protein matches data set . if the user wishes to query using protein accessions or identifiers from third - party sequence databases , various services are available that allow protein identifier cross - referencing , including the protein identifier cross reference service , picr ( http://www.ebi.ac.uk/tools/picr/ ) ( 20 ) and the uniprot i d mapping service ( http://www.uniprot.org/ ) . both of these services can be used to convert protein identifiers or accessions from a large number of protein sequence databases to uniparc sequence identifiers . the three interpro biomart data sources include matches to the full taxonomy range in uniprotkb or uniparc . in this respect , the interpro biomart is different in structure to the ensembl biomart ( http://www.ensembl.org/biomart/martview ) ( 17 , 18 ) which is organized into species - specific data sets . the interpro biomart is used to extend the functionality of the primary interpro web interface , providing biomart this allows data to be downloaded in tab- or comma - separated values format , suitable for computational analysis . the interpro biomart web service is the data source behind the interpro distributed annotation system ( das ) service ( 21 ) , available from http://www.ebi.ac.uk/das-srv/interpro/das . interpro , which contains all interpro member database signature matches to uniprotkb protein sequences.interpro - matches - overview that provides the maximum extent of the matches from all signatures that are integrated into a single interpro entry against uniprotkb protein sequences . supermatch matches described in the biomart uniprotkb protein matches data set.interpro - uniparc - matches that provides match information for protein sequences identified using uniparc identifiers.interpro - s4 is used to provide protein family classification to the new ebi search service and is therefore part of the wider programme of data integration at the ebi . interpro , which contains all interpro member database signature matches to uniprotkb protein sequences . interpro - matches - overview that provides the maximum extent of the matches from all signatures that are integrated into a single interpro entry against uniprotkb protein sequences . interpro - uniparc - matches that provides match information for protein sequences identified using uniparc identifiers . interpro - s4 is used to provide protein family classification to the new ebi search service and is therefore part of the wider programme of data integration at the ebi . in common with all biomart implementations , the interpro biomart enables the construction of simple queries as well as complex , multi - faceted queries where the data is filtered on several criteria . where multiple filters are applied , records are returned that meet all of the filter criteria ( i.e. and logic is applied across the filters ) . the user is able to specify precisely which data attributes should be returned , equivalent to columns in a spreadsheet . users should be aware that the structure of a biomart database , which is highly redundant to facilitate high query speed , can result in redundancy in the results reported . the presence of repeated rows of results in the output depends on the construction of the query and the structure of the underlying biomart tables . the authors therefore recommend the use of the unique results only option when querying the biomart , which removes repeated rows of results . which pfam signatures does interpro integrate into family entries? data setsfiltersattributesinterpro entry annotationinterpro entry type : familyinterpro entry accessionsource signature database : pfaminterpro entry short namesignature accessionsignature i d ( name ) the pfam database contains a broad spectrum of hidden markov models that can be used to predict both family classification and domain organization . the interpro curation team has integrated > 96% of pfam signatures into interpro at the time of writing . during integration , interpro assigns a type to an interpro entry and by extension , its signatures , dependent on what is being represented ( a family , domain , site or repeat ) . using the biomart , it is possible to return the full set of integrated pfam signatures that interpro considers to be of type family. this query can be easily modified to request signatures built by any of the member databases that fit into any of the available interpro entry types . repeat. each interpro entry has exactly one type and consequently all integrated member database signatures also have one type , as assigned by the interpro curation team . figure 3 illustrates selection of the interpro entry annotation data set . following selection of this data set , the user is able to select filters and attributes ( in whichever order they choose ) . figure 4 illustrates the selection of the two filters applied in this query , which will restrict the rows of data returned . figure 5 illustrates the selection of attributes , which are equivalent to the columns of a spreadsheet . initially , the user is presented with the first 10 matching rows of data , giving an opportunity to refine the query prior to requesting the full set of results . figure 4.building a filter with two components : include results for family entry types that comprise signatures from pfam . figure 5.selecting the attributes to be included in the biomart output ( equivalent to the columns of a spreadsheet ) . the ordering of the columns is determined by the order in which the attributes are selected . results button at the top of the interface provides the first 10 results matching the query , to allow the query to be modified or improved . selecting a dataset in the interpro biomart . building a filter with two components : include results for family entry types that comprise signatures from pfam . selecting the attributes to be included in the biomart output ( equivalent to the columns of a spreadsheet ) . the ordering of the columns is determined by the order in which the attributes are selected . results button at the top of the interface provides the first 10 results matching the query , to allow the query to be modified or improved . which go terms are mapped to prosite signatures in interpro ( i.e. can i retrieve a prosite2go mapping?) data setsfiltersattributesinterpro entry annotationsource signature database : prosite patterns and prosite profiles ( ctrl click to select both).interpro entry accessionsignature accessiongo idgo term namego root term ( process / component / function ) a major use of interpro is the association of go terms to proteins via the signatures that they match . interpro provides the interpro2go mappings as a file that can be downloaded from the ftp site ; however , it is difficult to extract subsets of information from this file . in the past , a frequent request from users was the provision of go term mapping information for a particular member database . with the advent of the biomart , it is now very easy to provide this information as illustrated above . query # 3 . which metabolic pathways are associated with proteins matching the interpro family ( cxcr4 , ipr001277)? data setsfiltersattributespathwaypathway stable idpathway nameinterpro entry annotationinterpro entry i d = ipr001277interpro entry accessioninterpro entry nameuniprotkb protein accessionuniprotkb protein i d ( name)source signature databasesignature accessionsignature i d ( name)match start positionmatch stop position the interpro biomart is federated with the reactome biomart ( 22 , 23 ) from which the reactions , pathways and biological processes and as such , can provide valuable biological insight if married to the data in interpro . ( ipr009138 ) been identified by mass spectrometry? data setsfiltersattributespridepride experiment accessionexperiment titlesample nametaxonomy term ( newt / ncbi taxon)taxonomy i d ( newt / ncbi taxon)tissue ontology term ( brenda)brenda i d ( tissue)cell type term ( cl)cl i d ( cell type)gene ontology term ( go)go i d ( gene ontology)interpro entriesinterpro entry i d = ipr009138interpro entry accessioninterpro entry name the interpro biomart is also federated with the pride biomart . pride is the proteomics identifications database , which contains identifications of proteins and peptides arising from mass spectrometry . the two biomarts are linked via uniprotkb protein accessions , so this query returns information about identifications of the proteins that match the member database signatures integrated into interpro entry ipr009138 . query # 1 . which pfam signatures does interpro integrate into family entries? data setsfiltersattributesinterpro entry annotationinterpro entry type : familyinterpro entry accessionsource signature database : pfaminterpro entry short namesignature accessionsignature i d ( name ) the pfam database contains a broad spectrum of hidden markov models that can be used to predict both family classification and domain organization . the interpro curation team has integrated > 96% of pfam signatures into interpro at the time of writing . during integration type to an interpro entry and by extension , its signatures , dependent on what is being represented ( a family , domain , site or repeat ) . using the biomart , it is possible to return the full set of integrated pfam signatures that interpro considers to be of type family. this query can be easily modified to request signatures built by any of the member databases that fit into any of the available interpro entry types . repeat. each interpro entry has exactly one type and consequently all integrated member database signatures also have one type , as assigned by the interpro curation team . following selection of this data set , the user is able to select filters and attributes ( in whichever order they choose ) . figure 4 illustrates the selection of the two filters applied in this query , which will restrict the rows of data returned . figure 5 illustrates the selection of attributes , which are equivalent to the columns of a spreadsheet . initially , the user is presented with the first 10 matching rows of data , giving an opportunity to refine the query prior to requesting the full set of results . figure 4.building a filter with two components : include results for family entry types that comprise signatures from pfam . figure 5.selecting the attributes to be included in the biomart output ( equivalent to the columns of a spreadsheet ) . the ordering of the columns is determined by the order in which the attributes are selected . results button at the top of the interface provides the first 10 results matching the query , to allow the query to be modified or improved . selecting a dataset in the interpro biomart . building a filter with two components : include results for family entry types that comprise signatures from pfam . selecting the attributes to be included in the biomart output ( equivalent to the columns of a spreadsheet ) . the ordering of the columns is determined by the order in which the attributes are selected . results button at the top of the interface provides the first 10 results matching the query , to allow the query to be modified or improved . which go terms are mapped to prosite signatures in interpro ( i.e. can i retrieve a prosite2go mapping?) data setsfiltersattributesinterpro entry annotationsource signature database : prosite patterns and prosite profiles ( ctrl click to select both).interpro entry accessionsignature accessiongo idgo term namego root term ( process / component / function ) a major use of interpro is the association of go terms to proteins via the signatures that they match . interpro2go mappings as a file that can be downloaded from the ftp site ; however , it is difficult to extract subsets of information from this file . in the past , a frequent request from users was the provision of go term mapping information for a particular member database . with the advent of the biomart , it is now very easy to provide this information as illustrated above . query # 3 . which metabolic pathways are associated with proteins matching the interpro family ( cxcr4 , ipr001277)? data setsfiltersattributespathwaypathway stable idpathway nameinterpro entry annotationinterpro entry i d = ipr001277interpro entry accessioninterpro entry nameuniprotkb protein accessionuniprotkb protein i d ( name)source signature databasesignature accessionsignature i d ( name)match start positionmatch stop position the interpro biomart is federated with the reactome biomart ( 22 , 23 ) from which the reactions , pathways and biological processes and as such , can provide valuable biological insight if married to the data in interpro . in which tissues have proteins matching the interpro family neural cell adhesion ( ipr009138 ) been identified by mass spectrometry? data setsfiltersattributespridepride experiment accessionexperiment titlesample nametaxonomy term ( newt / ncbi taxon)taxonomy i d ( newt / ncbi taxon)tissue ontology term ( brenda)brenda i d ( tissue)cell type term ( cl)cl i d ( cell type)gene ontology term ( go)go i d ( gene ontology)interpro entriesinterpro entry i d = ipr009138interpro entry accessioninterpro entry name the interpro biomart is also federated with the pride biomart . pride is the proteomics identifications database , which contains identifications of proteins and peptides arising from mass spectrometry . the two biomarts are linked via uniprotkb protein accessions , so this query returns information about identifications of the proteins that match the member database signatures integrated into interpro entry ipr009138 . the interpro biomart has proven a valuable addition to the interpro software infrastructure , supporting new tools , such as the interpro das service , as well as providing an efficient route to answer queries from the interpro user community . the biomart has furnished interpro with a web service , for which robust apis exist in several languages ( including both perl and java ) . additionally , biomart provides a substantial resource for bioinformaticians to query interpro , alongside the federated databases uniprotkb , reactome and pride . ( see table 1 , which describes these bioinformatics resources ) . table 1.external data sources included in the interpro biomartsourceurlbiomart urldescription of contentsuniprotkbhttp://www.uniprot.orghttp://www.ebi.ac.uk/ uniprot / biomart / martviewa comprehensive , high quality and freely accessible resource of protein sequence and functional information , comprising the human - curated swiss - prot data set and the automatically annotated trembl data set.pridehttp://www.ebi.ac.uk/pridehttp://www.ebi.ac.uk/pride/biomart/martviewa database of identifications of proteins and peptides , arising from mass spectrometry - based proteomics.reactome pathway databasehttp://www.reactome.orghttp://www.reactome.org / cgi - bin / marta human - curated database of biological pathways , focusing on human pathways , but providing automated prediction of pathways in other species . external data sources included in the interpro biomart it is intended to federate the interpro biomart with the new uniparc biomart that is under development at the ebi . this will allow the interpro biomart to be queried using identifiers and accessions from a large variety of protein sequence databases other than the uniprotkb , including several model organism databases . biotechnology and biological sciences research council 's bioinformatics and biological resources fund ( grant number bb / f010508/1 ) ; european union under the program fp7 capacities : scientific data repositories ; the working title for the project is improving protein annotation and co - ordination using technology ( impact ) ( grant number 213037 ) . funding for open access charge : european union under the program fp7 capacities : scientific data repositories ; the working title for the project is improving protein annotation and co - ordination using technology ( impact ) ( grant number 213037 ) .

the interpro biomart provides users with query - optimized access to predictions of family classification , protein domains and functional sites , based on a broad spectrum of integrated computational models ( signatures ) that are generated by the interpro member databases : gene3d , hamap , panther , pfam , pirsf , prints , prodom , prosite , smart , superfamily and tigrfams . these predictions are provided for all protein sequences from both the uniprot knowledge base and the uniparc protein sequence archive . the interpro biomart is supplementary to the primary interpro web interface ( http://www.ebi.ac.uk/interpro ) , providing a web service and the ability to build complex , custom queries that can efficiently return thousands of rows of data in a variety of formats . this article describes the information available from the interpro biomart and illustrates its utility with examples of how to build queries that return useful biological information.database url : http://www.ebi.ac.uk/interpro/biomart/martview .

Introduction Data content Services supported by the InterPro BioMart Query examples To demonstrate the utility of the InterPro BioMart, here we present several biologically relevant queries Discussion and future directions Funding

the interpro resource ( http://www.ebi.ac.uk/interpro ) ( 1 ) provides an integrated set of computational models ( or signatures ) for protein family classification and the prediction of structural and functional domains , sites and repeats . the predictive signatures are built by the 11 interpro member databases that , together with the interpro team at the ebi , comprise the interpro consortium . the member databases are gene3d ( 2 ) , hamap ( 3 ) , panther ( 4 ) , pfam ( 5 ) , pirsf ( 6 ) , prints ( 7 ) , prodom ( 8) , prosite ( 9 ) , smart ( 10 ) , superfamily ( 11 ) and tigrfams ( 12 ) . the interpro team at the ebi integrates the predictive signatures from these member databases into interpro entries. interpro entries are curated by a team of experts in a variety of fields in biology . the curation process includes the creation of entries , the structuring of the entry hierarchies , the provision of detailed abstracts describing each entry and the addition of useful cross - references to other databases and ontologies . this example entry comprises two member database signatures , one from pfam and the other from superfamily . in total , this interpro entry matches 2753 uniprotkb protein sequences . figure 1.an example human - curated interpro entry , illustrating the detailed description provided for the entry and cross references to the go and the member database signatures from which the entry is composed . an example human - curated interpro entry , illustrating the detailed description provided for the entry and cross references to the go and the member database signatures from which the entry is composed . the integration described above is useful because the individual member databases have distinct but overlapping interests and use a number of different algorithms and modeling techniques . from the perspective of the biologist or bioinformatician wishing to use these predictive techniques , interpro allows consideration of all of the available signatures from a single resource , without the need to be concerned with differences or overlap between the foci of the individual member databases . as well as integrating the member database signatures , interpro calculates matches to these signatures for the whole of the uniprot knowledge base ( uniprotkb , http://www.uniprot.org ) and the uniparc sequence archive ( 13 ) . figure 2 illustrates a set of matches to a single uniprotkb protein sequence , which matches three interpro entries . the matches of interpro signatures and entries to the sequences in uniprotkb are available from the main interpro website as well as from the interpro biomart , however , at the time of writing , the matches to uniparc sequences are only available from the biomart . it is expected that uniparc matches will be included in a future version of the main interpro website . the interpro biomart is built on the technology developed by the biomart project ( http://www.biomart.org ) ( 14 , 15 ) , a collaboration between the ontario institute for cancer research ( oicr ) and the european bioinformatics institute ( ebi ) . for this sequence , interpro provides a prediction of protein family membership , an overview of the domain organization and the details of matches to member database signatures . at the foot of the view can be seen associated go terms , based upon the calculated matches to interpro entries . for this sequence , interpro provides a prediction of protein family membership , an overview of the domain organization and the details of matches to member database signatures . at the foot of the view can be seen associated go terms , based upon the calculated matches to interpro entries . the adoption of biomart as a mechanism to share the data in interpro has been motivated by the benefits that biomart brings : the ability to build complex filters on the data ; the facility to select specifically which data types are returned ( equivalent to the columns of a spreadsheet ) ; the capacity of biomart to handle queries that return many thousands of rows of data and the provision of a web service with an associated data federation mechanism . the interpro biomart provides three data sources : interpro entry annotation , uniprotkb protein matches and uniparc protein matches. match information can be obtained from both the interpro entry annotation data source and the uniprotkb protein matches data source . interpro entry annotation data source focuses on descriptions of the interpro entries and the hierarchical relationships between them . uniprotkb protein matches data set is focused on the uniprotkb protein entity , allowing queries to be built based on attributes of the protein sequence , including options to filter on the taxonomic group annotated on the sequence . this data set also provides the opportunity to retrieve match information with respect to member database signatures as well as summarized match information , described as supermatch is determined where one or more member database signatures that have been integrated together into the same entry have overlapping matches to the protein in the same region of the sequence . supermatch are then calculated as the most extreme bounds of the matches of all the member databases signatures comprising the entry . uniparc protein matches data set provides equivalent information to the uniprotkb protein matches data set , coordinated on sequences included in the uniparc database , a non - redundant , historical archive of protein sequences extracted from public databases . at the time of writing , the uniparc database includes 25.6 million unique sequences ; the interpro match calculation pipeline is run against all of these sequences and the results are made available from this biomart data set . this service allows matches to be returned for sequences that are present in ( for example ) model organism protein sequence databases which are not yet represented in uniprotkb . for users interested in matches for specific protein sequences , this data set supports filtering by uniparc i d or sequence checksum ( crc-64 or md5 ) , as does the uniprotkb protein matches data set . if the user wishes to query using protein accessions or identifiers from third - party sequence databases , various services are available that allow protein identifier cross - referencing , including the protein identifier cross reference service , picr ( http://www.ebi.ac.uk/tools/picr/ ) ( 20 ) and the uniprot i d mapping service ( http://www.uniprot.org/ ) . both of these services can be used to convert protein identifiers or accessions from a large number of protein sequence databases to uniparc sequence identifiers . the three interpro biomart data sources include matches to the full taxonomy range in uniprotkb or uniparc . in this respect , the interpro biomart is different in structure to the ensembl biomart ( http://www.ensembl.org/biomart/martview ) ( 17 , 18 ) which is organized into species - specific data sets . the interpro biomart provides three data sources : interpro entry annotation , uniprotkb protein matches and uniparc protein matches. match information can be obtained from both the interpro entry annotation data source and the uniprotkb protein matches data source . interpro entry annotation data source focuses on descriptions of the interpro entries and the hierarchical relationships between them . uniprotkb protein matches data set is focused on the uniprotkb protein entity , allowing queries to be built based on attributes of the protein sequence , including options to filter on the taxonomic group annotated on the sequence . supermatch are then calculated as the most extreme bounds of the matches of all the member databases signatures comprising the entry . uniparc protein matches data set provides equivalent information to the uniprotkb protein matches data set , coordinated on sequences included in the uniparc database , a non - redundant , historical archive of protein sequences extracted from public databases . at the time of writing , the uniparc database includes 25.6 million unique sequences ; the interpro match calculation pipeline is run against all of these sequences and the results are made available from this biomart data set . this service allows matches to be returned for sequences that are present in ( for example ) model organism protein sequence databases which are not yet represented in uniprotkb . for users interested in matches for specific protein sequences , this data set supports filtering by uniparc i d or sequence checksum ( crc-64 or md5 ) , as does the uniprotkb protein matches data set . if the user wishes to query using protein accessions or identifiers from third - party sequence databases , various services are available that allow protein identifier cross - referencing , including the protein identifier cross reference service , picr ( http://www.ebi.ac.uk/tools/picr/ ) ( 20 ) and the uniprot i d mapping service ( http://www.uniprot.org/ ) . both of these services can be used to convert protein identifiers or accessions from a large number of protein sequence databases to uniparc sequence identifiers . the three interpro biomart data sources include matches to the full taxonomy range in uniprotkb or uniparc . in this respect , the interpro biomart is different in structure to the ensembl biomart ( http://www.ensembl.org/biomart/martview ) ( 17 , 18 ) which is organized into species - specific data sets . the interpro biomart is used to extend the functionality of the primary interpro web interface , providing biomart this allows data to be downloaded in tab- or comma - separated values format , suitable for computational analysis . the interpro biomart web service is the data source behind the interpro distributed annotation system ( das ) service ( 21 ) , available from http://www.ebi.ac.uk/das-srv/interpro/das . interpro , which contains all interpro member database signature matches to uniprotkb protein sequences.interpro - matches - overview that provides the maximum extent of the matches from all signatures that are integrated into a single interpro entry against uniprotkb protein sequences . supermatch matches described in the biomart uniprotkb protein matches data set.interpro - uniparc - matches that provides match information for protein sequences identified using uniparc identifiers.interpro - s4 is used to provide protein family classification to the new ebi search service and is therefore part of the wider programme of data integration at the ebi . interpro , which contains all interpro member database signature matches to uniprotkb protein sequences . interpro - matches - overview that provides the maximum extent of the matches from all signatures that are integrated into a single interpro entry against uniprotkb protein sequences . interpro - uniparc - matches that provides match information for protein sequences identified using uniparc identifiers . interpro - s4 is used to provide protein family classification to the new ebi search service and is therefore part of the wider programme of data integration at the ebi . in common with all biomart implementations , the interpro biomart enables the construction of simple queries as well as complex , multi - faceted queries where the data is filtered on several criteria . the user is able to specify precisely which data attributes should be returned , equivalent to columns in a spreadsheet . the presence of repeated rows of results in the output depends on the construction of the query and the structure of the underlying biomart tables . the authors therefore recommend the use of the unique results only option when querying the biomart , which removes repeated rows of results . data setsfiltersattributesinterpro entry annotationinterpro entry type : familyinterpro entry accessionsource signature database : pfaminterpro entry short namesignature accessionsignature i d ( name ) the pfam database contains a broad spectrum of hidden markov models that can be used to predict both family classification and domain organization . the interpro curation team has integrated > 96% of pfam signatures into interpro at the time of writing . using the biomart , it is possible to return the full set of integrated pfam signatures that interpro considers to be of type family. each interpro entry has exactly one type and consequently all integrated member database signatures also have one type , as assigned by the interpro curation team . figure 3 illustrates selection of the interpro entry annotation data set . figure 4 illustrates the selection of the two filters applied in this query , which will restrict the rows of data returned . figure 5 illustrates the selection of attributes , which are equivalent to the columns of a spreadsheet . initially , the user is presented with the first 10 matching rows of data , giving an opportunity to refine the query prior to requesting the full set of results . figure 5.selecting the attributes to be included in the biomart output ( equivalent to the columns of a spreadsheet ) . the ordering of the columns is determined by the order in which the attributes are selected . selecting a dataset in the interpro biomart . selecting the attributes to be included in the biomart output ( equivalent to the columns of a spreadsheet ) . the ordering of the columns is determined by the order in which the attributes are selected . interpro provides the interpro2go mappings as a file that can be downloaded from the ftp site ; however , it is difficult to extract subsets of information from this file . which metabolic pathways are associated with proteins matching the interpro family ( cxcr4 , ipr001277)? data setsfiltersattributespathwaypathway stable idpathway nameinterpro entry annotationinterpro entry i d = ipr001277interpro entry accessioninterpro entry nameuniprotkb protein accessionuniprotkb protein i d ( name)source signature databasesignature accessionsignature i d ( name)match start positionmatch stop position the interpro biomart is federated with the reactome biomart ( 22 , 23 ) from which the reactions , pathways and biological processes and as such , can provide valuable biological insight if married to the data in interpro . data setsfiltersattributespridepride experiment accessionexperiment titlesample nametaxonomy term ( newt / ncbi taxon)taxonomy i d ( newt / ncbi taxon)tissue ontology term ( brenda)brenda i d ( tissue)cell type term ( cl)cl i d ( cell type)gene ontology term ( go)go i d ( gene ontology)interpro entriesinterpro entry i d = ipr009138interpro entry accessioninterpro entry name the interpro biomart is also federated with the pride biomart . data setsfiltersattributesinterpro entry annotationinterpro entry type : familyinterpro entry accessionsource signature database : pfaminterpro entry short namesignature accessionsignature i d ( name ) the pfam database contains a broad spectrum of hidden markov models that can be used to predict both family classification and domain organization . the interpro curation team has integrated > 96% of pfam signatures into interpro at the time of writing . using the biomart , it is possible to return the full set of integrated pfam signatures that interpro considers to be of type family. this query can be easily modified to request signatures built by any of the member databases that fit into any of the available interpro entry types . each interpro entry has exactly one type and consequently all integrated member database signatures also have one type , as assigned by the interpro curation team . figure 4 illustrates the selection of the two filters applied in this query , which will restrict the rows of data returned . initially , the user is presented with the first 10 matching rows of data , giving an opportunity to refine the query prior to requesting the full set of results . figure 5.selecting the attributes to be included in the biomart output ( equivalent to the columns of a spreadsheet ) . selecting a dataset in the interpro biomart . selecting the attributes to be included in the biomart output ( equivalent to the columns of a spreadsheet ) . the ordering of the columns is determined by the order in which the attributes are selected . interpro2go mappings as a file that can be downloaded from the ftp site ; however , it is difficult to extract subsets of information from this file . which metabolic pathways are associated with proteins matching the interpro family ( cxcr4 , ipr001277)? data setsfiltersattributespathwaypathway stable idpathway nameinterpro entry annotationinterpro entry i d = ipr001277interpro entry accessioninterpro entry nameuniprotkb protein accessionuniprotkb protein i d ( name)source signature databasesignature accessionsignature i d ( name)match start positionmatch stop position the interpro biomart is federated with the reactome biomart ( 22 , 23 ) from which the reactions , pathways and biological processes and as such , can provide valuable biological insight if married to the data in interpro . in which tissues have proteins matching the interpro family neural cell adhesion ( ipr009138 ) been identified by mass spectrometry? data setsfiltersattributespridepride experiment accessionexperiment titlesample nametaxonomy term ( newt / ncbi taxon)taxonomy i d ( newt / ncbi taxon)tissue ontology term ( brenda)brenda i d ( tissue)cell type term ( cl)cl i d ( cell type)gene ontology term ( go)go i d ( gene ontology)interpro entriesinterpro entry i d = ipr009138interpro entry accessioninterpro entry name the interpro biomart is also federated with the pride biomart . the interpro biomart has proven a valuable addition to the interpro software infrastructure , supporting new tools , such as the interpro das service , as well as providing an efficient route to answer queries from the interpro user community . the biomart has furnished interpro with a web service , for which robust apis exist in several languages ( including both perl and java ) . additionally , biomart provides a substantial resource for bioinformaticians to query interpro , alongside the federated databases uniprotkb , reactome and pride . table 1.external data sources included in the interpro biomartsourceurlbiomart urldescription of contentsuniprotkbhttp://www.uniprot.orghttp://www.ebi.ac.uk/ uniprot / biomart / martviewa comprehensive , high quality and freely accessible resource of protein sequence and functional information , comprising the human - curated swiss - prot data set and the automatically annotated trembl data set.pridehttp://www.ebi.ac.uk/pridehttp://www.ebi.ac.uk/pride/biomart/martviewa database of identifications of proteins and peptides , arising from mass spectrometry - based proteomics.reactome pathway databasehttp://www.reactome.orghttp://www.reactome.org / cgi - bin / marta human - curated database of biological pathways , focusing on human pathways , but providing automated prediction of pathways in other species . external data sources included in the interpro biomart it is intended to federate the interpro biomart with the new uniparc biomart that is under development at the ebi . this will allow the interpro biomart to be queried using identifiers and accessions from a large variety of protein sequence databases other than the uniprotkb , including several model organism databases .

four cohorts contributed to the discovery meta - analysis through the contribution of phenotypic and gwas data . these included the framingham heart study ( n = 5,759 ) , precocious coronary artery disease ( procardis ) ( n = 3,259 ) , the fenland study ( n = 1,372 ) , and the diabetes genetics initiative ( dgi ) ( n = 311 ) , for a total of 10,701 participants . eleven cohorts contributed to the follow - up efforts ; these included metabolic syndrome in men ( metsim ) ( n = 5,122 ) , botnia prevalence , prediction and prevention of diabetes ( botnia - ppp ) ( n = 2,280 ) , helsinki birth cohort study ( hbcs ) ( n = 1,649 ) , the ely study ( n = 1,568 ) , the hertfordshire study ( n = 1,016 ) , uppsala longitudinal study of adult men ( ulsam ) ( n = 939 ) , relationship between insulin sensitivity and cardiovascular disease ( risc ) ( n = 914 ) , prospective investigation of the vasculature in uppsala seniors ( pivus ) ( n = 912 ) , segovia ( n = 911 ) , the greek health randomized aging study ( ghras ) ( n = 668 ) , and stockholm diabetes prevention program ( sdpp ) ( n = 399 ) , for a total of 16,378 participants ( with maximal sample for any one snp of 15,898 ) . we excluded individuals with known diabetes , on antidiabetic treatment , or with fasting glucose 7 mmol / l ( 3 ) ; all participants were of european descent . proinsulin ( pmol / l ) was measured from fasting whole blood , plasma , or serum or a combination of these using enzyme - linked immunosorbent or immunometric assays . fasting insulin ( pmol / l ) was measured using either enzyme - linked immunosorbent , immunofluorescent , or radioimmunometric assays ( supplementary table 1 ) . genome - wide commercial arrays ( affymetrix 500k , mips 50k , and illumina human1m/610k ) were used by the four discovery cohorts as described in supplementary table 1 . we aimed to identify genetic variants associated with high proinsulin levels relative to an individual s fasting insulin levels . this can be done by examining proinsulin - to - insulin ratios or by statistically adjusting proinsulin for fasting insulin . we chose the latter because the adjusted trait has comparable predictive value ( 18 ) and displayed better statistical performance in pilot studies and adequate heritability in the framingham heart study , one of the larger cohorts examined here ( h = 0.36 vs. 0.34 for the proinsulin - to - insulin ratio ) . in framingham , correlation between the adjusted trait and the ratio was 0.95 , and the quantile - quantile gwas plots were comparable . we used a linear regression model with natural log transformed fasting proinsulin as the dependent variable and genotypes as predictors , with adjustment for natural - log transformed fasting insulin values , sex , age , geographical covariates ( if applicable ) , and age squared ( framingham only ) to evaluate the association under an additive genetic model . association analysis was performed by individual studies using snptest ( 19 ) , stata ( 20 ) , plink ( 21 ) , or lmekin ( r kinship package ) software ( 22 ) . genome - wide association inflation coefficients were estimated for each discovery cohort using the genomic control ( gc ) method ( 23 ) and applied subsequently to each individual snp association test statistics to correct for cryptic relatedness . the gc value for the final meta - analysis of proinsulin adjusted for fasting insulin was 1.01 . the inverse - variance fixed effects meta - analysis method was used to evaluate the pooled regression estimates for additively coded snps using metal ( 24 ) . sex interaction effects were evaluated with a function in the gwama software ( 25 ) . we carried forward to stage 2 the most significant snp from each of 21 independent loci that showed association with proinsulin in stage 1 analyses at p < 1 10 . additionally , two snps near the p < 1 10 threshold ( in asap2 and a gene desert region ) were carried forward as a result of biological plausibility ( asap2 is involved in vesicular transport ) and/or consistency of direction of effect in all discovery stage 1 studies ( both loci ) . we genotyped these 23 variants in 11 additional stage 2 studies totaling 16,378 nondiabetic participants of european ancestry ( supplementary table 1 ; genotyping assays and conditions are available upon request ) . we meta - analyzed stage 1 and stage 2 results using inverse - variance weighted fixed effects meta - analysis methods , including up to 27,079 participants . additional analyses and expression and expression quantitative trait loci ( eqtl ) studies are described in the supplementary data . four cohorts contributed to the discovery meta - analysis through the contribution of phenotypic and gwas data . these included the framingham heart study ( n = 5,759 ) , precocious coronary artery disease ( procardis ) ( n = 3,259 ) , the fenland study ( n = 1,372 ) , and the diabetes genetics initiative ( dgi ) ( n = 311 ) , for a total of 10,701 participants . eleven cohorts contributed to the follow - up efforts ; these included metabolic syndrome in men ( metsim ) ( n = 5,122 ) , botnia prevalence , prediction and prevention of diabetes ( botnia - ppp ) ( n = 2,280 ) , helsinki birth cohort study ( hbcs ) ( n = 1,649 ) , the ely study ( n = 1,568 ) , the hertfordshire study ( n = 1,016 ) , uppsala longitudinal study of adult men ( ulsam ) ( n = 939 ) , relationship between insulin sensitivity and cardiovascular disease ( risc ) ( n = 914 ) , prospective investigation of the vasculature in uppsala seniors ( pivus ) ( n = 912 ) , segovia ( n = 911 ) , the greek health randomized aging study ( ghras ) ( n = 668 ) , and stockholm diabetes prevention program ( sdpp ) ( n = 399 ) , for a total of 16,378 participants ( with maximal sample for any one snp of 15,898 ) . we excluded individuals with known diabetes , on antidiabetic treatment , or with fasting glucose 7 mmol / l ( 3 ) ; all participants were of european descent . proinsulin ( pmol / l ) was measured from fasting whole blood , plasma , or serum or a combination of these using enzyme - linked immunosorbent or immunometric assays . fasting insulin ( pmol / l ) was measured using either enzyme - linked immunosorbent , immunofluorescent , or radioimmunometric assays ( supplementary table 1 ) . genome - wide commercial arrays ( affymetrix 500k , mips 50k , and illumina human1m/610k ) were used by the four discovery cohorts as described in supplementary table 1 . we aimed to identify genetic variants associated with high proinsulin levels relative to an individual s fasting insulin levels . this can be done by examining proinsulin - to - insulin ratios or by statistically adjusting proinsulin for fasting insulin . we chose the latter because the adjusted trait has comparable predictive value ( 18 ) and displayed better statistical performance in pilot studies and adequate heritability in the framingham heart study , one of the larger cohorts examined here ( h = 0.36 vs. 0.34 for the proinsulin - to - insulin ratio ) . in framingham , correlation between the adjusted trait and the ratio was 0.95 , and the quantile - quantile gwas plots were comparable . we used a linear regression model with natural log transformed fasting proinsulin as the dependent variable and genotypes as predictors , with adjustment for natural - log transformed fasting insulin values , sex , age , geographical covariates ( if applicable ) , and age squared ( framingham only ) to evaluate the association under an additive genetic model . association analysis was performed by individual studies using snptest ( 19 ) , stata ( 20 ) , plink ( 21 ) , or lmekin ( r kinship package ) software ( 22 ) . genome - wide association inflation coefficients were estimated for each discovery cohort using the genomic control ( gc ) method ( 23 ) and applied subsequently to each individual snp association test statistics to correct for cryptic relatedness . the gc value for the final meta - analysis of proinsulin adjusted for fasting insulin was 1.01 . the inverse - variance fixed effects meta - analysis method was used to evaluate the pooled regression estimates for additively coded snps using metal ( 24 ) . sex interaction effects were evaluated with a function in the gwama software ( 25 ) . we carried forward to stage 2 the most significant snp from each of 21 independent loci that showed association with proinsulin in stage 1 analyses at p < 1 10 . additionally , two snps near the p < 1 10 threshold ( in asap2 and a gene desert region ) were carried forward as a result of biological plausibility ( asap2 is involved in vesicular transport ) and/or consistency of direction of effect in all discovery stage 1 studies ( both loci ) . we genotyped these 23 variants in 11 additional stage 2 studies totaling 16,378 nondiabetic participants of european ancestry ( supplementary table 1 ; genotyping assays and conditions are available upon request ) . we meta - analyzed stage 1 and stage 2 results using inverse - variance weighted fixed effects meta - analysis methods , including up to 27,079 participants . additional analyses and expression and expression quantitative trait loci ( eqtl ) studies are described in the supplementary data . we conducted a two - stage association study in individuals of european descent ( total n = 27,079 , with n = 10,701 in the discovery stage ) . cohort and phenotype information can be found in supplementary table 1 , and the study design is outlined in supplementary fig . 3 . a total of 21 independent variants ( including two snps identified during conditional analyses , see below ) met our statistical threshold for follow - up ( p < 1 10 ; fig . 1 ) . the clean dataset showed no systematic deviation from the null expectation , with the exception of the tail of the distribution ( fig . directly genotyped and imputed snps are plotted with their meta - analysis p values ( as log10 values ) as a function of genomic position ( ncbi build 36 ) . the snps that achieved genome - wide significance ( p < 5 10 ) on follow - up are shown in red . insert : quantile - quantile ( q - q ) plot for fasting proinsulin adjusted for fasting insulin . the expected null distribution is plotted along the diagonal , the entire distribution of observed p values is plotted in blue , and a distribution that excludes the nine novel findings is plotted in red . we followed up 23 snps ( the 21 mentioned above plus 2 others that approached our significance threshold and were selected as a result of biological plausibility ; see research design and methods ) in 11 cohorts totaling up to 16,378 nondiabetic individuals of european descent ( table 1 and supplementary table 2 ) . joint meta - analysis of discovery and follow - up cohorts ( n = 27,079 ) revealed nine signals at eight loci reaching genome - wide significance ( p < 5 10 ) , of which two are novel ( sgsm2 , larp6 ) , five have previously been associated with glucose metabolism and/or t2d ( tcf7l2 , slc30a8 , madd , vps13c / c2cd4a / b , and arap1 ) , and one ( pcsk1 ) has been previously implicated in obesity and associated with proinsulin levels , although not at genome - wide significance ( table 1 and fig . 2 ) . adjusting for bmi , fasting glucose , or both did not attenuate these signals . of note , when adjusting for fasting glucose or both fasting glucose and bmi ( but not bmi alone ) , one other locus , snx7 , reached genome - wide significance ( p = 5.4 10 and 1.5 10 , respectively ) . loci associated with fasting proinsulin levels at genome - wide levels of statistical significance -coefficients for the effect allele are shown after adjustments for sex , age , geographic covariates ( if applicable ) , and age squared ( framingham only ) , besides the additional adjustments denoted in the column headings . the combined p values are shown for the joint analysis with the basic adjustments listed above . * this second snp in madd was selected for further testing based on a suggestive p value ( 8.6 10 ) when analyses for chromosome 11 were conditioned for the top two signals ( madd and arap1 ) . * * snx7 only reached genome - wide significance after adjusting for fasting glucose ( p = 5.4 10 ) . another signal ( rs306549 in ddx31 ) reached genome - wide significance in women ( p = 2.0 10 ) but not in men ( p = 0.17 ) ; see text for details . regional plots of eight genomic regions containing novel genome - wide significant associations . for each region , directly genotyped and imputed snps are plotted with their meta - analysis p values ( as log10 values ) as a function of genomic position ( ncbi build 36 ) . in each panel , the stage 1 discovery snp taken forward to stage 2 follow - up is represented by a purple diamond ( with global meta - analysis p value ) , with its stage 1 discovery p value denoted by a red diamond with bolded borders . estimated recombination rates ( taken from hapmap ) are plotted to reflect the local ld structure around the associated snps and their correlated proxies ( according to a white to red scale from r = 0 to 1 , based on pairwise r values from hapmap ceu ) . a : arap1 region ; b : madd region ; c : pcsk1 region ; d : tcf7l2 region ; e : vps13c / c2cd4a / b region ; f : slc30a8 region ; g : larp6 region ; h : sgsm2 region . conditional analyses on the two strongest signals revealed that the madd locus harbors two independent signals 19 kb apart ( rs10501320 and rs10838687 ; r = 0.068 in hapmap ceu ) , whereas a second independent signal near arap1 did not replicate ( fig . 2b , table 1 , and supplementary table 2 ) . among the nine replicated snps , individual loci explained between 0.2 and 1.4% of the variance in proinsulin in the discovery samples and up to 2.3% of the variance in the follow - up samples . together , the nine genome - wide significant snps explained between 5.4 and 7.7% of the proinsulin variance in the discovery samples and 8.1% of the variance in the risc cohort , one of the few follow - up cohorts with genotypes available for all nine snps . we noted some degree of heterogeneity in our joint meta - analyses ( table 1 ) . part of the heterogeneity arose from the metsim sample , which enrolled only men ; exclusion of this cohort from our meta - analysis reduced the heterogeneity . we also stratified our analyses by sex and tested for a snp sex interaction ( 26 ) . our overall findings remained essentially unchanged after sex stratification , and heterogeneity was attenuated ( e.g. , i = 77.2% , heterogeneity p = 1.9 10 for combined men and women , whereas i = 64.6% , heterogeneity p = 4.5 10 [ men ] and i = 55.6% , heterogeneity p = 0.01 [ women ] in stratified analyses ) . furthermore , tests for interaction with sex among snps that reached our follow - up significance threshold revealed a locus ( rs306549 in ddx31 ) where a genome - wide significant association was seen in women ( p = 2.0 10 ; supplementary fig . although removal of the metsim cohort improved the heterogeneity score and produced nominal significance for the association in men ( p = 0.02 ) , the effect size remained threefold stronger in women than in men ( -coefficient 0.0427 vs. 0.0165 , respectively ) . to provide further reassurance regarding any residual heterogeneity , we repeated our meta - analyses based on p values ( rather than -coefficients ) and meta - analyzed the resulting z scores . our findings were essentially unchanged , suggesting that heterogeneity in the -estimates across cohorts has not produced spurious results . proinsulin is initially cleaved to 32,33-split proinsulin and further to insulin and c - peptide before secretion ( supplementary fig . 1 ) ; we were therefore interested in the effects of the nine top snps on these traits . the proinsulin - raising alleles of each snp were consistently associated with higher 32,33-split proinsulin levels , with effect sizes following the rank order of proinsulin effect sizes . nearly all associations reached nominal conventional levels of statistical significance in this smaller dataset of 4,1036,343 individuals with measures of 32,33-split proinsulin levels ( all p < 1.5 10 , with the exception of the conditional signal at madd ) . the insulinogenic index ( 27 ) , which measures dynamic insulin secretion during the first 30 min after an oral glucose load and was available in 14,956 subjects , showed nominal associations for four loci . of these , the proinsulin - raising alleles were associated with a lower insulinogenic index at vps13c / c2cd4a / b , tcf7l2 , and slc30a8 and higher at arap1 ( table 2 ) . association of proinsulin loci with insulin - processing traits -coefficients are adjusted for age , sex , and study - specific covariates ( if applicable ) . we detected no nominal associations with fasting c - peptide ( p > 0.05 ) . given the differences in hepatic clearance of insulin and c - peptide , we also performed sensitivity analyses to account for any possible impact this may have had on our results . we adjusted proinsulin levels for fasting c - peptide rather than fasting insulin in two cohorts ( ely and botnia - ppp ) ; comparison of -estimates showed that the majority of loci had very similar effect sizes and the same rank order was preserved , arguing against noticeable discrepancies between the two adjustment schemes . to clarify potential mechanisms , the top nine signals ( arap1 , two at madd , pcsk1 , tcf7l2 , vps13c / c2cd4a / b , slc30a8 , larp6 , and sgsm2 ) were also examined in relation to other glucometabolic traits ( fasting and 2-h postload glucose and insulin , homeostasis model assessment estimates of -cell function [ homa - b ] and insulin resistance [ homa - ir ] , glycated hemoglobin [ a1c ] , t2d , and bmi [ table 3 ] ) . we investigated results available from magic meta - analyses of gwas of glycemic traits ( 35 ) and obtained t2d and bmi results in collaboration with the diabetes genetics replication and meta - analysis ( diagram ) ( 29 ) and genomewide investigation of anthropometric measures ( giant ) ( 30 ) consortia , respectively . nominal associations ( p < 0.05 ) were found for fasting glucose ( with the proinsulin - raising allele increasing fasting glucose levels at madd , slc30a8 , tcf7l2 , and vps13c / c2cd4a / b and decreasing fasting glucose levels at arap1 and pcsk1 ) , fasting insulin ( increased levels at arap1 , larp6 , and sgsm2 and decreased levels at tcf7l2 ) , homa - b ( decreased at madd , slc30a8 , vps13c / c2cd4a / b , and tcf7l2 and increased at pcsk1 , arap1 , and larp6 ) , insulin resistance as measured by homa - ir ( increased at larp6 and sgsm2 and decreased at tcf7l2 ) , and 2-h postload glucose ( decreased at slc30a8 and vps13c / c2cd4a / b and increased at arap1 and tcf7l2 ) . we detected no significant associations for 2-h postload insulin or insulin sensitivity as estimated by the matsuda index ( 31 ) ( table 3 ) . association of proinsulin loci with other glycemic traits -coefficients are adjusted for age , sex , and study - specific covariates ( if applicable ) . or is for t2d only as a dichotomous trait . associations with t2d were confirmed for four known t2d loci ( slc30a8 , arap1 , vps13c / c2cd4a / b , and tcf7l2 ; table 3 ) . counterintuitively , the proinsulin - raising allele of arap1 ( formerly known as centd2 and reported as such in diagram+ ) ( 29 ) was associated with a lower fasting glucose ( 0.019 mg / dl per a allele ; p = 1.7 10 ) , lower a1c ( 0.023% ; p = 0.02 ) , and a lower risk of t2d ( odds ratio [ or ] 0.88 ; p = 7.8 10 ; table 3 ) . the two novel loci ( larp6 and sgsm2 ) did not show significant associations with t2d ( or [ 95% ci ] : 1.01 [ 0.951.07 ] and 1.01 [ 0.961.08 ] , respectively ) , indicating that if they increase t2d risk they do so to an extent confined within the bounds of narrow 95% ci . we used mach ( 32 ) or impute ( 19 ) applied to the 1000 genomes ceu reference panel ( www.1000genomes.org ) to carry out imputation of 8 million autosomal snps with minor allele frequency > 1% . analysis of 1000 genomes - imputed data in the four discovery cohorts indicates that although there are low - frequency ( 15% ) genetic variants that influence levels of circulating proinsulin , these are found in the same loci that contain common proinsulin - influencing variants , and none of them yield substantially stronger signals than the index snp at each locus ( supplementary fig . 5 ) . using current databases of copy number variants ( 33 ) and the snap software ( http://www.broadinstitute.org/mpg/snap/index.php ; ceu , hapmap release 22 ) , we checked whether any of the proinsulin - associated snps were within 500 kb and in linkage disequilibrium ( ld ) with any of the snps known to tag copy number variants in the human genome . no copy number variant tag snps with r > 0.3 were found within 500 kb of our lead snps . to guide identification of the gene responsible for each association signal , we also examined the gene expression profile of selected genes in each associated region across a range of human tissues , including islets and fluorescence - activated cell ( fac)-sorted -cells ( fig . we defined 1-mb intervals around the lead snp at each locus and prioritized biologically plausible genes as gleaned from the literature ( see box in supplementary data ) . 3f ) . however , at the larp6 locus , ct62 is expressed exclusively in testis , likely excluding it as a relevant gene in this context . at the arap1 locus , stard10 is expressed more strongly in pancreatic and islet tissue than any other tissue type ; similarly , at the vps13c locus both c2cd4a and c2cd4b demonstrate higher expression in pancreas and islets than all other tissue types . expression profiles of biologically plausible genes within each associated locus across a range of human tissue types , including islet preparations from three donors . a : arap1 region ; b : madd region ; c : vps13c / c2cd4a / b region ; d : larp6 region ; e : sgsm2 region . f : expression levels of genes near the proinsulin - associated variants in human fac - sorted -cells . data are expression means sd of the relative expression measured by quantitative pcr obtained from three human nondiabetic donors . we also studied the expression of the transcript for the gene closest to the index snp at each of the nine replicated loci in human islets isolated from 55 nondiabetic and 9 diabetic individuals . of the nine loci , pcsk1 ( p = 0.02 ) and madd ( p = 0.07 ) demonstrated 3545% lower expression in subjects with t2d compared with control subjects . we evaluated whether any of the associated snps was in strong ld with a potentially causal variant . we used snpper ( 34 ) to classify all snps in strong ld with the lead snp ( r 0.8 ) within a 1-mb region . we found that pcsk1 rs6235 codes for a nonsynonymous variant ( s690 t ) , which is in perfect ld with rs6234 , another missense variant ( q665e ) ; both were predicted to be nondamaging by polyphen ( 35 ) and sift ( 36 ) . at slc30a8 , the proinsulin - associated snp rs11558471 is a perfect proxy for the known t2d - associated snp rs13266634 , encoding r325w . the t allele ( encoding tryptophan ) is predicted to be benign by polyphen , but damaging by sift . we found no other strong ( r > 0.8 ) correlations in hapmap ceu with potentially functional snps within 1 mb of the lead signals . we also tested whether any of the proinsulin - associated snps might influence proximal ( cis ) expression of human transcripts , in tissues available to us that had been paired to genetic data . we found a significant association ( p = 0.01 permutation threshold ) of rs1549318 with expression levels of larp6 in adipose tissue . snp rs1549318 is located 37 kb from larp6 , and the proinsulin - raising t allele is associated with lower levels of expression . analysis of an eqtl database from human liver indicated that the proinsulin - raising a allele of the lead snp at the sgsm2 locus ( rs179456 ) was associated with increased liver expression of trps1 ( p = 0.004 ) . we constructed unweighted and weighted genotype scores composed of the nine genome - wide significant proinsulin - raising alleles , with weights defined by the -coefficients from our replication meta - analysis , and tested the association of these scores with cad in the coronary artery disease genome - wide replication and meta - analysis ( cardiogram ) ( 37 ) ( n = 22,000 cad case subjects and 60,000 control subjects ) and c4d ( 38 ) ( n = 15,420 cad case subjects and 15,062 control subjects ) datasets . neither weighted nor unweighted genotype scores reached nominal significance in either dataset ( p = 0.47 and 0.81 for unweighted and weighted scores in cardiogram , respectively ; p = 0.60 and 0.43 for unweighted and weighted scores in c4d , respectively ) . we conducted a two - stage association study in individuals of european descent ( total n = 27,079 , with n = 10,701 in the discovery stage ) . cohort and phenotype information can be found in supplementary table 1 , and the study design is outlined in supplementary fig . 3 . a total of 21 independent variants ( including two snps identified during conditional analyses , see below ) met our statistical threshold for follow - up ( p < 1 10 ; fig . 1 ) . the clean dataset showed no systematic deviation from the null expectation , with the exception of the tail of the distribution ( fig . directly genotyped and imputed snps are plotted with their meta - analysis p values ( as log10 values ) as a function of genomic position ( ncbi build 36 ) . the snps that achieved genome - wide significance ( p < 5 10 ) on follow - up are shown in red . insert : quantile - quantile ( q - q ) plot for fasting proinsulin adjusted for fasting insulin . the expected null distribution is plotted along the diagonal , the entire distribution of observed p values is plotted in blue , and a distribution that excludes the nine novel findings is plotted in red . we followed up 23 snps ( the 21 mentioned above plus 2 others that approached our significance threshold and were selected as a result of biological plausibility ; see research design and methods ) in 11 cohorts totaling up to 16,378 nondiabetic individuals of european descent ( table 1 and supplementary table 2 ) . joint meta - analysis of discovery and follow - up cohorts ( n = 27,079 ) revealed nine signals at eight loci reaching genome - wide significance ( p < 5 10 ) , of which two are novel ( sgsm2 , larp6 ) , five have previously been associated with glucose metabolism and/or t2d ( tcf7l2 , slc30a8 , madd , vps13c / c2cd4a / b , and arap1 ) , and one ( pcsk1 ) has been previously implicated in obesity and associated with proinsulin levels , although not at genome - wide significance ( table 1 and fig . 2 ) . adjusting for bmi , fasting glucose , or both did not attenuate these signals . of note , when adjusting for fasting glucose or both fasting glucose and bmi ( but not bmi alone ) , one other locus , snx7 , reached genome - wide significance ( p = 5.4 10 and 1.5 10 , respectively ) . loci associated with fasting proinsulin levels at genome - wide levels of statistical significance -coefficients for the effect allele are shown after adjustments for sex , age , geographic covariates ( if applicable ) , and age squared ( framingham only ) , besides the additional adjustments denoted in the column headings . the combined p values are shown for the joint analysis with the basic adjustments listed above . * this second snp in madd was selected for further testing based on a suggestive p value ( 8.6 10 ) when analyses for chromosome 11 were conditioned for the top two signals ( madd and arap1 ) . * snx7 only reached genome - wide significance after adjusting for fasting glucose ( p = 5.4 10 ) . another signal ( rs306549 in ddx31 ) reached genome - wide significance in women ( p = 2.0 10 ) but not in men ( p = 0.17 ) ; see text for details . regional plots of eight genomic regions containing novel genome - wide significant associations . for each region , directly genotyped and imputed snps are plotted with their meta - analysis p values ( as log10 values ) as a function of genomic position ( ncbi build 36 ) . in each panel , the stage 1 discovery snp taken forward to stage 2 follow - up is represented by a purple diamond ( with global meta - analysis p value ) , with its stage 1 discovery p value denoted by a red diamond with bolded borders . estimated recombination rates ( taken from hapmap ) are plotted to reflect the local ld structure around the associated snps and their correlated proxies ( according to a white to red scale from r = 0 to 1 , based on pairwise r values from hapmap ceu ) . a : arap1 region ; b : madd region ; c : pcsk1 region ; d : tcf7l2 region ; e : vps13c / c2cd4a / b region ; f : slc30a8 region ; g : larp6 region ; h : sgsm2 region . conditional analyses on the two strongest signals revealed that the madd locus harbors two independent signals 19 kb apart ( rs10501320 and rs10838687 ; r = 0.068 in hapmap ceu ) , whereas a second independent signal near arap1 did not replicate ( fig . 2b , table 1 , and supplementary table 2 ) . among the nine replicated snps , individual loci explained between 0.2 and 1.4% of the variance in proinsulin in the discovery samples and up to 2.3% of the variance in the follow - up samples . together , the nine genome - wide significant snps explained between 5.4 and 7.7% of the proinsulin variance in the discovery samples and 8.1% of the variance in the risc cohort , one of the few follow - up cohorts with genotypes available for all nine snps . we noted some degree of heterogeneity in our joint meta - analyses ( table 1 ) . part of the heterogeneity arose from the metsim sample , which enrolled only men ; exclusion of this cohort from our meta - analysis reduced the heterogeneity . we also stratified our analyses by sex and tested for a snp sex interaction ( 26 ) . our overall findings remained essentially unchanged after sex stratification , and heterogeneity was attenuated ( e.g. , i = 77.2% , heterogeneity p = 1.9 10 for combined men and women , whereas i = 64.6% , heterogeneity p = 4.5 10 [ men ] and i = 55.6% , heterogeneity p = 0.01 [ women ] in stratified analyses ) . furthermore , tests for interaction with sex among snps that reached our follow - up significance threshold revealed a locus ( rs306549 in ddx31 ) where a genome - wide significant association was seen in women ( p = 2.0 10 ; supplementary fig . although removal of the metsim cohort improved the heterogeneity score and produced nominal significance for the association in men ( p = 0.02 ) , the effect size remained threefold stronger in women than in men ( -coefficient 0.0427 vs. 0.0165 , respectively ) . to provide further reassurance regarding any residual heterogeneity , we repeated our meta - analyses based on p values ( rather than -coefficients ) and meta - analyzed the resulting z scores . our findings were essentially unchanged , suggesting that heterogeneity in the -estimates across cohorts has not produced spurious results . proinsulin is initially cleaved to 32,33-split proinsulin and further to insulin and c - peptide before secretion ( supplementary fig . 1 ) ; we were therefore interested in the effects of the nine top snps on these traits . the proinsulin - raising alleles of each snp were consistently associated with higher 32,33-split proinsulin levels , with effect sizes following the rank order of proinsulin effect sizes . nearly all associations reached nominal conventional levels of statistical significance in this smaller dataset of 4,1036,343 individuals with measures of 32,33-split proinsulin levels ( all p < 1.5 10 , with the exception of the conditional signal at madd ) . the insulinogenic index ( 27 ) , which measures dynamic insulin secretion during the first 30 min after an oral glucose load and was available in 14,956 subjects , showed nominal associations for four loci . of these , the proinsulin - raising alleles were associated with a lower insulinogenic index at vps13c / c2cd4a / b , tcf7l2 , and slc30a8 and higher at arap1 ( table 2 ) . association of proinsulin loci with insulin - processing traits -coefficients are adjusted for age , sex , and study - specific covariates ( if applicable ) . we detected no nominal associations with fasting c - peptide ( p > 0.05 ) . given the differences in hepatic clearance of insulin and c - peptide , we also performed sensitivity analyses to account for any possible impact this may have had on our results . we adjusted proinsulin levels for fasting c - peptide rather than fasting insulin in two cohorts ( ely and botnia - ppp ) ; comparison of -estimates showed that the majority of loci had very similar effect sizes and the same rank order was preserved , arguing against noticeable discrepancies between the two adjustment schemes . to clarify potential mechanisms , the top nine signals ( arap1 , two at madd , pcsk1 , tcf7l2 , vps13c / c2cd4a / b , slc30a8 , larp6 , and sgsm2 ) were also examined in relation to other glucometabolic traits ( fasting and 2-h postload glucose and insulin , homeostasis model assessment estimates of -cell function [ homa - b ] and insulin resistance [ homa - ir ] , glycated hemoglobin [ a1c ] , t2d , and bmi [ table 3 ] ) . we investigated results available from magic meta - analyses of gwas of glycemic traits ( 35 ) and obtained t2d and bmi results in collaboration with the diabetes genetics replication and meta - analysis ( diagram ) ( 29 ) and genomewide investigation of anthropometric measures ( giant ) ( 30 ) consortia , respectively . nominal associations ( p < 0.05 ) were found for fasting glucose ( with the proinsulin - raising allele increasing fasting glucose levels at madd , slc30a8 , tcf7l2 , and vps13c / c2cd4a / b and decreasing fasting glucose levels at arap1 and pcsk1 ) , fasting insulin ( increased levels at arap1 , larp6 , and sgsm2 and decreased levels at tcf7l2 ) , homa - b ( decreased at madd , slc30a8 , vps13c / c2cd4a / b , and tcf7l2 and increased at pcsk1 , arap1 , and larp6 ) , insulin resistance as measured by homa - ir ( increased at larp6 and sgsm2 and decreased at tcf7l2 ) , and 2-h postload glucose ( decreased at slc30a8 and vps13c / c2cd4a / b and increased at arap1 and tcf7l2 ) . we detected no significant associations for 2-h postload insulin or insulin sensitivity as estimated by the matsuda index ( 31 ) ( table 3 ) . association of proinsulin loci with other glycemic traits -coefficients are adjusted for age , sex , and study - specific covariates ( if applicable ) . or is for t2d only as a dichotomous trait . associations with t2d were confirmed for four known t2d loci ( slc30a8 , arap1 , vps13c / c2cd4a / b , and tcf7l2 ; table 3 ) . counterintuitively , the proinsulin - raising allele of arap1 ( formerly known as centd2 and reported as such in diagram+ ) ( 29 ) was associated with a lower fasting glucose ( 0.019 mg / dl per a allele ; p = 1.7 10 ) , lower a1c ( 0.023% ; p = 0.02 ) , and a lower risk of t2d ( odds ratio [ or ] 0.88 ; p = 7.8 10 ; table 3 ) . the two novel loci ( larp6 and sgsm2 ) did not show significant associations with t2d ( or [ 95% ci ] : 1.01 [ 0.951.07 ] and 1.01 [ 0.961.08 ] , respectively ) , indicating that if they increase t2d risk they do so to an extent confined within the bounds of narrow 95% ci . we used mach ( 32 ) or impute ( 19 ) applied to the 1000 genomes ceu reference panel ( www.1000genomes.org ) to carry out imputation of 8 million autosomal snps with minor allele frequency > 1% . analysis of 1000 genomes - imputed data in the four discovery cohorts indicates that although there are low - frequency ( 15% ) genetic variants that influence levels of circulating proinsulin , these are found in the same loci that contain common proinsulin - influencing variants , and none of them yield substantially stronger signals than the index snp at each locus ( supplementary fig . 5 ) . using current databases of copy number variants ( 33 ) and the snap software ( http://www.broadinstitute.org/mpg/snap/index.php ; ceu , hapmap release 22 ) , we checked whether any of the proinsulin - associated snps were within 500 kb and in linkage disequilibrium ( ld ) with any of the snps known to tag copy number variants in the human genome . no copy number variant tag snps with r > 0.3 were found within 500 kb of our lead snps . to guide identification of the gene responsible for each association signal , we also examined the gene expression profile of selected genes in each associated region across a range of human tissues , including islets and fluorescence - activated cell ( fac)-sorted -cells ( fig . we defined 1-mb intervals around the lead snp at each locus and prioritized biologically plausible genes as gleaned from the literature ( see box in supplementary data ) . however , at the larp6 locus , ct62 is expressed exclusively in testis , likely excluding it as a relevant gene in this context . at the arap1 locus , stard10 is expressed more strongly in pancreatic and islet tissue than any other tissue type ; similarly , at the vps13c locus both c2cd4a and c2cd4b demonstrate higher expression in pancreas and islets than all other tissue types . expression profiles of biologically plausible genes within each associated locus across a range of human tissue types , including islet preparations from three donors . a : arap1 region ; b : madd region ; c : vps13c / c2cd4a / b region ; d : larp6 region ; e : sgsm2 region . f : expression levels of genes near the proinsulin - associated variants in human fac - sorted -cells . data are expression means sd of the relative expression measured by quantitative pcr obtained from three human nondiabetic donors . we also studied the expression of the transcript for the gene closest to the index snp at each of the nine replicated loci in human islets isolated from 55 nondiabetic and 9 diabetic individuals . of the nine loci , pcsk1 ( p = 0.02 ) and madd ( p = 0.07 ) demonstrated 3545% lower expression in subjects with t2d compared with control subjects . we evaluated whether any of the associated snps was in strong ld with a potentially causal variant . we used snpper ( 34 ) to classify all snps in strong ld with the lead snp ( r 0.8 ) within a 1-mb region . we found that pcsk1 rs6235 codes for a nonsynonymous variant ( s690 t ) , which is in perfect ld with rs6234 , another missense variant ( q665e ) ; both were predicted to be nondamaging by polyphen ( 35 ) and sift ( 36 ) . at slc30a8 , the proinsulin - associated snp rs11558471 is a perfect proxy for the known t2d - associated snp rs13266634 , encoding r325w . the t allele ( encoding tryptophan ) is predicted to be benign by polyphen , but damaging by sift . we found no other strong ( r > 0.8 ) correlations in hapmap ceu with potentially functional snps within 1 mb of the lead signals . we also tested whether any of the proinsulin - associated snps might influence proximal ( cis ) expression of human transcripts , in tissues available to us that had been paired to genetic data . we found a significant association ( p = 0.01 permutation threshold ) of rs1549318 with expression levels of larp6 in adipose tissue . snp rs1549318 is located 37 kb from larp6 , and the proinsulin - raising t allele is associated with lower levels of expression . analysis of an eqtl database from human liver indicated that the proinsulin - raising a allele of the lead snp at the sgsm2 locus ( rs179456 ) was associated with increased liver expression of trps1 ( p = 0.004 ) . we constructed unweighted and weighted genotype scores composed of the nine genome - wide significant proinsulin - raising alleles , with weights defined by the -coefficients from our replication meta - analysis , and tested the association of these scores with cad in the coronary artery disease genome - wide replication and meta - analysis ( cardiogram ) ( 37 ) ( n = 22,000 cad case subjects and 60,000 control subjects ) and c4d ( 38 ) ( n = 15,420 cad case subjects and 15,062 control subjects ) datasets . neither weighted nor unweighted genotype scores reached nominal significance in either dataset ( p = 0.47 and 0.81 for unweighted and weighted scores in cardiogram , respectively ; p = 0.60 and 0.43 for unweighted and weighted scores in c4d , respectively ) . we report the first meta - analysis of genome - wide association datasets for circulating fasting proinsulin . we adjusted proinsulin for fasting insulin levels , aiming to capture an increase in proinsulin relative to the nonspecific activation of the insulin processing pathway induced by generalized insulin resistance ( supplementary fig . 2 ) . loci that simply influence insulin resistance are typically sought by a gwas for fasting insulin or more sophisticated measures of insulin sensitivity ( 3,4,6 ) . thus , we hoped to identify loci that indicate the inability of the -cell to process proinsulin adequately in response to metabolic demands . we have identified nine signals at eight loci associated with higher proinsulin levels ( see box in supplementary data ) . two of these loci ( larp6 and sgsm2 ) have not been previously related to metabolic traits . a 10th signal emerged after sex - stratified analyses ; an explanation for the female - specific genome - wide significant association at ddx31 requires fine - mapping to identify the causal gene . although the function of the ddx31 gene product is unknown , other members of the dead - box protein family have been implicated in sex - specific processes such as spermatogenesis ( 39 ) . we have also replicated at the genome - wide level previously reported nominal associations of madd , tcf7l2 , vps13c / c2cd4a / b , slc30a8 , and pcsk1 with proinsulin ( 6,1417,40 ) . the knowledge that tcf7l2 , slc30a8 , vps13c / c2cd4a / b , and arap1 are established t2d loci provides reassurance that a quest for genetic determinants of proinsulin can serve to identify disease - associated signals . interestingly , the proinsulin - raising alleles at tcf7l2 , slc30a8 , and vps13c / c2cd4a / b cause impairment of -cell function , as estimated by homa - b and the insulinogenic index . by raising proinsulin but lowering insulin secretion , these loci point to defects in the insulin processing and secretion pathway , distal to the first enzymatic step . such a hypothesis is consistent with postulated modes of action for tcf7l2 ( 41 ) and slc30a8 ( 42 ) ; vps13c , by influencing protein trafficking across membrane compartments , could also affect the same process . further fine - mapping and functional experiments will be required to establish the precise mechanism at this locus . arap1 , which harbors the strongest proinsulin association , provides an intriguing counterpoint . under its previous designation of centd2 it was recently associated with t2d ( 29 ) ; however , the t2d - associated allele is associated with lower proinsulin levels , as well as lower -cell function ( homa - b and insulinogenic index ) . this suggests that the genetic defect that gives rise to t2d at this locus causes a generalized downregulation of insulin secretion ( e.g. , through a reduction in -cell mass / function or very early defects in insulin processing ) and stands in contrast with tcf7l2 , slc30a8 , and vps13c / c2cd4a / b . a corollary of the divergent effect of these loci on t2d is that both disproportionate elevations and reductions in proinsulin can indicate -cell dysfunction . of the genes that lie within 1 mb of the arap1 association signal , we have demonstrated islet expression in the four strong biological candidates we examined ( arap1 , inppl1 , stard10 , and rab6a ) ; however , expression of stard10 was much higher in pancreas than in any other human tissue , and of all genes tested at the arap1 locus stard10 was expressed most strongly in islets , indicating that the role of its protein product in the transfer of phospholipids to membranes may be particularly relevant to this cell type . larp6 is a ribonucleoprotein identified in the current study as a novel locus associated with increased fasting proinsulin levels . it is involved in the regulation of translation and subcellular localization of collagen i , in a manner dependent upon both the rna - binding and la domains ( 43 ) . the associated snp rs1549318 is located within a region of high ld , which spans the gene and includes a number of snps within the rna - binding domain . although the link between larp6 and proinsulin levels is not clear , it is nominally associated with fasting insulin and homa - ir , but not t2d . it may therefore represent a marker of insulin resistance and perhaps other related common dysmetabolic conditions . in previous publications we have reported the association of c2cd4b with fasting glucose ( 3 ) and that of the nearby locus vps13c with 2-h glucose ( 4 ) ; c2cd4b is also associated with t2d in japanese ( 44 ) , with supportive evidence found in europeans ( 3,44 ) . here the strongest association with proinsulin reported here ( rs4502156 ) and those associated with fasting glucose and 2-h glucose may represent independent signals , since they are all in relatively weak ld in hapmap ceu europeans : rs4502156 versus rs11071657 ( best fasting glucose signal ) , r = 0.306 ; rs4502156 vs. rs17271305 ( best 2-h glucose signal ) , r = 0.450 ; and rs11071657 versus rs17271305 , r = 0.287 . on the other hand , in europeans our proinsulin - associated snp is in strong ld ( r = 0.967 ) with the t2d - associated snp reported by yamauchi et al . although four strong biological candidates ( c2cd4a , c2cd4b , vps13c , and rora , a gene that encodes a member of the nr1 subfamily of nuclear hormone receptors ) are expressed in fac - sorted -cells , the relative expression of the first two is much higher in islets than in other human tissues , again suggesting that these two genes , encoding nuclear factors that are upregulated in response to inflammation , may be particularly relevant to endocrine pancreatic function . the genome - wide association of a missense variant in pcsk1 with fasting proinsulin also serves as a positive control . pcsk1 encodes the protein prohormone convertase 1/3 ( pc1 ) , which is the first enzyme in the proinsulin processing pathway , where it cleaves proinsulin to 32,33-split proinsulin ( supplementary fig . 1 ) . a related enzyme , pc2 , acts on 32,33-split proinsulin in the second processing step . people deficient in pc1 become obese at an early age and exhibit pituitary hypofunction because of the lack of several mature peptide hormones ( 45 ) , whereas pc2-null mice demonstrate increased levels of 32,33-split proinsulin ( 46 ) . the rs6235 snp reported here results in the substitution of a serine residue for threonine at position 690 of the molecule ; the minor allele ( thr ) is associated with higher proinsulin levels . a nominal association of the same allele with higher proinsulin levels has recently been reported ( 40 ) ; its association with higher bmi is only nominal here , but confirms a previous report ( 47 ) . this specific amino acid change has been shown not to affect enzyme catalysis or maturation of the protein in vitro ( 47 ) , but the cooh terminus of the protein ( where s690 t is located , adjacent to a conserved proline residue ) is known to direct the correct subcellular targeting of the protein as well as stabilizing and partially inhibiting pc1 . although one might expect lower levels of the reaction product ( 32,33-split proinsulin ) in carriers of the risk allele , the potential diversion of the substrate down its alternate path ( giving rise to 65,66-split proinsulin , whose assay typically has 60% cross - reactivity with 32,33-split proinsulin ) requires further study . alternatively , if changes in the activity of pc1 also affect that of pc2 ( for instance , by competing for inhibitory peptides ) one might see reductions in the catalytic function of both enzymes and accumulation of both proinsulin and 32,33-split proinsulin . because of the reported relationship between proinsulin levels and coronary events ( 1113 ) , the identification of genetic determinants of proinsulin levels might help shed light on whether hyperproinsulinemia is a mediator of cad or a byproduct of a shared etiological mechanism . if hyperproinsulinemia is causally associated with an increased risk of cad , one might expect that snps that specifically and selectively raise proinsulin levels should increase the risk of cad given an adequately powered study . we have not observed such an effect for a genotype score constructed with the genome - wide significant proinsulin association signals . assuming conservative approximations of the reported effect sizes of proinsulin on cad ( or 1.5 per 1-sd increase in proinsulin ) ( 12,13 ) , and of the nine snps reported here on circulating proinsulin ( 5% ) , a cad cohort like cardiogram has 99% power to detect an effect of proinsulin snps on cad . the absence of statistical significance argues against a direct etiological role of proinsulin on cad . in summary , we have identified nine loci that associate with fasting proinsulin levels . several of these loci increase risk of t2d ; interestingly , both proinsulin - raising and lowering alleles can lead to t2d through decreases in insulin secretion , indicating defects distal or proximal to the first enzymatic step in proinsulin conversion , respectively . other genetic determinants of proinsulin levels do not necessarily lead to higher t2d risk , suggesting that it is not a mere elevation in proinsulin , but rather the specific impairment in proinsulin processing and the reaction of the -cell to this defect that determine whether ultimately -cell insufficiency will cause pathological hyperglycemia . the direct elevation of fasting proinsulin out of proportion to fasting insulin does not seem to increase risk of cad .

objectiveproinsulin is a precursor of mature insulin and c - peptide . higher circulating proinsulin levels are associated with impaired -cell function , raised glucose levels , insulin resistance , and type 2 diabetes ( t2d ) . studies of the insulin processing pathway could provide new insights about t2d pathophysiology.research design and methodswe have conducted a meta - analysis of genome - wide association tests of 2.5 million genotyped or imputed single nucleotide polymorphisms ( snps ) and fasting proinsulin levels in 10,701 nondiabetic adults of european ancestry , with follow - up of 23 loci in up to 16,378 individuals , using additive genetic models adjusted for age , sex , fasting insulin , and study - specific covariates.resultsnine snps at eight loci were associated with proinsulin levels ( p < 5 108 ) . two loci ( larp6 and sgsm2 ) have not been previously related to metabolic traits , one ( madd ) has been associated with fasting glucose , one ( pcsk1 ) has been implicated in obesity , and four ( tcf7l2 , slc30a8 , vps13c / c2cd4a / b , and arap1 , formerly centd2 ) increase t2d risk . the proinsulin - raising allele of arap1 was associated with a lower fasting glucose ( p = 1.7 104 ) , improved -cell function ( p = 1.1 105 ) , and lower risk of t2d ( odds ratio 0.88 ; p = 7.8 106 ) . notably , pcsk1 encodes the protein prohormone convertase 1/3 , the first enzyme in the insulin processing pathway . a genotype score composed of the nine proinsulin - raising alleles was not associated with coronary disease in two large case - control datasets.conclusionswe have identified nine genetic variants associated with fasting proinsulin . our findings illuminate the biology underlying glucose homeostasis and t2d development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis .

RESEARCH DESIGN AND METHODS Cohort/study description. Proinsulin and insulin measurements. Genotyping. Statistical analyses. Follow-up SNP selection and analysis. RESULTS Genome-wide association meta-analysis (stage 1). Follow-up studies (stage 2) and global (stage 1 + stage 2) meta-analysis for 23 loci. Heterogeneity and sex-stratified analyses. Exploration of proinsulin processing mechanisms. Association with other glycemic traits. Fine-mapping, copy number variants, and tissue expression. Functional exploration. CAD. DISCUSSION

eleven cohorts contributed to the follow - up efforts ; these included metabolic syndrome in men ( metsim ) ( n = 5,122 ) , botnia prevalence , prediction and prevention of diabetes ( botnia - ppp ) ( n = 2,280 ) , helsinki birth cohort study ( hbcs ) ( n = 1,649 ) , the ely study ( n = 1,568 ) , the hertfordshire study ( n = 1,016 ) , uppsala longitudinal study of adult men ( ulsam ) ( n = 939 ) , relationship between insulin sensitivity and cardiovascular disease ( risc ) ( n = 914 ) , prospective investigation of the vasculature in uppsala seniors ( pivus ) ( n = 912 ) , segovia ( n = 911 ) , the greek health randomized aging study ( ghras ) ( n = 668 ) , and stockholm diabetes prevention program ( sdpp ) ( n = 399 ) , for a total of 16,378 participants ( with maximal sample for any one snp of 15,898 ) . we chose the latter because the adjusted trait has comparable predictive value ( 18 ) and displayed better statistical performance in pilot studies and adequate heritability in the framingham heart study , one of the larger cohorts examined here ( h = 0.36 vs. 0.34 for the proinsulin - to - insulin ratio ) . we used a linear regression model with natural log transformed fasting proinsulin as the dependent variable and genotypes as predictors , with adjustment for natural - log transformed fasting insulin values , sex , age , geographical covariates ( if applicable ) , and age squared ( framingham only ) to evaluate the association under an additive genetic model . eleven cohorts contributed to the follow - up efforts ; these included metabolic syndrome in men ( metsim ) ( n = 5,122 ) , botnia prevalence , prediction and prevention of diabetes ( botnia - ppp ) ( n = 2,280 ) , helsinki birth cohort study ( hbcs ) ( n = 1,649 ) , the ely study ( n = 1,568 ) , the hertfordshire study ( n = 1,016 ) , uppsala longitudinal study of adult men ( ulsam ) ( n = 939 ) , relationship between insulin sensitivity and cardiovascular disease ( risc ) ( n = 914 ) , prospective investigation of the vasculature in uppsala seniors ( pivus ) ( n = 912 ) , segovia ( n = 911 ) , the greek health randomized aging study ( ghras ) ( n = 668 ) , and stockholm diabetes prevention program ( sdpp ) ( n = 399 ) , for a total of 16,378 participants ( with maximal sample for any one snp of 15,898 ) . we chose the latter because the adjusted trait has comparable predictive value ( 18 ) and displayed better statistical performance in pilot studies and adequate heritability in the framingham heart study , one of the larger cohorts examined here ( h = 0.36 vs. 0.34 for the proinsulin - to - insulin ratio ) . we used a linear regression model with natural log transformed fasting proinsulin as the dependent variable and genotypes as predictors , with adjustment for natural - log transformed fasting insulin values , sex , age , geographical covariates ( if applicable ) , and age squared ( framingham only ) to evaluate the association under an additive genetic model . the snps that achieved genome - wide significance ( p < 5 10 ) on follow - up are shown in red . joint meta - analysis of discovery and follow - up cohorts ( n = 27,079 ) revealed nine signals at eight loci reaching genome - wide significance ( p < 5 10 ) , of which two are novel ( sgsm2 , larp6 ) , five have previously been associated with glucose metabolism and/or t2d ( tcf7l2 , slc30a8 , madd , vps13c / c2cd4a / b , and arap1 ) , and one ( pcsk1 ) has been previously implicated in obesity and associated with proinsulin levels , although not at genome - wide significance ( table 1 and fig . of note , when adjusting for fasting glucose or both fasting glucose and bmi ( but not bmi alone ) , one other locus , snx7 , reached genome - wide significance ( p = 5.4 10 and 1.5 10 , respectively ) . loci associated with fasting proinsulin levels at genome - wide levels of statistical significance -coefficients for the effect allele are shown after adjustments for sex , age , geographic covariates ( if applicable ) , and age squared ( framingham only ) , besides the additional adjustments denoted in the column headings . in each panel , the stage 1 discovery snp taken forward to stage 2 follow - up is represented by a purple diamond ( with global meta - analysis p value ) , with its stage 1 discovery p value denoted by a red diamond with bolded borders . among the nine replicated snps , individual loci explained between 0.2 and 1.4% of the variance in proinsulin in the discovery samples and up to 2.3% of the variance in the follow - up samples . together , the nine genome - wide significant snps explained between 5.4 and 7.7% of the proinsulin variance in the discovery samples and 8.1% of the variance in the risc cohort , one of the few follow - up cohorts with genotypes available for all nine snps . the proinsulin - raising alleles of each snp were consistently associated with higher 32,33-split proinsulin levels , with effect sizes following the rank order of proinsulin effect sizes . nearly all associations reached nominal conventional levels of statistical significance in this smaller dataset of 4,1036,343 individuals with measures of 32,33-split proinsulin levels ( all p < 1.5 10 , with the exception of the conditional signal at madd ) . of these , the proinsulin - raising alleles were associated with a lower insulinogenic index at vps13c / c2cd4a / b , tcf7l2 , and slc30a8 and higher at arap1 ( table 2 ) . association of proinsulin loci with insulin - processing traits -coefficients are adjusted for age , sex , and study - specific covariates ( if applicable ) . to clarify potential mechanisms , the top nine signals ( arap1 , two at madd , pcsk1 , tcf7l2 , vps13c / c2cd4a / b , slc30a8 , larp6 , and sgsm2 ) were also examined in relation to other glucometabolic traits ( fasting and 2-h postload glucose and insulin , homeostasis model assessment estimates of -cell function [ homa - b ] and insulin resistance [ homa - ir ] , glycated hemoglobin [ a1c ] , t2d , and bmi [ table 3 ] ) . nominal associations ( p < 0.05 ) were found for fasting glucose ( with the proinsulin - raising allele increasing fasting glucose levels at madd , slc30a8 , tcf7l2 , and vps13c / c2cd4a / b and decreasing fasting glucose levels at arap1 and pcsk1 ) , fasting insulin ( increased levels at arap1 , larp6 , and sgsm2 and decreased levels at tcf7l2 ) , homa - b ( decreased at madd , slc30a8 , vps13c / c2cd4a / b , and tcf7l2 and increased at pcsk1 , arap1 , and larp6 ) , insulin resistance as measured by homa - ir ( increased at larp6 and sgsm2 and decreased at tcf7l2 ) , and 2-h postload glucose ( decreased at slc30a8 and vps13c / c2cd4a / b and increased at arap1 and tcf7l2 ) . association of proinsulin loci with other glycemic traits -coefficients are adjusted for age , sex , and study - specific covariates ( if applicable ) . associations with t2d were confirmed for four known t2d loci ( slc30a8 , arap1 , vps13c / c2cd4a / b , and tcf7l2 ; table 3 ) . counterintuitively , the proinsulin - raising allele of arap1 ( formerly known as centd2 and reported as such in diagram+ ) ( 29 ) was associated with a lower fasting glucose ( 0.019 mg / dl per a allele ; p = 1.7 10 ) , lower a1c ( 0.023% ; p = 0.02 ) , and a lower risk of t2d ( odds ratio [ or ] 0.88 ; p = 7.8 10 ; table 3 ) . the two novel loci ( larp6 and sgsm2 ) did not show significant associations with t2d ( or [ 95% ci ] : 1.01 [ 0.951.07 ] and 1.01 [ 0.961.08 ] , respectively ) , indicating that if they increase t2d risk they do so to an extent confined within the bounds of narrow 95% ci . analysis of 1000 genomes - imputed data in the four discovery cohorts indicates that although there are low - frequency ( 15% ) genetic variants that influence levels of circulating proinsulin , these are found in the same loci that contain common proinsulin - influencing variants , and none of them yield substantially stronger signals than the index snp at each locus ( supplementary fig . analysis of an eqtl database from human liver indicated that the proinsulin - raising a allele of the lead snp at the sgsm2 locus ( rs179456 ) was associated with increased liver expression of trps1 ( p = 0.004 ) . we constructed unweighted and weighted genotype scores composed of the nine genome - wide significant proinsulin - raising alleles , with weights defined by the -coefficients from our replication meta - analysis , and tested the association of these scores with cad in the coronary artery disease genome - wide replication and meta - analysis ( cardiogram ) ( 37 ) ( n = 22,000 cad case subjects and 60,000 control subjects ) and c4d ( 38 ) ( n = 15,420 cad case subjects and 15,062 control subjects ) datasets . the snps that achieved genome - wide significance ( p < 5 10 ) on follow - up are shown in red . joint meta - analysis of discovery and follow - up cohorts ( n = 27,079 ) revealed nine signals at eight loci reaching genome - wide significance ( p < 5 10 ) , of which two are novel ( sgsm2 , larp6 ) , five have previously been associated with glucose metabolism and/or t2d ( tcf7l2 , slc30a8 , madd , vps13c / c2cd4a / b , and arap1 ) , and one ( pcsk1 ) has been previously implicated in obesity and associated with proinsulin levels , although not at genome - wide significance ( table 1 and fig . of note , when adjusting for fasting glucose or both fasting glucose and bmi ( but not bmi alone ) , one other locus , snx7 , reached genome - wide significance ( p = 5.4 10 and 1.5 10 , respectively ) . loci associated with fasting proinsulin levels at genome - wide levels of statistical significance -coefficients for the effect allele are shown after adjustments for sex , age , geographic covariates ( if applicable ) , and age squared ( framingham only ) , besides the additional adjustments denoted in the column headings . in each panel , the stage 1 discovery snp taken forward to stage 2 follow - up is represented by a purple diamond ( with global meta - analysis p value ) , with its stage 1 discovery p value denoted by a red diamond with bolded borders . among the nine replicated snps , individual loci explained between 0.2 and 1.4% of the variance in proinsulin in the discovery samples and up to 2.3% of the variance in the follow - up samples . together , the nine genome - wide significant snps explained between 5.4 and 7.7% of the proinsulin variance in the discovery samples and 8.1% of the variance in the risc cohort , one of the few follow - up cohorts with genotypes available for all nine snps . the proinsulin - raising alleles of each snp were consistently associated with higher 32,33-split proinsulin levels , with effect sizes following the rank order of proinsulin effect sizes . of these , the proinsulin - raising alleles were associated with a lower insulinogenic index at vps13c / c2cd4a / b , tcf7l2 , and slc30a8 and higher at arap1 ( table 2 ) . association of proinsulin loci with insulin - processing traits -coefficients are adjusted for age , sex , and study - specific covariates ( if applicable ) . to clarify potential mechanisms , the top nine signals ( arap1 , two at madd , pcsk1 , tcf7l2 , vps13c / c2cd4a / b , slc30a8 , larp6 , and sgsm2 ) were also examined in relation to other glucometabolic traits ( fasting and 2-h postload glucose and insulin , homeostasis model assessment estimates of -cell function [ homa - b ] and insulin resistance [ homa - ir ] , glycated hemoglobin [ a1c ] , t2d , and bmi [ table 3 ] ) . nominal associations ( p < 0.05 ) were found for fasting glucose ( with the proinsulin - raising allele increasing fasting glucose levels at madd , slc30a8 , tcf7l2 , and vps13c / c2cd4a / b and decreasing fasting glucose levels at arap1 and pcsk1 ) , fasting insulin ( increased levels at arap1 , larp6 , and sgsm2 and decreased levels at tcf7l2 ) , homa - b ( decreased at madd , slc30a8 , vps13c / c2cd4a / b , and tcf7l2 and increased at pcsk1 , arap1 , and larp6 ) , insulin resistance as measured by homa - ir ( increased at larp6 and sgsm2 and decreased at tcf7l2 ) , and 2-h postload glucose ( decreased at slc30a8 and vps13c / c2cd4a / b and increased at arap1 and tcf7l2 ) . association of proinsulin loci with other glycemic traits -coefficients are adjusted for age , sex , and study - specific covariates ( if applicable ) . associations with t2d were confirmed for four known t2d loci ( slc30a8 , arap1 , vps13c / c2cd4a / b , and tcf7l2 ; table 3 ) . counterintuitively , the proinsulin - raising allele of arap1 ( formerly known as centd2 and reported as such in diagram+ ) ( 29 ) was associated with a lower fasting glucose ( 0.019 mg / dl per a allele ; p = 1.7 10 ) , lower a1c ( 0.023% ; p = 0.02 ) , and a lower risk of t2d ( odds ratio [ or ] 0.88 ; p = 7.8 10 ; table 3 ) . the two novel loci ( larp6 and sgsm2 ) did not show significant associations with t2d ( or [ 95% ci ] : 1.01 [ 0.951.07 ] and 1.01 [ 0.961.08 ] , respectively ) , indicating that if they increase t2d risk they do so to an extent confined within the bounds of narrow 95% ci . analysis of an eqtl database from human liver indicated that the proinsulin - raising a allele of the lead snp at the sgsm2 locus ( rs179456 ) was associated with increased liver expression of trps1 ( p = 0.004 ) . we constructed unweighted and weighted genotype scores composed of the nine genome - wide significant proinsulin - raising alleles , with weights defined by the -coefficients from our replication meta - analysis , and tested the association of these scores with cad in the coronary artery disease genome - wide replication and meta - analysis ( cardiogram ) ( 37 ) ( n = 22,000 cad case subjects and 60,000 control subjects ) and c4d ( 38 ) ( n = 15,420 cad case subjects and 15,062 control subjects ) datasets . we report the first meta - analysis of genome - wide association datasets for circulating fasting proinsulin . we adjusted proinsulin for fasting insulin levels , aiming to capture an increase in proinsulin relative to the nonspecific activation of the insulin processing pathway induced by generalized insulin resistance ( supplementary fig . we have identified nine signals at eight loci associated with higher proinsulin levels ( see box in supplementary data ) . two of these loci ( larp6 and sgsm2 ) have not been previously related to metabolic traits . we have also replicated at the genome - wide level previously reported nominal associations of madd , tcf7l2 , vps13c / c2cd4a / b , slc30a8 , and pcsk1 with proinsulin ( 6,1417,40 ) . the knowledge that tcf7l2 , slc30a8 , vps13c / c2cd4a / b , and arap1 are established t2d loci provides reassurance that a quest for genetic determinants of proinsulin can serve to identify disease - associated signals . interestingly , the proinsulin - raising alleles at tcf7l2 , slc30a8 , and vps13c / c2cd4a / b cause impairment of -cell function , as estimated by homa - b and the insulinogenic index . under its previous designation of centd2 it was recently associated with t2d ( 29 ) ; however , the t2d - associated allele is associated with lower proinsulin levels , as well as lower -cell function ( homa - b and insulinogenic index ) . , through a reduction in -cell mass / function or very early defects in insulin processing ) and stands in contrast with tcf7l2 , slc30a8 , and vps13c / c2cd4a / b . in previous publications we have reported the association of c2cd4b with fasting glucose ( 3 ) and that of the nearby locus vps13c with 2-h glucose ( 4 ) ; c2cd4b is also associated with t2d in japanese ( 44 ) , with supportive evidence found in europeans ( 3,44 ) . pcsk1 encodes the protein prohormone convertase 1/3 ( pc1 ) , which is the first enzyme in the proinsulin processing pathway , where it cleaves proinsulin to 32,33-split proinsulin ( supplementary fig .

therefore , on a conventional mammogram , fat appears dark , whereas fibroglandular tissue appears white . in 1976 , wolfe was one of the first to demonstrate an association between breast density and the risk of developing breast cancer . this association was confirmed by mccormack et al . in a large meta - analysis including 42 studies , they showed that the risk of developing breast cancer is increased in dense breasts . the magnitude of this risk can be as high as 4.6-fold for the most dense breasts compared with the least dense category . recently , boyd et al . found similar results , showing that women with fibroglandular tissue in more than 75% of the mammogram had an increased risk of developing breast cancer when compared with women with less than 10% breast density in the mammogram ( odds ratio 4.7 ) . in addition to this association , several studies have shown that the sensitivity of the mammogram for detecting breast cancer is decreased in dense breasts [ 5 , 6 ] . for example , carney et al . demonstrated in a study of 463,372 screening mammograms that the sensitivity and specificity for fatty breast mammography were 88 and 97% respectively . however , these numbers decreased in extremely dense breasts ( sensitivity 62% , specificity 90% ) . several qualitative methods to assess breast density have been proposed in the past years , for example the wolfe and tabar classifications [ 2 , 7 ] . in 2003 , the breast imaging reporting and data system ( bi - rads ) quantitative classification was introduced . of these different breast density classification systems , bi - rads guidelines recommend an assessment of breast composition to be included in the analysis of the mammogram . according to the quantitative bi - rads breast density classification , breast density on mammogram can be divided into four categories : ( 1 ) almost entirely fat ( 24% ) , ( 2 ) scattered fibroglandular densities ( 2549% ) , ( 3 ) heterogeneously dense ( 5074% ) , and ( 4 ) extremely dense ( 75% ) . in the past nonetheless , they have not yet attained a strong hold in screening or clinical settings , mainly because these systems are time and labour intensive and require dedicated software programs and operator training . in screening and clinical settings , radiologists therefore generally assess breast density in a visual manner ( eyeballing ) as part of the total evaluation of the mammogram . in this study , we aimed to evaluate the accuracy of visual assessment of breast density ( on standard digital mammograms ) for both experienced and inexperienced readers as compared to the semi - automated assessment of breast density using a dedicated software program . consecutive digital mammograms of 200 women ( mean age 51.6 years , range 23.991.2 years ) were included . for this study , ethics approval and informed consent for the use of the ( coded ) images was waived according to dutch law . all images were acquired on a dedicated mammography system ( senographe essential , ge healthcare , waukesha , wi , usa ) . the standard craniocaudal ( cc ) and mediolateral oblique ( mlo ) imaging projections were used . additionally excluded were mammograms of breasts that had undergone surgery of any kind , for instance lumpectomy because of breast cancer or benign breast diseases or breast reduction . in the netherlands , research covered by the medical research involving human subjects act must be submitted to an accredited medical ethics committee for approval . however , the act does not cover retrospective research using ( coded ) data from patient s medical record or patient images . therefore , our medical ethics committee concluded that the research proposal of the current study does not , under dutch law , require medical ethics approval because there is no extra burden placed on research subjects for this study ( decision number : metc 11 - 4 - 049 ) . breast density was visually assessed on our dedicated mammographic workstations by an experienced mammoradiologist ( c.b . , 18 years experience ) and by an ( inexperienced ) senior resident in radiology ( c.f . breast densities had to be scored in one of the four quantitative bi - rads categories . both readers did not receive any specific training in assessing breast density and were blinded to each other s results , as well as to the results of the semi - automated analysis . breast density was analysed by using a so - called operator - dependent thresholding technique ( leica qwin version 3 , leica microsystems , cambridge , uk ) . this approach is an extensively evaluated method for assessing breast density on standard mammograms [ 1012 ] . more importantly , the association between increased risk for breast cancer in dense breasts has been demonstrated using the thresholding approach . in short , this technique translates the brightness of each pixel in a digitised mammographic image into a grey - level value . the grey - level value of this pixel is obtained , and appropriate threshold grey - level values are selected by the same operator to create an overlay representing the fibroglandular tissue within the breast . next , the total breast area is automatically identified , since regions outside of the breast tissue area are completely black , i.e. they have a grey - level pixel value of 0 . in the mediolateral oblique projections of the breast , the number of pixels is calculated for both the colour overlay of the fibroglandular tissue and the total area of the breast . breast density is presented as the area percentage of the fibroglandular tissue within the breast and is calculated by dividing the number of pixels in the colour overlay by the number of pixels in the total breast area and multiplying by 100 ( fig . 1 ) . the semi - automated analysis was performed by a experienced operator and radiologist ( m.b.i.l . , 5 years of experience using the thresholding approach and this software program ) , who was blinded to the results of both mammography readers . intra - observer reproducibility was assessed by re - analysing a subgroup of randomly selected mammograms ( n = 50 ) with an appropriate interval between these analyses of > 6 months . for this purpose , patients received a different coding . to determine inter - observer reproducibility , the same sample of mammograms was evaluated by an inexperienced operator , m.j.l . ( no experience in using the thresholding technique and the software programme , and receiving only 1 h of operator training by m.b.i.l . ) . the latter was blinded to the results of both mammography readers and the semi - automated analysis performed.fig . d graphic overlay of ( b ) and ( c ) . in this particular example , the mammographic breast density was 22% ( bi - rads density category 1 ) semi - automated detection of mammographic breast density . d graphic overlay of ( b ) and ( c ) . in this particular example , the mammographic breast density was 22% ( bi - rads density category 1 ) statistical analyses were performed by using spss 17.0 ( spss , chicago , il , usa ) and the sas statistical package ( sas institute , cary , nc , usa ) . the correlation between breast densities of both breasts ( as determined by the semi - automated analysis ) and of both cc and mlo projections was assessed by calculating the intra - class correlation coefficient ( icc ) . for the final analysis , the cc projection was used , since it avoids the need for additional segmentation to exclude the pectoral muscle in the mlo projection , which might create bias within the measurements . mean values of the breast density in the cc projections were assessed and translated into one of the four corresponding quantitative bi - rads categories . the inter- and intra - observer reproducibility of the semi - automated analyses was also calculated by using the icc . bland - altman plots were used to show the agreement between the various semi - automated analyses . furthermore , the agreement of the quantitative bi - rads categorisation between the experienced and inexperienced reader was compared and expressed as the weighted kappa value . finally , the accuracy of this quantitative bi - rads categorisation between the readers was evaluated ( with the semi - automated analysis as reference standard ) and again expressed as weighted kappa value . consecutive digital mammograms of 200 women ( mean age 51.6 years , range 23.991.2 years ) were included . for this study , ethics approval and informed consent for the use of the ( coded ) images was waived according to dutch law . all images were acquired on a dedicated mammography system ( senographe essential , ge healthcare , waukesha , wi , usa ) . the standard craniocaudal ( cc ) and mediolateral oblique ( mlo ) imaging projections were used . additionally excluded were mammograms of breasts that had undergone surgery of any kind , for instance lumpectomy because of breast cancer or benign breast diseases or breast reduction . in the netherlands , research covered by the medical research involving human subjects act must be submitted to an accredited medical ethics committee for approval . however , the act does not cover retrospective research using ( coded ) data from patient s medical record or patient images . therefore , our medical ethics committee concluded that the research proposal of the current study does not , under dutch law , require medical ethics approval because there is no extra burden placed on research subjects for this study ( decision number : metc 11 - 4 - 049 ) . breast density was visually assessed on our dedicated mammographic workstations by an experienced mammoradiologist ( c.b . , 18 years experience ) and by an ( inexperienced ) senior resident in radiology ( c.f . breast densities had to be scored in one of the four quantitative bi - rads categories . both readers did not receive any specific training in assessing breast density and were blinded to each other s results , as well as to the results of the semi - automated analysis . breast density was analysed by using a so - called operator - dependent thresholding technique ( leica qwin version 3 , leica microsystems , cambridge , uk ) . this approach is an extensively evaluated method for assessing breast density on standard mammograms [ 1012 ] . more importantly , the association between increased risk for breast cancer in dense breasts has been demonstrated using the thresholding approach . in short , this technique translates the brightness of each pixel in a digitised mammographic image into a grey - level value . the grey - level value of this pixel is obtained , and appropriate threshold grey - level values are selected by the same operator to create an overlay representing the fibroglandular tissue within the breast . next , the total breast area is automatically identified , since regions outside of the breast tissue area are completely black , i.e. they have a grey - level pixel value of 0 . in the mediolateral oblique projections of the breast , the number of pixels is calculated for both the colour overlay of the fibroglandular tissue and the total area of the breast . breast density is presented as the area percentage of the fibroglandular tissue within the breast and is calculated by dividing the number of pixels in the colour overlay by the number of pixels in the total breast area and multiplying by 100 ( fig . 1 ) . the semi - automated analysis was performed by a experienced operator and radiologist ( m.b.i.l . , 5 years of experience using the thresholding approach and this software program ) , who was blinded to the results of both mammography readers . intra - observer reproducibility was assessed by re - analysing a subgroup of randomly selected mammograms ( n = 50 ) with an appropriate interval between these analyses of > 6 months . for this purpose , patients received a different coding . to determine inter - observer reproducibility , the same sample of mammograms was evaluated by an inexperienced operator , m.j.l . ( no experience in using the thresholding technique and the software programme , and receiving only 1 h of operator training by m.b.i.l . ) . the latter was blinded to the results of both mammography readers and the semi - automated analysis performed.fig . d graphic overlay of ( b ) and ( c ) . in this particular example , the mammographic breast density was 22% ( bi - rads density category 1 ) semi - automated detection of mammographic breast density . d graphic overlay of ( b ) and ( c ) . in this particular example , the mammographic breast density was 22% ( bi - rads density category 1 ) statistical analyses were performed by using spss 17.0 ( spss , chicago , il , usa ) and the sas statistical package ( sas institute , cary , nc , usa ) . the correlation between breast densities of both breasts ( as determined by the semi - automated analysis ) and of both cc and mlo projections was assessed by calculating the intra - class correlation coefficient ( icc ) . for the final analysis , the cc projection was used , since it avoids the need for additional segmentation to exclude the pectoral muscle in the mlo projection , which might create bias within the measurements . mean values of the breast density in the cc projections were assessed and translated into one of the four corresponding quantitative bi - rads categories . the inter- and intra - observer reproducibility of the semi - automated analyses was also calculated by using the icc . bland - altman plots were used to show the agreement between the various semi - automated analyses . furthermore , the agreement of the quantitative bi - rads categorisation between the experienced and inexperienced reader was compared and expressed as the weighted kappa value . finally , the accuracy of this quantitative bi - rads categorisation between the readers was evaluated ( with the semi - automated analysis as reference standard ) and again expressed as weighted kappa value . using the semi - automated analysis , the icc of the left - sided breast density in both cc and mlo projection was excellent : 0.91 [ 95% confidence interval ( ci ) 0.880.93 ] . similar excellent results were acquired for the right breast in both projections : icc 0.91 ( 95% ci 0.880.93 ) . the breast densities of the left and right breast were comparable ( icc 0.92 , 95% ci 0.890.94 ) in the cc projection . for the mlo projection , the icc for the breast densities of the left and right breast was excellent ( 0.91 , 95% ci 0.890.93 ) . all these results were highly significant ( all p < 0.0001 ) and comparable with results previously published . bland - altman plots of all these analyses showed very good agreement ( fig . 2 ) . although there is a slightly larger disagreement for more dense breasts , the observed differences are still acceptable.fig . 2bland - altman plots for the semi - automated analyses of breast densities of both breasts in two projections . intra - class correlation coefficients ( icc ) of the measurements were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the difference bland - altman plots for the semi - automated analyses of breast densities of both breasts in two projections . intra - class correlation coefficients ( icc ) of the measurements were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the difference reproducibility of the semi - automated analysis in a subset of mammograms ( n = 50 ) proved to be good as well . the icc of both cc and mlo projections of the left and right breast were all highly significant ( p < 0.0001 ) with values ranging from 0.82 to 0.88 . even more interesting is the inter - observer comparison of the semi - automated analysis . in these analyses , the icc of both cc and mlo projections of the left and right breast were also highly significant ( p < 0.0001 ) with values ranging from 0.80 to 0.88 . although the observed differences in breast densities were slightly higher than in the initial semi - automated analyses , bland - altman plots still showed good agreement of all these analyses with acceptable differences in breast density measurements ( figs . 3 and 4).fig . intra - class correlation coefficients ( icc ) were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the differencesfig . intra - class correlation coefficients ( icc ) of the measurements were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the differences intra - observer agreement of the semi - automated analyses . intra - class correlation coefficients ( icc ) were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the differences inter - observer agreement of the semi - automated analyses . intra - class correlation coefficients ( icc ) of the measurements were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the differences table 1 shows the inter - observer agreement of the quantitative bi - rads classification of both mammographic readers . there was a disagreement between the quantitative bi - rads categorisations of the experienced and inexperienced reader . in 83 of the 200 cases ( 42% ) , the agreement of the experienced and inexperienced reader was therefore only moderate with a kappa value of 0.52 ( table 1).table 1inter - reader agreement of breast density according to bi - rads classifications . results of the experienced reader are presented in the columns , results of the inexperienced reader are presented in the rowsexperienced readerbi - rads 1bi - rads 2bi - rads 3bi - rads 4totalinexperienced readerbi - rads 166 ( 33.0%)2 ( 1.0%)2 ( 1.0%)0 ( 0.0%)70 ( 35.0%)bi - rads 219 ( 9.5%)36 ( 18.0%)1 ( 0.5%)0 ( 0.0%)56 ( 28.0%)bi - rads 35 ( 2.5%)38 ( 19.0%)10 ( 5.0%)0 ( 0.0%)53 ( 26.5%)bi - rads 41 ( 0.5%)3 ( 1.5%)14 ( 7.0%)3 ( 1.5%)21 ( 10.5%)total91 ( 45.5%)79 ( 39.5%)27 ( 13.5%)3 ( 1.5%)200 ( 100.0%)the overall weighted kappa was 0.521 , a moderate value ( 95% confidence interval 0.4460.597 ) inter - reader agreement of breast density according to bi - rads classifications . results of the experienced reader are presented in the columns , results of the inexperienced reader are presented in the rows the overall weighted kappa was 0.521 , a moderate value ( 95% confidence interval 0.4460.597 ) when compared with the results of the semi - automated analyses , the experienced reader agreed with the quantitative bi - rads category in 58.5% of the cases . the classification was overestimated in 35.5% of the cases and underestimated in 6.0% of the cases . in comparison , the inexperienced reader agreed less ( 42.0% ) and generally overestimated the quantitative bi - rads classification more than the experienced reader ( 56.0% ) . agreement between the classification of both readers versus the semi - automated analysis was poor to moderate with weighted kappa values of 0.367 ( experienced reader ) and 0.232 ( inexperienced reader , table 2).table 2classification of the results of the experienced and inexperienced readers and the software analysis . the agreement of the respective readers results and the semi - automated software is presented as the weighted kappa valueexperienced readerinexperienced readersoftware analysistotal patients classified200 ( 100.0%)200 ( 100.0%)200 ( 100.0%)bi - rads 1 classification ( % ) 91 ( 45.5%)70 ( 35.0%)127 ( 63.5%)bi - rads 2 classification ( % ) 79 ( 39.5%)56 ( 28.0%)68 ( 34.0%)bi - rads 3 classification ( % ) 27 ( 13.5%)53 ( 26.5%)5 ( 2.5%)bi - rads 4 classification ( % ) 3 ( 1.5%)21 ( 10.5%)0 ( 0.0%)correct classification ( % ) 117 ( 58.5%)84 ( 42.0%)classification overestimated ( % ) 71 ( 35.5%)112 ( 56.0%)classification underestimated ( % ) 12 ( 6.0%)4 ( 2.0%)weighted kappa0.3670.232 classification of the results of the experienced and inexperienced readers and the software analysis . the agreement of the respective readers results and the semi - automated software is presented as the weighted kappa value in this study , reliability of visual assessment of breast density for both experienced and inexperienced readers was evaluated , as compared to the semi - automated assessment of breast density using a dedicated software program . our results showed that there was disagreement between the quantitative bi - rads categorisation of the experienced and inexperienced readers . when compared to the semi - automated analysis , the experienced reader agreed with the quantitative bi - rads classification in 58.5% of the cases . the classification was overestimated in 35.5% of the cases , and underestimated in 6.0% of the cases . furthermore , the semi - automated assessment of breast density showed good intra- and interobserver reproducibility . breast density is an important risk factor for breast cancer development , independent of other breast cancer risk factors . also , breast cancer is more difficult to detect in mammographically dense breasts [ 5 , 6 ] . in our institution , mammograms are evaluated by radiologists and/or ( supervised ) residents , using the bi - rads classification for breast density . however , our study results showed disagreement between radiologist ( experienced reader ) and resident ( inexperienced reader ) and that breast density is frequently overrated ( even by a highly experienced reader ) . these findings are in line with previously published results . in the majority of cases , although this may seem a negligible overestimation , a speculative ( but nonetheless plausible ) assumption is that overrating breast density might lead to more imaging [ e.g. additional mammographic projections , ultrasound , or contrast - enhanced magnetic resonance ( mr ) mammography ] , more costs , and more patient anxiety . due to the improvements in mr mammography , it is worth considering which patients are at increased risk of developing breast cancer ( i.e. patients with mammographically dense breasts ) and who might benefit from a shorter screening interval or additional mr mammography . despite the fact that this information is enclosed within the images , it is not used in current clinical settings or screening to identify high - risk patients , since the ( visual ) bi - rads density classification is not suitable for the expression of breast cancer risk . based on our current observations ( which show a substantial disagreement between the visual and semi - automated assessment of breast density ) , we would prefer a ( workstation ) integrated ( semi-)automated analysis of breast density to identify patients at high risk for developing breast cancer or in whom breast cancer is likely to be missed . for this purpose , several ( semi-)automated systems to assess breast density have been proposed in the past . of these , the so - called thresholding approach using ( commercially available ) software packages has been extensively studied in the past and is therefore frequently used in quantitative assessment of breast density . using this thresholding technique , boyd et al . showed that women with mammographic breast density 75% had an increased risk of developing breast cancer when compared to women with breast density less than 10% ( odds ratio 4.7 , 95% confidence interval 3.07.4 ) . we also used a thresholding approach using the leica qwin software package and showed similar results in breast densities of both breasts and in both projections . in addition , our software demonstrated a good intra - observer agreement ( fig . a major advantage of our semi - automated analysis is that even analysts with hardly any experience using our software can achieve good reproducibility ( fig . 4 ) . the assessment of the mammographic breast density using this technique is time and labour intensive . for example , our time for the assessment of breast density in a single patient study was estimated to be 58 min . recently , stone et al . demonstrated that assessing breast density of one breast in one projection is sufficient this suggestion is supported by the findings of our current study and enables the inclusion of women that have undergone unilateral breast surgery of any kind . furthermore , the thresholding approach requires proper operator - training to use the software , although our current results ( based on our software program ) suggest otherwise . in screening or clinical settings , this software needs to be integrated in the mammographic work stations . because of these disadvantages , mammographic breast density is usually assessed visually , especially in screening / clinical settings and large studies in which a great number of mammograms need to be evaluated . performed a similar study using a fully automated software package . in this study mammograms of 65 women however , on close inspection , five of the radiologists had already interacted with the software programme , leaving two radiologists ( albeit experienced in mammography reading ) untrained for the breast density analysis . the breast density was overestimated by these two radiologists in 37% of the cases , as compared to 36% overestimation by the experienced radiologist in our study . so although it might be difficult to generalise our study results based on the results of two readers , they are in line with previously published results . in addition , there were other interesting differences in the analyses performed by us and martin et al . in the study of martin et al . , 6% of the images could not be analysed due to technical difficulties versus no technical difficulties in our study . furthermore , the 95% limits of agreement between the trained radiologists and the automated analyses of the study of martin et al . were rather large : 16% to + 27% . visual inspection of the bland - altman plots of our study show that our limits of agreement were much smaller ( fig . 2 ) . our study included relatively small numbers of dense breasts , i.e. quantitative bi - rads categories 3 and 4 . the study of martin et al . described similar results and included 15 additional women with dense breasts in their study after their initial inclusion of 50 women . previously published larger studies show slightly higher numbers of dense breasts in their populations , presumably owing to the larger size of the populations used when compared to our study population . for our study , we opted not to include additional women with dense breasts to prevent inclusion bias . there is no accurate way to determine breast density other than histopathologic analysis of mastectomy specimens . it is obvious that these specimens are not available for this study , and previously published studies assessing breast density with various computerised methods also lack this ( true ) golden standard . as we have shown in this study , our software programme acquired similar results to other , more validated programmes . this is why we have chosen to use our software programme as the reference standard ( not golden standard ) to which we compared the visual assessment of breast density . a second limitation of our study is that the association between mammographically dense breasts and risk of developing breast cancer has not yet been demonstrated with our software programme . due to the comparability of acquired results with more validated software programs , we expect that breast density ( as assessed by our software program ) is also associated with increased breast cancer risk . however , larger studies using our software programme are in progress to prove this hypothesis . finally , our analysis remains a semi - automated ( and not fully automated ) technique , requiring input from an operator and is therefore at risk of introducing observer - dependent bias . recently , a commercially available software tool ( quantra , hologic , bedford , ma , usa ) to automatically assess breast density was compared to the cumulus software program . this study showed a strong density correlation between both breasts and for both methods , suggesting that fully automated assessment of breast density could also aid in breast cancer risk estimation . this was confirmed in another study by pinker et al . , demonstrating that breast density , as assessed with this automated analysis , was strongly associated with breast cancer risk in women younger than 50 years , but not older than 50 years . in line with these developments , another fully automated software program was launched earlier this year : volpara ( matakina international , wellington , new zealand ) . however , the major drawback of these recently available programs is their limited availability on only ge or hologic digital mammography systems . in summary , our results showed that there is a disagreement in quantitative bi - rads breast density classification between experienced and inexperienced readers and the semi - automated software analysis . in order to accurately assess breast density in a reproducible and observer - independent manner , we would recommend the use of an integrated software tool , which can be applied in both screening and clinical settings . this semi - automated analysis of breast density might aid in identifying patients at high risk for developing breast cancer and/or patients who can benefit from additional mr mammography because of an unreliable mammogram . currently , the thresholding approach to assess breast density on standard mammograms is preferable , for instance as assessed by the dedicated software program used in this study or any other commercially available software package . in order to rapidly assess breast density and to include patients who have undergone mastectomy , assessment of a single breast in one projection ( preferably the cc projection ) is sufficient . this proposal is also supported by our current findings . in turn , future studies could investigate whether patients with mammographically dense breasts ( i.e. those at high risk of developing breast cancer ) can benefit from this accurate breast density assessment , for instance by shortening the screening interval or by performing additional ( mr ) imaging . the results of our study showed that visual assessment of breast density on mammograms is inaccurate and observer - dependent . semi - automated analysis of breast density reduces inter - observer variation of breast density classification on mammograms .

objectivesvisual inspection is generally used to assess breast density . our study aim was to compare visual assessment of breast density of experienced and inexperienced readers with semi - automated analysis of breast density.methodsbreast density was assessed by an experienced and an inexperienced reader in 200 mammograms and scored according to the quantitative bi - rads classification . breast density was also assessed by dedicated software using a semi - automated thresholding technique . agreement between breast density classification of both readers as well as agreement between their assessment versus the semi - automated analysis as reference standard was expressed as the weighted kappa value.resultsusing the semi - automated analysis , agreement between breast density measurements of both breasts in both projections was excellent ( icc > 0.9 , p < 0.0001 ) . reproducibility of the semi - automated analysis was excellent ( icc > 0.8 , p < 0.0001 ) . the experienced reader correctly classified the bi - rads breast density classification in 58.5% of the cases . classification was overestimated in 35.5% of the cases and underestimated in 6.0% of the cases . results of the inexperienced reader were less accurate . agreement between the classification of both readers versus the semi - automated analysis was considered only moderate with weighted kappa values of 0.367 ( experienced reader ) and 0.232 ( inexperienced reader).conclusionvisual assessment of breast density on mammograms is inaccurate and observer - dependent .

Introduction Materials and methods Study inclusion Statistical analysis Results Discussion Conclusion

in 1976 , wolfe was one of the first to demonstrate an association between breast density and the risk of developing breast cancer . found similar results , showing that women with fibroglandular tissue in more than 75% of the mammogram had an increased risk of developing breast cancer when compared with women with less than 10% breast density in the mammogram ( odds ratio 4.7 ) . several qualitative methods to assess breast density have been proposed in the past years , for example the wolfe and tabar classifications [ 2 , 7 ] . in 2003 , the breast imaging reporting and data system ( bi - rads ) quantitative classification was introduced . of these different breast density classification systems , bi - rads guidelines recommend an assessment of breast composition to be included in the analysis of the mammogram . according to the quantitative bi - rads breast density classification , breast density on mammogram can be divided into four categories : ( 1 ) almost entirely fat ( 24% ) , ( 2 ) scattered fibroglandular densities ( 2549% ) , ( 3 ) heterogeneously dense ( 5074% ) , and ( 4 ) extremely dense ( 75% ) . in screening and clinical settings , radiologists therefore generally assess breast density in a visual manner ( eyeballing ) as part of the total evaluation of the mammogram . in this study , we aimed to evaluate the accuracy of visual assessment of breast density ( on standard digital mammograms ) for both experienced and inexperienced readers as compared to the semi - automated assessment of breast density using a dedicated software program . breast density was visually assessed on our dedicated mammographic workstations by an experienced mammoradiologist ( c.b . , 18 years experience ) and by an ( inexperienced ) senior resident in radiology ( c.f . breast densities had to be scored in one of the four quantitative bi - rads categories . both readers did not receive any specific training in assessing breast density and were blinded to each other s results , as well as to the results of the semi - automated analysis . breast density was analysed by using a so - called operator - dependent thresholding technique ( leica qwin version 3 , leica microsystems , cambridge , uk ) . this approach is an extensively evaluated method for assessing breast density on standard mammograms [ 1012 ] . breast density is presented as the area percentage of the fibroglandular tissue within the breast and is calculated by dividing the number of pixels in the colour overlay by the number of pixels in the total breast area and multiplying by 100 ( fig . the semi - automated analysis was performed by a experienced operator and radiologist ( m.b.i.l . , 5 years of experience using the thresholding approach and this software program ) , who was blinded to the results of both mammography readers . the latter was blinded to the results of both mammography readers and the semi - automated analysis performed.fig . in this particular example , the mammographic breast density was 22% ( bi - rads density category 1 ) semi - automated detection of mammographic breast density . in this particular example , the mammographic breast density was 22% ( bi - rads density category 1 ) statistical analyses were performed by using spss 17.0 ( spss , chicago , il , usa ) and the sas statistical package ( sas institute , cary , nc , usa ) . the correlation between breast densities of both breasts ( as determined by the semi - automated analysis ) and of both cc and mlo projections was assessed by calculating the intra - class correlation coefficient ( icc ) . mean values of the breast density in the cc projections were assessed and translated into one of the four corresponding quantitative bi - rads categories . the inter- and intra - observer reproducibility of the semi - automated analyses was also calculated by using the icc . bland - altman plots were used to show the agreement between the various semi - automated analyses . furthermore , the agreement of the quantitative bi - rads categorisation between the experienced and inexperienced reader was compared and expressed as the weighted kappa value . finally , the accuracy of this quantitative bi - rads categorisation between the readers was evaluated ( with the semi - automated analysis as reference standard ) and again expressed as weighted kappa value . breast density was visually assessed on our dedicated mammographic workstations by an experienced mammoradiologist ( c.b . , 18 years experience ) and by an ( inexperienced ) senior resident in radiology ( c.f . breast densities had to be scored in one of the four quantitative bi - rads categories . both readers did not receive any specific training in assessing breast density and were blinded to each other s results , as well as to the results of the semi - automated analysis . breast density was analysed by using a so - called operator - dependent thresholding technique ( leica qwin version 3 , leica microsystems , cambridge , uk ) . breast density is presented as the area percentage of the fibroglandular tissue within the breast and is calculated by dividing the number of pixels in the colour overlay by the number of pixels in the total breast area and multiplying by 100 ( fig . the semi - automated analysis was performed by a experienced operator and radiologist ( m.b.i.l . , 5 years of experience using the thresholding approach and this software program ) , who was blinded to the results of both mammography readers . to determine inter - observer reproducibility , the same sample of mammograms was evaluated by an inexperienced operator , m.j.l . the latter was blinded to the results of both mammography readers and the semi - automated analysis performed.fig . in this particular example , the mammographic breast density was 22% ( bi - rads density category 1 ) semi - automated detection of mammographic breast density . in this particular example , the mammographic breast density was 22% ( bi - rads density category 1 ) statistical analyses were performed by using spss 17.0 ( spss , chicago , il , usa ) and the sas statistical package ( sas institute , cary , nc , usa ) . the correlation between breast densities of both breasts ( as determined by the semi - automated analysis ) and of both cc and mlo projections was assessed by calculating the intra - class correlation coefficient ( icc ) . mean values of the breast density in the cc projections were assessed and translated into one of the four corresponding quantitative bi - rads categories . the inter- and intra - observer reproducibility of the semi - automated analyses was also calculated by using the icc . bland - altman plots were used to show the agreement between the various semi - automated analyses . furthermore , the agreement of the quantitative bi - rads categorisation between the experienced and inexperienced reader was compared and expressed as the weighted kappa value . finally , the accuracy of this quantitative bi - rads categorisation between the readers was evaluated ( with the semi - automated analysis as reference standard ) and again expressed as weighted kappa value . using the semi - automated analysis , the icc of the left - sided breast density in both cc and mlo projection was excellent : 0.91 [ 95% confidence interval ( ci ) 0.880.93 ] . for the mlo projection , the icc for the breast densities of the left and right breast was excellent ( 0.91 , 95% ci 0.890.93 ) . all these results were highly significant ( all p < 0.0001 ) and comparable with results previously published . 2bland - altman plots for the semi - automated analyses of breast densities of both breasts in two projections . intra - class correlation coefficients ( icc ) of the measurements were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the difference bland - altman plots for the semi - automated analyses of breast densities of both breasts in two projections . intra - class correlation coefficients ( icc ) of the measurements were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the difference reproducibility of the semi - automated analysis in a subset of mammograms ( n = 50 ) proved to be good as well . the icc of both cc and mlo projections of the left and right breast were all highly significant ( p < 0.0001 ) with values ranging from 0.82 to 0.88 . even more interesting is the inter - observer comparison of the semi - automated analysis . in these analyses , the icc of both cc and mlo projections of the left and right breast were also highly significant ( p < 0.0001 ) with values ranging from 0.80 to 0.88 . although the observed differences in breast densities were slightly higher than in the initial semi - automated analyses , bland - altman plots still showed good agreement of all these analyses with acceptable differences in breast density measurements ( figs . intra - class correlation coefficients ( icc ) were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the differencesfig . intra - class correlation coefficients ( icc ) of the measurements were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the differences intra - observer agreement of the semi - automated analyses . intra - class correlation coefficients ( icc ) were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the differences inter - observer agreement of the semi - automated analyses . intra - class correlation coefficients ( icc ) of the measurements were highly significant ( all p < 0.0001 ) . solid lines represent the mean of the differences between the two analyses , dotted lines represent the boundaries of two times the standard deviation of the differences table 1 shows the inter - observer agreement of the quantitative bi - rads classification of both mammographic readers . there was a disagreement between the quantitative bi - rads categorisations of the experienced and inexperienced reader . in 83 of the 200 cases ( 42% ) , the agreement of the experienced and inexperienced reader was therefore only moderate with a kappa value of 0.52 ( table 1).table 1inter - reader agreement of breast density according to bi - rads classifications . results of the experienced reader are presented in the columns , results of the inexperienced reader are presented in the rowsexperienced readerbi - rads 1bi - rads 2bi - rads 3bi - rads 4totalinexperienced readerbi - rads 166 ( 33.0%)2 ( 1.0%)2 ( 1.0%)0 ( 0.0%)70 ( 35.0%)bi - rads 219 ( 9.5%)36 ( 18.0%)1 ( 0.5%)0 ( 0.0%)56 ( 28.0%)bi - rads 35 ( 2.5%)38 ( 19.0%)10 ( 5.0%)0 ( 0.0%)53 ( 26.5%)bi - rads 41 ( 0.5%)3 ( 1.5%)14 ( 7.0%)3 ( 1.5%)21 ( 10.5%)total91 ( 45.5%)79 ( 39.5%)27 ( 13.5%)3 ( 1.5%)200 ( 100.0%)the overall weighted kappa was 0.521 , a moderate value ( 95% confidence interval 0.4460.597 ) inter - reader agreement of breast density according to bi - rads classifications . results of the experienced reader are presented in the columns , results of the inexperienced reader are presented in the rows the overall weighted kappa was 0.521 , a moderate value ( 95% confidence interval 0.4460.597 ) when compared with the results of the semi - automated analyses , the experienced reader agreed with the quantitative bi - rads category in 58.5% of the cases . the classification was overestimated in 35.5% of the cases and underestimated in 6.0% of the cases . in comparison , the inexperienced reader agreed less ( 42.0% ) and generally overestimated the quantitative bi - rads classification more than the experienced reader ( 56.0% ) . agreement between the classification of both readers versus the semi - automated analysis was poor to moderate with weighted kappa values of 0.367 ( experienced reader ) and 0.232 ( inexperienced reader , table 2).table 2classification of the results of the experienced and inexperienced readers and the software analysis . the agreement of the respective readers results and the semi - automated software is presented as the weighted kappa valueexperienced readerinexperienced readersoftware analysistotal patients classified200 ( 100.0%)200 ( 100.0%)200 ( 100.0%)bi - rads 1 classification ( % ) 91 ( 45.5%)70 ( 35.0%)127 ( 63.5%)bi - rads 2 classification ( % ) 79 ( 39.5%)56 ( 28.0%)68 ( 34.0%)bi - rads 3 classification ( % ) 27 ( 13.5%)53 ( 26.5%)5 ( 2.5%)bi - rads 4 classification ( % ) 3 ( 1.5%)21 ( 10.5%)0 ( 0.0%)correct classification ( % ) 117 ( 58.5%)84 ( 42.0%)classification overestimated ( % ) 71 ( 35.5%)112 ( 56.0%)classification underestimated ( % ) 12 ( 6.0%)4 ( 2.0%)weighted kappa0.3670.232 classification of the results of the experienced and inexperienced readers and the software analysis . the agreement of the respective readers results and the semi - automated software is presented as the weighted kappa value in this study , reliability of visual assessment of breast density for both experienced and inexperienced readers was evaluated , as compared to the semi - automated assessment of breast density using a dedicated software program . our results showed that there was disagreement between the quantitative bi - rads categorisation of the experienced and inexperienced readers . when compared to the semi - automated analysis , the experienced reader agreed with the quantitative bi - rads classification in 58.5% of the cases . the classification was overestimated in 35.5% of the cases , and underestimated in 6.0% of the cases . furthermore , the semi - automated assessment of breast density showed good intra- and interobserver reproducibility . in our institution , mammograms are evaluated by radiologists and/or ( supervised ) residents , using the bi - rads classification for breast density . however , our study results showed disagreement between radiologist ( experienced reader ) and resident ( inexperienced reader ) and that breast density is frequently overrated ( even by a highly experienced reader ) . despite the fact that this information is enclosed within the images , it is not used in current clinical settings or screening to identify high - risk patients , since the ( visual ) bi - rads density classification is not suitable for the expression of breast cancer risk . based on our current observations ( which show a substantial disagreement between the visual and semi - automated assessment of breast density ) , we would prefer a ( workstation ) integrated ( semi-)automated analysis of breast density to identify patients at high risk for developing breast cancer or in whom breast cancer is likely to be missed . for this purpose , several ( semi-)automated systems to assess breast density have been proposed in the past . of these , the so - called thresholding approach using ( commercially available ) software packages has been extensively studied in the past and is therefore frequently used in quantitative assessment of breast density . we also used a thresholding approach using the leica qwin software package and showed similar results in breast densities of both breasts and in both projections . a major advantage of our semi - automated analysis is that even analysts with hardly any experience using our software can achieve good reproducibility ( fig . the assessment of the mammographic breast density using this technique is time and labour intensive . for example , our time for the assessment of breast density in a single patient study was estimated to be 58 min . demonstrated that assessing breast density of one breast in one projection is sufficient this suggestion is supported by the findings of our current study and enables the inclusion of women that have undergone unilateral breast surgery of any kind . in this study mammograms of 65 women however , on close inspection , five of the radiologists had already interacted with the software programme , leaving two radiologists ( albeit experienced in mammography reading ) untrained for the breast density analysis . the breast density was overestimated by these two radiologists in 37% of the cases , as compared to 36% overestimation by the experienced radiologist in our study . furthermore , the 95% limits of agreement between the trained radiologists and the automated analyses of the study of martin et al . visual inspection of the bland - altman plots of our study show that our limits of agreement were much smaller ( fig . quantitative bi - rads categories 3 and 4 . previously published larger studies show slightly higher numbers of dense breasts in their populations , presumably owing to the larger size of the populations used when compared to our study population . this is why we have chosen to use our software programme as the reference standard ( not golden standard ) to which we compared the visual assessment of breast density . due to the comparability of acquired results with more validated software programs , we expect that breast density ( as assessed by our software program ) is also associated with increased breast cancer risk . finally , our analysis remains a semi - automated ( and not fully automated ) technique , requiring input from an operator and is therefore at risk of introducing observer - dependent bias . recently , a commercially available software tool ( quantra , hologic , bedford , ma , usa ) to automatically assess breast density was compared to the cumulus software program . this study showed a strong density correlation between both breasts and for both methods , suggesting that fully automated assessment of breast density could also aid in breast cancer risk estimation . , demonstrating that breast density , as assessed with this automated analysis , was strongly associated with breast cancer risk in women younger than 50 years , but not older than 50 years . in summary , our results showed that there is a disagreement in quantitative bi - rads breast density classification between experienced and inexperienced readers and the semi - automated software analysis . in order to accurately assess breast density in a reproducible and observer - independent manner , we would recommend the use of an integrated software tool , which can be applied in both screening and clinical settings . this semi - automated analysis of breast density might aid in identifying patients at high risk for developing breast cancer and/or patients who can benefit from additional mr mammography because of an unreliable mammogram . currently , the thresholding approach to assess breast density on standard mammograms is preferable , for instance as assessed by the dedicated software program used in this study or any other commercially available software package . in order to rapidly assess breast density and to include patients who have undergone mastectomy , assessment of a single breast in one projection ( preferably the cc projection ) is sufficient . the results of our study showed that visual assessment of breast density on mammograms is inaccurate and observer - dependent . semi - automated analysis of breast density reduces inter - observer variation of breast density classification on mammograms .

numerous reports have described the fabrication of electrospun polymer nanofibers applicable in tissue engineering for the regeneration of skin , bone , heart , cornea , the nervous system , and other tissues.13 the specific morphology of nanofibrous nonwoven materials makes them suitable for wound dressings.3,4 their high porosity , consisting of relatively small pores , enables good permeability for oxygen and water but protects wounds from dehydration and bacterial penetration.3,5 moreover , the large surface area and high pore volume of nanofibers promote the migration of keratinocytes on the wound surface and may play a role in accelerating the healing process . nanofibers composed of natural polymers , such as chitin,6 chitosan7 or collagen,8 or synthetic polymers or their blends , such as polyurethane ( pur),9,10 polyvinyl alcohol ( pva),10 polyacrylonitrile,10 or polyethersulfone,11 have been specifically engineered as dressings for wound healing or as tissue engineered scaffolds for skin substitutes . in addition to physical protection of the wound site , nanofibrous materials have also been developed for the local delivery of therapeutic agents , such as antibiotics,12 anesthetics,13,14 or growth factors.7,15 various processing techniques have been used to produce nanofibers.1,1618 however , the electrospinning technique allows the production of continuous polymeric nanofibers and provides numerous opportunities to modify and control their morphological parameters , such as surface area , fiber diameter , the porosity of the nanofibrous layer ( fiber density ) , basis weight,19 or internal fiber structure.18,20,21 nanospider electrospinning technology is based on a needleless method in which polymeric jets are spontaneously formed from liquid surfaces22,23 and allows the production of fibers with diameters ranging from tens of nanometers to tens of micrometers . nanospider ( elmarco , liberec , czech republic ) technology is very flexible , enabling the formation of nanofibrous materials from various polymers.24 the process provides high production capacity , stability , and easy maintenance compared to other known industrial - scale technologies based on needles or capillary spinners.25,26 the incorporation of various additional substances , such as pharmacologically active compounds , in the nanofibers can be accomplished by simply adding the substances to the polymer solution . in our previous work27 using nanospider technology , we demonstrated that the immunosuppressive drug cyclosporine a can be incorporated directly into electrospun nanofibers without any loss of its pharmacological activity . to achieve drug release from nanofibers , two basic delivery designs are known : matrices and reservoirs . in the matrix - type of carriers , the drug is homogeneously dispersed in the material of the nanofiber , and its release is based on solid - state diffusion or a desorption mechanism;13,28,29 however , such drug - loaded systems tends to have a strong burst release within the first hours . in the second design , reservoir - type structures are formed by a drug - loaded core and a covering polymer shell.21,28,30 the core - shell structure enables better control of the drug release profile by adjusting the shell s properties , such as its microstructure , thickness , and degradability . on the other hand , some of the complications that persist during nanofiber preparation do not allow for easy large - scale production . several papers have described the preparation and testing of multi - layer polymer nanofibrous structures containing a middle layer that incorporates a pharmacologically active compound , covered by other nanofibrous layers of a different polymer.3133 chen et al31 prepared biodegradable sandwich - structured nanofibers from poly(d , l)-lactide - co - glycolide and collagen with incorporated vancomycin , gentamicin , and lidocaine . they described the conditions of nanofibrous mat preparation by classical jet electrospinning and the in vitro release of the drugs , as well as their effect on the in vivo healing process in rat wounds . kim et al32 observed rhodamine and peptide release from multi - layered pcl / polyethylene oxide / pcl nanofibers . they found a decrease of the initial burst release with increasing thickness of the covering layer and a slower release of compounds from sandwiches with thicker covering layers . nevertheless , all of these authors prepared nanofibers by classical electrospinning based on needle or capillary spinners . in contrast , needleless electrospinning allows the sequential deposition of nanofibrous layers , and therefore , the preparation of compact sandwich structures in which the middle drug - loaded layer is covered by layers of a different polymer . these layers , with a large surface area and a microporous structure , improve the mechanical properties , promote wound healing , and also contribute to controlled drug release to the surrounding tissue . combining more layers of the required composition , porosity , thickness , fiber diameter , and/or drug concentration , it is possible to obtain a nanofibrous material with properties suitable for specific applications . the aim of this study was to employ needleless electrospinning for the preparation of multi - layer nanofibrous structures consisting of a drug - loaded middle layer and covering layers of various thicknesses , enabling the controlled release of the drug . the middle layer was made from crosslinked or noncrosslinked pva nanofibers containing the wide spectrum antibiotic gentamicin , while the covering layers were made from pur . the morphology of the prepared nanofibers was observed using scanning electron microscopy ( sem ) and characterized by brunauer , emmet , and teller ( bet ) nitrogen adsorption / desorption measurements and mercury porosimetry . gentamicin release was quantified in vitro using high - performance liquid chromatography / mass spectrometry ( hplc / ms ) , and its bactericidal effect was confirmed on staphylococcus aureus and pseudomonas aeruginosa cultures . the effect of the preparation conditions on the morphology of the nanofibers and subsequent gentamicin release was evaluated . polyvinyl alcohol ( pva ) ( type z 220 , viscosity of 4 wt% water solution at 20c 11.515 mpa.s , saponification degree 90.592.5 mol% ) was supplied by nippon gohsei ( osaka , japan ) . pur ( estane 5714 f1 , viscosity of 15 wt% solution in thf 600900 mpa.s ) was obtained from lubrizol corp ( wickliffe , oh ) . n , n - dimethylacetamide ( sigma - aldrich , st louis , mo ) and toluene and phosphoric acid ( penta a.s . , prague , czech republic ) were obtained in analytical reagent grade and used as received . pva and pur nanofibers were prepared using nanospider technology.22 the process parameters were individually optimized for each polymer . pva nanofibrous layers were made at a basis weight of 0.8 g / m , while the covering pur layers had basis weights of 1.5 , 4.4 , and 14.7 g / m . layers of each polymer intended for mercury porosimetry and nitrogen adsorption / desorption measurements were prepared separately in thicknesses ( basis weights ) similar to the sandwich structures . pva was dissolved in a water / phosphoric acid mixture at a polymer concentration of 11 wt% . the distance between the spinning electrode and the collector was 13 cm ; the spinning electrode rotated at 2 rpm , and the high voltage supply was 4555 kv / cm . gentamicin , at a concentration of 10 wt% , was added directly to the pva electrospun mixture . the pva layers were crosslinked thermally in a drying oven at 145c for 15 minutes . pur was dissolved in an n , n - dimethylacetamide / toluene mixture at a ratio of 2:1 ( w / w ) and a polymer concentration of 10 wt% . the electrospinning conditions were : 4 rpm for the spinning electrode , a 15 cm distance between the electrodes , and a 5 kv / cm electric field strength . the relative humidity was kept lower than 30% due to the risk of solvent explosion . the pur / pva / pur multi - layered sandwich structures were prepared by multi - step deposition of each layer . all layers were prepared on the same nanospider machine ; only the process parameters were varied according to the polymer type as described above . nonwoven polypropylene material ( atex , milan , italy ) the different thickness of each layer ( g / m ) results from the speed of the rotating collecting electrode . in the case of the pva layer with a basis weight of 0.8 g / m , the speed was 50 mm / min ; for the pur layers , the speeds were as follows : 1.5 g / m = 110 mm / min , 4.4 g / m = 30 mm / min , and 14.7 g / m = 18 mm / min with double electrospinning . the structure of the prepared nanofibers was observed using a ts 5130 vega scanning electron microscope , tescan ( brno , czech republic ) . the fiber diameters and the thicknesses of the nanofibrous layers were determined as mean values of 30 measurements in various locations . single layers of pva and pur nanofibers were prepared to determine porosity and surface area . mercury porosimetry measurements were made using an autopore iv 9500 porosimeter ( micromeritics , norcross , ga ) , and specific surface areas were calculated from nitrogen absorption / desorption isotherms recorded on an asap 2020 apparatus ( micromeritics ) . gentamicin release from the nanofibrous mats was investigated under laboratory conditions and by in vitro bacteriological assay . laboratory experiments were performed on agar dishes ( caso - agar , mercoplate ; merck and co , whitehouse station , nj ) in order to evaluate the time course of the drug release from the nanofibrous mats . discs of 8 mm in diameter were cut from the nanofibrous mats and placed on agar previously wetted with 0.5 ml of distilled water , 1 day before the release experiment . after various time periods , the discs were removed , immersed into distilled water ( 1 ml each ) and shaken for 2 days to rinse out the remaining gentamicin . the concentration of gentamicin in solution was quantified by an hplc - ms liquid chromatograph series 1200 with a triple quad lc/ ms 6460 tandem mass spectrometer ( agilent technologies , waldbroon , germany ) . the amount of gentamicin released into the agar was calculated as the difference between the initial and residual amounts of gentamicin in the disc . the drug release from each nanofibrous sample was determined 15 times and the average values given . the hplc - ms apparatus was also used to evaluate the stability of gentamicin during the electrospinning process . the in vitro antimicrobial properties of electrospun layers containing gentamicin were evaluated from inhibitory zone measurements against the gram - positive organism staphylococcus aureus ( atcc 6538 ) and the gram - negative organism pseudomonas aeruginosa ( atcc 27853 ) . the bacterial strains were obtained from the czech collection of microorganisms , brno , czech republic . a gelatin pellet infused with the bacterial strain was incubated in 9 ml of liquid media ( tryptic soy broth , mecrotube ; merck and co ) at 37c for 24 hours , and then diluted 1000-fold in a phosphate buffer . an aliquot of the suspension ( 0.7 ml ) was uniformly spread onto an agar plate ( caso - agar , mercoplate ; merck and co ) and allowed to dry for several minutes . the nanofibrous mats ( 6-mm diameter discs ) were placed on the agar plates and incubated at 37c . the release profiles of the gentamicin from the nanofibrous layered mats were evaluated using the inhibition of s. aureus after 1 , 3 , 6 , 24 , and 48 hours of treatment . upon removal from the agar plate , the nanofibrous discs were placed on a new agar plate to further evaluate the inhibitory effect from the residual gentamicin in the sample . the plates were incubated overnight at 37c , and the zones of inhibition were measured from photomicrographs using imagej software . to eliminate potential variability between the samples during the manufacturing process , the area of inhibition was calculated as ( area of inhibition / area of inhibition after 48 hours ) 100 , as the area of inhibition after 48 hours was found to be maximal ; also , no residual inhibitory activity was detected in the samples after 48 hours of drug release . antimicrobial susceptibility testing discs ( oxoid , cambridge , uk ) containing 10 , 30 , or 120 of gentamicin were used as standards . the multilayered nanofibrous mats were prepared in a multi - step process , changing the process parameters and evaluating the area weight after each step . the images did not reveal any artifacts or heterogeneities , and uniform nanofibrous structures were found . from the sem images , the fiber diameters were calculated as the mean value of 30 measurements at various locations . the mean fiber diameter obtained for pva fibers was 185 70 nm , while in the case of pur fibers it was 420 150 nm . the fiber diameters differed between pva and pur nanofibers ; however , the diameters were similar for samples of the same polymer , regardless of the layer thickness . this is due to the method of preparation , by which the fiber thickness is particularly influenced by the viscosity of the solution , the strength of the electrostatic field , and the distance between the electrodes ; the mat s thickness is driven by the speed of rotation of the collecting electrode and the duration of fiber deposition , while the other parameters remain unchanged . the structures of the three - layered nanofibrous pur/ pva / pur mats evaluated by sem are depicted in figure 1 . it can be seen that fracturing the samples in liquid nitrogen successfully maintained the inner sandwich structure and the original thickness of each layer . on the other hand , some secondary changes in the nanofibers , especially local melting of the fibers , can be seen . the mean values of the layer thicknesses calculated from the sem measurements are given in table 1 . it should be mentioned that the actual thickness of the nanofibrous layers in the subsequent drug release experiments , as well as in their potential in vivo application , will certainly differ from the values obtained by sem in a vacuum / dry state , due to the absorption of water under aqueous conditions . nitrogen adsorption / desorption bet measurements and mercury porosimetry were carried out with single pva and pur layers of the same basis weight as in the sandwich structures , and the results obtained are summarized in table 1 . bet measurements of specific surface areas revealed higher values in the case of the pva fibers ( 12.8 m / g ) compared to the pur ones ( between 7.5 m / g and 9.1 m / g ) , which corresponds to the smaller diameter of the pva fibers . similar to the fiber diameters , comparing pur layers of various thicknesses revealed that the surface areas were not influenced by the basis weight . the porosity of the pva nanofibers ( 85.8% ) determined by mercury porosimetry was higher than the porosity of the pur nanofibrous samples ( 70.7%78.9% ; see table 1 ) . the porosity of the pur layers slightly decreased with increasing basis weight , which was caused by the preparation procedure in which the fibers were deposited stepwise for a certain length of time . with the increasing deposition time needed to obtain thicker layers , a certain level of compression of a whole layer can occur , ie , the formed nanofibrous layer is more compact . mercury porosimetry , as well as bet measurements , proceeds on single layers , and therefore , the obtained values may be slightly different from those of real sandwich structures . nevertheless , the measurements of single layers provided us with more detailed information about morphological parameters depending on polymer type or layer thickness . an important finding was that sem images of the sandwich structures did not show any noticeable difference between the upper and lower pur nanofibrous layers ( see figure 1 ) , so the stepwise deposition of subsequent layers did not cause any significant compression of the lower layers . pore size distribution curves ( not depicted ) obtained from mercury porosimetry measurements did not show any pores less than tens of nanometers in size , suggesting that there is no porosity inside the pur and pva fibers and that the whole pore volume is given by the inter - fiber space . the relatively low values of specific surface areas ( see table 1 ) obtained by bet measurements also correspond to this finding . no morphological differences were observed between samples consisting of crosslinked or noncrosslinked pva layers . it can be noted that the additional crosslinking of the pva layers had no effect on any structural parameters of the resulting nanofibrous mats . also , no statistically significant differences in morphological parameters were observed between nanofibers prepared with 10 wt% gentamicin and without the drug . gentamicin release experiments were carried out with multi - layer nanofibrous mats consisting of a crosslinked or noncrosslinked pva layer of basis weight 0.8 g / m loaded with 10 wt% gentamicin and outer pur layers with basis weights of 1.5 , 4.4 , or 14.7 g / m . the concentration of the released gentamicin was assessed using hplc - ms . before starting the gentamicin release experiments , it was crucial to prove the stability of the drug during the incorporation procedure . ms spectra of fresh gentamicin before incorporation were compared with the spectra of gentamicin released from sandwich nanofibers consisting of crosslinked pva nanofibers and 14.7 g / m pur covering layers . the mass spectra did not show any differences , suggesting that the structure of gentamicin remained unchanged . the antimicrobial effectiveness of gentamicin released from crosslinked and noncrosslinked pva layers as well as from pur / pva / pur sandwiches was evaluated from the inhibition zones of s. aureus and p. aeruginosa after 1 , 2 , and 6 days of culture . clear inhibitory zones were observed with both strains around all of the gentamicin - loaded mats , while no inhibition was found around control nanofibers without the drug . the inhibitory zones on s. aureus cultures were comparable with those of gentamicin standard discs that released a similar amount of gentamicin ( figure 2 ) . smaller inhibitory zones were found on p. aeruginosa cultures when compared to s. aureus ; nevertheless , the inhibitory activity of gentamicin against p. aeruginosa was still preserved ( figure 2d ) . these tests proved that gentamicin survives hard electrospinning conditions , such as exposure to phosphoric acid , a high electrostatic field , and an elevated temperature during pva crosslinking , in terms of maintaining its structure and antibacterial activity . the gentamicin release experiments were first carried out by immersing samples of the nanofibrous mats ( pieces of ca 0.1 g ) in an excess of water ( 10 ml ) . figure 3 shows the dependence of the released gentamicin ( in percentage ) on time . it can be seen that the release of almost 90% of the total drug amount occurred within 1 hour . an effect of the various thicknesses of the covering pur layers was observed only for very short exposure times ( 10 and 30 minutes ) . for longer exposure times , regardless of the pur layer thickness , the amount of the released drug was similar ( 90%100% ) . no significant differences in the release profiles were found by comparing samples with a noncrosslinked or crosslinked pva layer . in the case of all tested samples , the flow of a liquid medium is greater and it can easily penetrate through a nanofibrous structure . the middle drug - loaded layer is easily accessible and the release of gentamicin as a water - soluble drug is thus accelerated . moreover , the excess water and lower gentamicin concentration in solution contribute to a steeper concentration gradient , and , consequently , faster drug diffusion , respectively . it is necessary to note that this experimental arrangement does not simulate the actual conditions of drug release in potential applications , particularly in wound healing . therefore , it would be desirable to perform the experiments in such a way that would more closely approximate real drug release into surrounding living tissue . unfortunately , this aspect has often been neglected by other authors , and most published reports rely on monitoring drug release into a large volume of liquid media . so , in the next step , release experiments were carried out by placing the sample discs on wet agar plates . the discs were removed from the agar after various time periods , and the residual gentamicin was washed out into water and quantified by hplc - ms analysis . the amount of gentamicin released to the agar was calculated from the differences between the initial and the residual amounts . in the case of samples with gentamicin incorporated into sandwiches with a noncrosslinked pva layer , the faster release was influenced by the degradation of the pva layer and the subsequent easier dissolution of the gentamicin . the release profiles are depicted in figure 4 . in the case of the thinnest covering pur nanofibrous layers ( 1.5 g / m ) , the entire quantity of gentamicin was released within 1 hour of sample application onto the agar plate . there was no apparent difference between the amount of residual gentamicin in nanofibers applied to the agar plate for 1 hour and those applied for any longer time period . a similar gentamicin release profile was observed for crosslinked pva nanofibers without pur layers ( not depicted ) . a significant difference was found in the case of samples covered by 4.4 g / m or 14.7 g / m pur layers . sandwiches with 4.4 g / m pur layers retained about 10% of the gentamicin after 24 hours of application , while those with 14.7 g / m pur layers retained 30% after 24 hours and still contained about 20% of gentamicin even after 48 hours . in accordance with the results discussed above , the antibacterial experiments were carried out only with sandwiches based on a crosslinked drug - loaded pva layer . the gentamicin release profiles were evaluated from the area of inhibition of s. aureus after various time periods ( figure 5 ) . after 1 hour , the largest inhibitory area ( 85% ) was found for 1.5 g / m pur sandwiches , indicating the largest amount of released gentamicin , while the inhibitory zones were smaller around the thicker nanofibrous sandwiches with outer pur layers of 4.4 g / m ( 59% ) or 14.7 g / m ( 56% ) ( figure 5a ) . after 6 hours of treatment , the inhibitory zones achieved their maximal size around all samples and did not increase further with time . the residual gentamicin in the samples was evaluated by applying the removed discs onto fresh bacterial cultures . no inhibition zones were observed around the 1.5 g / m pur layered mats , while the inhibition zones confirmed gentamicin retention in the 4.4 g / m and 14.7 g / m pur layered mats even after 24 hours ( figure 5b ) . bacterial inhibition , however , requires a certain limiting inhibitory concentration of gentamicin , and drug release is therefore not detectable in the more distant areas around the nanofibrous mats where the gentamicin concentration is below the level needed to exert an inhibitory effect . it can be concluded that the 1.5 g / m pur layers are too thin to influence the release of gentamicin and to prolong its retention in the nanofibrous mats . prolonged drug release was found for thicker covering layers of 4.4 g / m or 14.7 g / m thicknesses , but even so , more than 70% of the gentamicin was released within 24 hours . the prolongation of the gentamicin release was influenced by two factors : ( 1 ) since a covering pur layer with a higher basis weight is thicker , the gentamicin molecules must travel a longer distance from the surface of the pva nanofibers to the free volume ; and ( 2 ) thicker nanofibrous mats are more compact with a lower porosity than thinner ones ( see the column porosity in table 1 ) . consequently , migrating molecules of the released drug must travel through a nanofibrous layer with smaller pores . gibson et al34 studied water vapor diffusion and gas convection through nanofibrous mats and found a linear dependence of flow resistance on the thickness of the nanofibrous mats . this dependence was also mentioned by kim et al32 in the case of rhodamine and peptide release from multilayered pcl / polyethylene oxide / pcl nanofibers into a liquid environment . unlike these authors , we have expressed the thickness of the nanofibrous layers in basis weight because , according to our experience , thicknesses expressed in micrometers can vary significantly due to the compression of nanofibrous mats during manipulation or in in vitro / in vivo experiments . thickness expressed in micrometers is also dependent on the environment whether the sample is in dry conditions or immersed in water or a saline solution . the values obtained by sem measurements performed under vacuum / dry conditions may differ considerably from the real thickness of nanofiber mats in water / wet conditions during biological experiments or in medical applications . obviously , these changes affect the thickness of nanofibers as well as their total porosity and the distribution of the pore sizes , particularly the mean and most frequent pore radius . the release of the incorporated compound is also significantly dependent on the conditions of the experiment its release into a liquid environment is different when compared to its release into a solid or heterogeneous environment , such as biological tissues in a living organism . we are aware that a pva nanofibrous layer with a basis weight of 0.8 g / m and 10 wt% gentamicin was used to model gentamicin release from sandwiches of various thicknesses , but that this does not correspond to the actual therapeutic dosage of gentamicin commonly used for local application . the required therapeutic dose of gentamicin or other drugs can be easily obtained by adjusting the thickness ( basis weight ) of the middle drug - loaded layer or by adjusting the drug s concentration in the electrospun mixture . nevertheless , the thickness of the layers needs to be further optimized to control the drug release rate and to maintain the mechanical properties or other features of multi - layer nanofibrous mats . in our previous studies,35,36 we demonstrated that nanofibers produced by needleless technology represent a convenient scaffold for cell cultivation and subsequent transfer to damaged tissue . the multilayered nanofibers studied in this work may offer a promising combination of such inherent properties of electrospun nanofibers with controlled drug release for developing suitable scaffolds for wound dressing applications . moreover , the use of nanospider technology represents a relatively simple and easily controllable way to fabricate such nanofibrous scaffolds in mass production , which is advantageous when compared to other methods . the multilayered nanofibrous mats were prepared in a multi - step process , changing the process parameters and evaluating the area weight after each step . the images did not reveal any artifacts or heterogeneities , and uniform nanofibrous structures were found . from the sem images , the fiber diameters were calculated as the mean value of 30 measurements at various locations . the mean fiber diameter obtained for pva fibers was 185 70 nm , while in the case of pur fibers it was 420 150 nm . the fiber diameters differed between pva and pur nanofibers ; however , the diameters were similar for samples of the same polymer , regardless of the layer thickness . this is due to the method of preparation , by which the fiber thickness is particularly influenced by the viscosity of the solution , the strength of the electrostatic field , and the distance between the electrodes ; the mat s thickness is driven by the speed of rotation of the collecting electrode and the duration of fiber deposition , while the other parameters remain unchanged . the structures of the three - layered nanofibrous pur/ pva / pur mats evaluated by sem are depicted in figure 1 . it can be seen that fracturing the samples in liquid nitrogen successfully maintained the inner sandwich structure and the original thickness of each layer . on the other hand , some secondary changes in the nanofibers , especially local melting of the fibers , can be seen . the mean values of the layer thicknesses calculated from the sem measurements are given in table 1 . it should be mentioned that the actual thickness of the nanofibrous layers in the subsequent drug release experiments , as well as in their potential in vivo application , will certainly differ from the values obtained by sem in a vacuum / dry state , due to the absorption of water under aqueous conditions . nitrogen adsorption / desorption bet measurements and mercury porosimetry were carried out with single pva and pur layers of the same basis weight as in the sandwich structures , and the results obtained are summarized in table 1 . bet measurements of specific surface areas revealed higher values in the case of the pva fibers ( 12.8 m / g ) compared to the pur ones ( between 7.5 m / g and 9.1 m / g ) , which corresponds to the smaller diameter of the pva fibers . similar to the fiber diameters , comparing pur layers of various thicknesses revealed that the surface areas were not influenced by the basis weight . the porosity of the pva nanofibers ( 85.8% ) determined by mercury porosimetry was higher than the porosity of the pur nanofibrous samples ( 70.7%78.9% ; see table 1 ) . the porosity of the pur layers slightly decreased with increasing basis weight , which was caused by the preparation procedure in which the fibers were deposited stepwise for a certain length of time . with the increasing deposition time needed to obtain thicker layers , a certain level of compression of a whole layer can occur , ie , the formed nanofibrous layer is more compact . mercury porosimetry , as well as bet measurements , proceeds on single layers , and therefore , the obtained values may be slightly different from those of real sandwich structures . nevertheless , the measurements of single layers provided us with more detailed information about morphological parameters depending on polymer type or layer thickness . an important finding was that sem images of the sandwich structures did not show any noticeable difference between the upper and lower pur nanofibrous layers ( see figure 1 ) , so the stepwise deposition of subsequent layers did not cause any significant compression of the lower layers . pore size distribution curves ( not depicted ) obtained from mercury porosimetry measurements did not show any pores less than tens of nanometers in size , suggesting that there is no porosity inside the pur and pva fibers and that the whole pore volume is given by the inter - fiber space . the relatively low values of specific surface areas ( see table 1 ) obtained by bet measurements also correspond to this finding . no morphological differences were observed between samples consisting of crosslinked or noncrosslinked pva layers . it can be noted that the additional crosslinking of the pva layers had no effect on any structural parameters of the resulting nanofibrous mats . also , no statistically significant differences in morphological parameters were observed between nanofibers prepared with 10 wt% gentamicin and without the drug . gentamicin release experiments were carried out with multi - layer nanofibrous mats consisting of a crosslinked or noncrosslinked pva layer of basis weight 0.8 g / m loaded with 10 wt% gentamicin and outer pur layers with basis weights of 1.5 , 4.4 , or 14.7 g / m . the concentration of the released gentamicin was assessed using hplc - ms . before starting the gentamicin release experiments , it was crucial to prove the stability of the drug during the incorporation procedure . ms spectra of fresh gentamicin before incorporation were compared with the spectra of gentamicin released from sandwich nanofibers consisting of crosslinked pva nanofibers and 14.7 g / m pur covering layers . the mass spectra did not show any differences , suggesting that the structure of gentamicin remained unchanged . the antimicrobial effectiveness of gentamicin released from crosslinked and noncrosslinked pva layers as well as from pur / pva / pur sandwiches was evaluated from the inhibition zones of s. aureus and p. aeruginosa after 1 , 2 , and 6 days of culture . clear inhibitory zones were observed with both strains around all of the gentamicin - loaded mats , while no inhibition was found around control nanofibers without the drug . the inhibitory zones on s. aureus cultures were comparable with those of gentamicin standard discs that released a similar amount of gentamicin ( figure 2 ) . smaller inhibitory zones were found on p. aeruginosa cultures when compared to s. aureus ; nevertheless , the inhibitory activity of gentamicin against p. aeruginosa was still preserved ( figure 2d ) . these tests proved that gentamicin survives hard electrospinning conditions , such as exposure to phosphoric acid , a high electrostatic field , and an elevated temperature during pva crosslinking , in terms of maintaining its structure and antibacterial activity . the gentamicin release experiments were first carried out by immersing samples of the nanofibrous mats ( pieces of ca 0.1 g ) in an excess of water ( 10 ml ) . figure 3 shows the dependence of the released gentamicin ( in percentage ) on time . it can be seen that the release of almost 90% of the total drug amount occurred within 1 hour . an effect of the various thicknesses of the covering pur layers was observed only for very short exposure times ( 10 and 30 minutes ) . for longer exposure times , regardless of the pur layer thickness , the amount of the released drug was similar ( 90%100% ) . no significant differences in the release profiles were found by comparing samples with a noncrosslinked or crosslinked pva layer . in the case of all tested samples , the flow of a liquid medium is greater and it can easily penetrate through a nanofibrous structure . the middle drug - loaded layer is easily accessible and the release of gentamicin as a water - soluble drug is thus accelerated . moreover , the excess water and lower gentamicin concentration in solution contribute to a steeper concentration gradient , and , consequently , faster drug diffusion , respectively . it is necessary to note that this experimental arrangement does not simulate the actual conditions of drug release in potential applications , particularly in wound healing . therefore , it would be desirable to perform the experiments in such a way that would more closely approximate real drug release into surrounding living tissue . unfortunately , this aspect has often been neglected by other authors , and most published reports rely on monitoring drug release into a large volume of liquid media . so , in the next step , release experiments were carried out by placing the sample discs on wet agar plates . the discs were removed from the agar after various time periods , and the residual gentamicin was washed out into water and quantified by hplc - ms analysis . the amount of gentamicin released to the agar was calculated from the differences between the initial and the residual amounts . in the case of samples with gentamicin incorporated into sandwiches with a noncrosslinked pva layer , the faster release was influenced by the degradation of the pva layer and the subsequent easier dissolution of the gentamicin . the release profiles are depicted in figure 4 . in the case of the thinnest covering pur nanofibrous layers ( 1.5 g / m ) , the entire quantity of gentamicin was released within 1 hour of sample application onto the agar plate . there was no apparent difference between the amount of residual gentamicin in nanofibers applied to the agar plate for 1 hour and those applied for any longer time period . a similar gentamicin release profile was observed for crosslinked pva nanofibers without pur layers ( not depicted ) . a significant difference was found in the case of samples covered by 4.4 g / m or 14.7 g / m pur layers . sandwiches with 4.4 g / m pur layers retained about 10% of the gentamicin after 24 hours of application , while those with 14.7 g / m pur layers retained 30% after 24 hours and still contained about 20% of gentamicin even after 48 hours . in accordance with the results discussed above , the antibacterial experiments were carried out only with sandwiches based on a crosslinked drug - loaded pva layer . the gentamicin release profiles were evaluated from the area of inhibition of s. aureus after various time periods ( figure 5 ) . after 1 hour , the largest inhibitory area ( 85% ) was found for 1.5 g / m pur sandwiches , indicating the largest amount of released gentamicin , while the inhibitory zones were smaller around the thicker nanofibrous sandwiches with outer pur layers of 4.4 g / m ( 59% ) or 14.7 g / m ( 56% ) ( figure 5a ) . after 6 hours of treatment , the inhibitory zones achieved their maximal size around all samples and did not increase further with time . the residual gentamicin in the samples was evaluated by applying the removed discs onto fresh bacterial cultures . no inhibition zones were observed around the 1.5 g / m pur layered mats , while the inhibition zones confirmed gentamicin retention in the 4.4 g / m and 14.7 g / m pur layered mats even after 24 hours ( figure 5b ) . bacterial inhibition , however , requires a certain limiting inhibitory concentration of gentamicin , and drug release is therefore not detectable in the more distant areas around the nanofibrous mats where the gentamicin concentration is below the level needed to exert an inhibitory effect . it can be concluded that the 1.5 g / m pur layers are too thin to influence the release of gentamicin and to prolong its retention in the nanofibrous mats . prolonged drug release was found for thicker covering layers of 4.4 g / m or 14.7 g / m thicknesses , but even so , more than 70% of the gentamicin was released within 24 hours . the prolongation of the gentamicin release was influenced by two factors : ( 1 ) since a covering pur layer with a higher basis weight is thicker , the gentamicin molecules must travel a longer distance from the surface of the pva nanofibers to the free volume ; and ( 2 ) thicker nanofibrous mats are more compact with a lower porosity than thinner ones ( see the column consequently , migrating molecules of the released drug must travel through a nanofibrous layer with smaller pores . gibson et al34 studied water vapor diffusion and gas convection through nanofibrous mats and found a linear dependence of flow resistance on the thickness of the nanofibrous mats . this dependence was also mentioned by kim et al32 in the case of rhodamine and peptide release from multilayered pcl / polyethylene oxide / pcl nanofibers into a liquid environment . unlike these authors , we have expressed the thickness of the nanofibrous layers in basis weight because , according to our experience , thicknesses expressed in micrometers can vary significantly due to the compression of nanofibrous mats during manipulation or in in vitro / in vivo experiments . thickness expressed in micrometers is also dependent on the environment whether the sample is in dry conditions or immersed in water or a saline solution . the values obtained by sem measurements performed under vacuum / dry conditions may differ considerably from the real thickness of nanofiber mats in water / wet conditions during biological experiments or in medical applications . obviously , these changes affect the thickness of nanofibers as well as their total porosity and the distribution of the pore sizes , particularly the mean and most frequent pore radius . the release of the incorporated compound is also significantly dependent on the conditions of the experiment its release into a liquid environment is different when compared to its release into a solid or heterogeneous environment , such as biological tissues in a living organism . we are aware that a pva nanofibrous layer with a basis weight of 0.8 g / m and 10 wt% gentamicin was used to model gentamicin release from sandwiches of various thicknesses , but that this does not correspond to the actual therapeutic dosage of gentamicin commonly used for local application . the required therapeutic dose of gentamicin or other drugs can be easily obtained by adjusting the thickness ( basis weight ) of the middle drug - loaded layer or by adjusting the drug s concentration in the electrospun mixture . nevertheless , the thickness of the layers needs to be further optimized to control the drug release rate and to maintain the mechanical properties or other features of multi - layer nanofibrous mats . in our previous studies,35,36 we demonstrated that nanofibers produced by needleless technology represent a convenient scaffold for cell cultivation and subsequent transfer to damaged tissue . the multilayered nanofibers studied in this work may offer a promising combination of such inherent properties of electrospun nanofibers with controlled drug release for developing suitable scaffolds for wound dressing applications . moreover , the use of nanospider technology represents a relatively simple and easily controllable way to fabricate such nanofibrous scaffolds in mass production , which is advantageous when compared to other methods . in the present study , needleless electrospinning technology was successfully used for the preparation of pur / pva / pur multi - layer nanofibrous mats with gentamicin incorporated into the middle pva layer . this method enables effective large - scale production of nanofibrous sandwiches for various medical applications . morphological characterization of the sandwiches revealed highly homogeneous nanofibrous structures with tight connection between the individual nanofiber layers . hplc / ms analysis , as well as inhibition of bacterial growth , proved the stability of the gentamicin after its incorporation into the nanofibers during electrospinning . nanofibrous multi - layer drug carriers seem to be promising materials for use in various medical applications .

polyvinyl alcohol nanofibers incorporating the wide spectrum antibiotic gentamicin were prepared by nanospider needleless technology . a polyvinyl alcohol layer , serving as a drug reservoir , was covered from both sides by polyurethane layers of various thicknesses . the multilayered structure of the nanofibers was observed using scanning electron microscopy , the porosity was characterized by mercury porosimetry , and nitrogen adsorption / desorption measurements were used to determine specific surface areas . the stability of the gentamicin released from the electrospun layers was proved by high - performance liquid chromatography ( hplc ) and inhibition of bacterial growth . drug release was investigated using in vitro experiments with hplc / ms quantification , while the antimicrobial efficacy was evaluated on gram - positive staphylococcus aureus and gram - negative pseudomonas aeruginosa . both experiments proved that the released gentamicin retained its activity and showed that the retention of the drug in the nanofibers was prolonged with the increasing thickness of the covering layers .

Introduction Materials and methods Results and discussion Preparation and morphological characterization of nanofibrous mats Drug release from nanofibrous mats Conclusion

in addition to physical protection of the wound site , nanofibrous materials have also been developed for the local delivery of therapeutic agents , such as antibiotics,12 anesthetics,13,14 or growth factors.7,15 various processing techniques have been used to produce nanofibers.1,1618 however , the electrospinning technique allows the production of continuous polymeric nanofibers and provides numerous opportunities to modify and control their morphological parameters , such as surface area , fiber diameter , the porosity of the nanofibrous layer ( fiber density ) , basis weight,19 or internal fiber structure.18,20,21 nanospider electrospinning technology is based on a needleless method in which polymeric jets are spontaneously formed from liquid surfaces22,23 and allows the production of fibers with diameters ranging from tens of nanometers to tens of micrometers . nanospider ( elmarco , liberec , czech republic ) technology is very flexible , enabling the formation of nanofibrous materials from various polymers.24 the process provides high production capacity , stability , and easy maintenance compared to other known industrial - scale technologies based on needles or capillary spinners.25,26 the incorporation of various additional substances , such as pharmacologically active compounds , in the nanofibers can be accomplished by simply adding the substances to the polymer solution . in the matrix - type of carriers , the drug is homogeneously dispersed in the material of the nanofiber , and its release is based on solid - state diffusion or a desorption mechanism;13,28,29 however , such drug - loaded systems tends to have a strong burst release within the first hours . in the second design , reservoir - type structures are formed by a drug - loaded core and a covering polymer shell.21,28,30 the core - shell structure enables better control of the drug release profile by adjusting the shell s properties , such as its microstructure , thickness , and degradability . they found a decrease of the initial burst release with increasing thickness of the covering layer and a slower release of compounds from sandwiches with thicker covering layers . in contrast , needleless electrospinning allows the sequential deposition of nanofibrous layers , and therefore , the preparation of compact sandwich structures in which the middle drug - loaded layer is covered by layers of a different polymer . the aim of this study was to employ needleless electrospinning for the preparation of multi - layer nanofibrous structures consisting of a drug - loaded middle layer and covering layers of various thicknesses , enabling the controlled release of the drug . the middle layer was made from crosslinked or noncrosslinked pva nanofibers containing the wide spectrum antibiotic gentamicin , while the covering layers were made from pur . the morphology of the prepared nanofibers was observed using scanning electron microscopy ( sem ) and characterized by brunauer , emmet , and teller ( bet ) nitrogen adsorption / desorption measurements and mercury porosimetry . gentamicin release was quantified in vitro using high - performance liquid chromatography / mass spectrometry ( hplc / ms ) , and its bactericidal effect was confirmed on staphylococcus aureus and pseudomonas aeruginosa cultures . the effect of the preparation conditions on the morphology of the nanofibers and subsequent gentamicin release was evaluated . pva nanofibrous layers were made at a basis weight of 0.8 g / m , while the covering pur layers had basis weights of 1.5 , 4.4 , and 14.7 g / m . layers of each polymer intended for mercury porosimetry and nitrogen adsorption / desorption measurements were prepared separately in thicknesses ( basis weights ) similar to the sandwich structures . nonwoven polypropylene material ( atex , milan , italy ) the different thickness of each layer ( g / m ) results from the speed of the rotating collecting electrode . in the case of the pva layer with a basis weight of 0.8 g / m , the speed was 50 mm / min ; for the pur layers , the speeds were as follows : 1.5 g / m = 110 mm / min , 4.4 g / m = 30 mm / min , and 14.7 g / m = 18 mm / min with double electrospinning . the structure of the prepared nanofibers was observed using a ts 5130 vega scanning electron microscope , tescan ( brno , czech republic ) . single layers of pva and pur nanofibers were prepared to determine porosity and surface area . mercury porosimetry measurements were made using an autopore iv 9500 porosimeter ( micromeritics , norcross , ga ) , and specific surface areas were calculated from nitrogen absorption / desorption isotherms recorded on an asap 2020 apparatus ( micromeritics ) . gentamicin release from the nanofibrous mats was investigated under laboratory conditions and by in vitro bacteriological assay . laboratory experiments were performed on agar dishes ( caso - agar , mercoplate ; merck and co , whitehouse station , nj ) in order to evaluate the time course of the drug release from the nanofibrous mats . the hplc - ms apparatus was also used to evaluate the stability of gentamicin during the electrospinning process . the in vitro antimicrobial properties of electrospun layers containing gentamicin were evaluated from inhibitory zone measurements against the gram - positive organism staphylococcus aureus ( atcc 6538 ) and the gram - negative organism pseudomonas aeruginosa ( atcc 27853 ) . the release profiles of the gentamicin from the nanofibrous layered mats were evaluated using the inhibition of s. aureus after 1 , 3 , 6 , 24 , and 48 hours of treatment . upon removal from the agar plate , the nanofibrous discs were placed on a new agar plate to further evaluate the inhibitory effect from the residual gentamicin in the sample . to eliminate potential variability between the samples during the manufacturing process , the area of inhibition was calculated as ( area of inhibition / area of inhibition after 48 hours ) 100 , as the area of inhibition after 48 hours was found to be maximal ; also , no residual inhibitory activity was detected in the samples after 48 hours of drug release . this is due to the method of preparation , by which the fiber thickness is particularly influenced by the viscosity of the solution , the strength of the electrostatic field , and the distance between the electrodes ; the mat s thickness is driven by the speed of rotation of the collecting electrode and the duration of fiber deposition , while the other parameters remain unchanged . on the other hand , some secondary changes in the nanofibers , especially local melting of the fibers , can be seen . it should be mentioned that the actual thickness of the nanofibrous layers in the subsequent drug release experiments , as well as in their potential in vivo application , will certainly differ from the values obtained by sem in a vacuum / dry state , due to the absorption of water under aqueous conditions . nitrogen adsorption / desorption bet measurements and mercury porosimetry were carried out with single pva and pur layers of the same basis weight as in the sandwich structures , and the results obtained are summarized in table 1 . bet measurements of specific surface areas revealed higher values in the case of the pva fibers ( 12.8 m / g ) compared to the pur ones ( between 7.5 m / g and 9.1 m / g ) , which corresponds to the smaller diameter of the pva fibers . similar to the fiber diameters , comparing pur layers of various thicknesses revealed that the surface areas were not influenced by the basis weight . the porosity of the pva nanofibers ( 85.8% ) determined by mercury porosimetry was higher than the porosity of the pur nanofibrous samples ( 70.7%78.9% ; see table 1 ) . with the increasing deposition time needed to obtain thicker layers , a certain level of compression of a whole layer can occur , ie , the formed nanofibrous layer is more compact . mercury porosimetry , as well as bet measurements , proceeds on single layers , and therefore , the obtained values may be slightly different from those of real sandwich structures . the concentration of the released gentamicin was assessed using hplc - ms . before starting the gentamicin release experiments , it was crucial to prove the stability of the drug during the incorporation procedure . ms spectra of fresh gentamicin before incorporation were compared with the spectra of gentamicin released from sandwich nanofibers consisting of crosslinked pva nanofibers and 14.7 g / m pur covering layers . the antimicrobial effectiveness of gentamicin released from crosslinked and noncrosslinked pva layers as well as from pur / pva / pur sandwiches was evaluated from the inhibition zones of s. aureus and p. aeruginosa after 1 , 2 , and 6 days of culture . clear inhibitory zones were observed with both strains around all of the gentamicin - loaded mats , while no inhibition was found around control nanofibers without the drug . figure 3 shows the dependence of the released gentamicin ( in percentage ) on time . an effect of the various thicknesses of the covering pur layers was observed only for very short exposure times ( 10 and 30 minutes ) . for longer exposure times , regardless of the pur layer thickness , the amount of the released drug was similar ( 90%100% ) . in the case of samples with gentamicin incorporated into sandwiches with a noncrosslinked pva layer , the faster release was influenced by the degradation of the pva layer and the subsequent easier dissolution of the gentamicin . in the case of the thinnest covering pur nanofibrous layers ( 1.5 g / m ) , the entire quantity of gentamicin was released within 1 hour of sample application onto the agar plate . sandwiches with 4.4 g / m pur layers retained about 10% of the gentamicin after 24 hours of application , while those with 14.7 g / m pur layers retained 30% after 24 hours and still contained about 20% of gentamicin even after 48 hours . the gentamicin release profiles were evaluated from the area of inhibition of s. aureus after various time periods ( figure 5 ) . after 1 hour , the largest inhibitory area ( 85% ) was found for 1.5 g / m pur sandwiches , indicating the largest amount of released gentamicin , while the inhibitory zones were smaller around the thicker nanofibrous sandwiches with outer pur layers of 4.4 g / m ( 59% ) or 14.7 g / m ( 56% ) ( figure 5a ) . no inhibition zones were observed around the 1.5 g / m pur layered mats , while the inhibition zones confirmed gentamicin retention in the 4.4 g / m and 14.7 g / m pur layered mats even after 24 hours ( figure 5b ) . bacterial inhibition , however , requires a certain limiting inhibitory concentration of gentamicin , and drug release is therefore not detectable in the more distant areas around the nanofibrous mats where the gentamicin concentration is below the level needed to exert an inhibitory effect . prolonged drug release was found for thicker covering layers of 4.4 g / m or 14.7 g / m thicknesses , but even so , more than 70% of the gentamicin was released within 24 hours . the prolongation of the gentamicin release was influenced by two factors : ( 1 ) since a covering pur layer with a higher basis weight is thicker , the gentamicin molecules must travel a longer distance from the surface of the pva nanofibers to the free volume ; and ( 2 ) thicker nanofibrous mats are more compact with a lower porosity than thinner ones ( see the column porosity in table 1 ) . unlike these authors , we have expressed the thickness of the nanofibrous layers in basis weight because , according to our experience , thicknesses expressed in micrometers can vary significantly due to the compression of nanofibrous mats during manipulation or in in vitro / in vivo experiments . we are aware that a pva nanofibrous layer with a basis weight of 0.8 g / m and 10 wt% gentamicin was used to model gentamicin release from sandwiches of various thicknesses , but that this does not correspond to the actual therapeutic dosage of gentamicin commonly used for local application . the required therapeutic dose of gentamicin or other drugs can be easily obtained by adjusting the thickness ( basis weight ) of the middle drug - loaded layer or by adjusting the drug s concentration in the electrospun mixture . nevertheless , the thickness of the layers needs to be further optimized to control the drug release rate and to maintain the mechanical properties or other features of multi - layer nanofibrous mats . this is due to the method of preparation , by which the fiber thickness is particularly influenced by the viscosity of the solution , the strength of the electrostatic field , and the distance between the electrodes ; the mat s thickness is driven by the speed of rotation of the collecting electrode and the duration of fiber deposition , while the other parameters remain unchanged . on the other hand , some secondary changes in the nanofibers , especially local melting of the fibers , can be seen . it should be mentioned that the actual thickness of the nanofibrous layers in the subsequent drug release experiments , as well as in their potential in vivo application , will certainly differ from the values obtained by sem in a vacuum / dry state , due to the absorption of water under aqueous conditions . nitrogen adsorption / desorption bet measurements and mercury porosimetry were carried out with single pva and pur layers of the same basis weight as in the sandwich structures , and the results obtained are summarized in table 1 . bet measurements of specific surface areas revealed higher values in the case of the pva fibers ( 12.8 m / g ) compared to the pur ones ( between 7.5 m / g and 9.1 m / g ) , which corresponds to the smaller diameter of the pva fibers . similar to the fiber diameters , comparing pur layers of various thicknesses revealed that the surface areas were not influenced by the basis weight . the porosity of the pva nanofibers ( 85.8% ) determined by mercury porosimetry was higher than the porosity of the pur nanofibrous samples ( 70.7%78.9% ; see table 1 ) . with the increasing deposition time needed to obtain thicker layers , a certain level of compression of a whole layer can occur , ie , the formed nanofibrous layer is more compact . mercury porosimetry , as well as bet measurements , proceeds on single layers , and therefore , the obtained values may be slightly different from those of real sandwich structures . before starting the gentamicin release experiments , it was crucial to prove the stability of the drug during the incorporation procedure . ms spectra of fresh gentamicin before incorporation were compared with the spectra of gentamicin released from sandwich nanofibers consisting of crosslinked pva nanofibers and 14.7 g / m pur covering layers . the antimicrobial effectiveness of gentamicin released from crosslinked and noncrosslinked pva layers as well as from pur / pva / pur sandwiches was evaluated from the inhibition zones of s. aureus and p. aeruginosa after 1 , 2 , and 6 days of culture . clear inhibitory zones were observed with both strains around all of the gentamicin - loaded mats , while no inhibition was found around control nanofibers without the drug . figure 3 shows the dependence of the released gentamicin ( in percentage ) on time . an effect of the various thicknesses of the covering pur layers was observed only for very short exposure times ( 10 and 30 minutes ) . for longer exposure times , regardless of the pur layer thickness , the amount of the released drug was similar ( 90%100% ) . in the case of samples with gentamicin incorporated into sandwiches with a noncrosslinked pva layer , the faster release was influenced by the degradation of the pva layer and the subsequent easier dissolution of the gentamicin . in the case of the thinnest covering pur nanofibrous layers ( 1.5 g / m ) , the entire quantity of gentamicin was released within 1 hour of sample application onto the agar plate . sandwiches with 4.4 g / m pur layers retained about 10% of the gentamicin after 24 hours of application , while those with 14.7 g / m pur layers retained 30% after 24 hours and still contained about 20% of gentamicin even after 48 hours . after 1 hour , the largest inhibitory area ( 85% ) was found for 1.5 g / m pur sandwiches , indicating the largest amount of released gentamicin , while the inhibitory zones were smaller around the thicker nanofibrous sandwiches with outer pur layers of 4.4 g / m ( 59% ) or 14.7 g / m ( 56% ) ( figure 5a ) . no inhibition zones were observed around the 1.5 g / m pur layered mats , while the inhibition zones confirmed gentamicin retention in the 4.4 g / m and 14.7 g / m pur layered mats even after 24 hours ( figure 5b ) . bacterial inhibition , however , requires a certain limiting inhibitory concentration of gentamicin , and drug release is therefore not detectable in the more distant areas around the nanofibrous mats where the gentamicin concentration is below the level needed to exert an inhibitory effect . prolonged drug release was found for thicker covering layers of 4.4 g / m or 14.7 g / m thicknesses , but even so , more than 70% of the gentamicin was released within 24 hours . the prolongation of the gentamicin release was influenced by two factors : ( 1 ) since a covering pur layer with a higher basis weight is thicker , the gentamicin molecules must travel a longer distance from the surface of the pva nanofibers to the free volume ; and ( 2 ) thicker nanofibrous mats are more compact with a lower porosity than thinner ones ( see the column consequently , migrating molecules of the released drug must travel through a nanofibrous layer with smaller pores . unlike these authors , we have expressed the thickness of the nanofibrous layers in basis weight because , according to our experience , thicknesses expressed in micrometers can vary significantly due to the compression of nanofibrous mats during manipulation or in in vitro / in vivo experiments . we are aware that a pva nanofibrous layer with a basis weight of 0.8 g / m and 10 wt% gentamicin was used to model gentamicin release from sandwiches of various thicknesses , but that this does not correspond to the actual therapeutic dosage of gentamicin commonly used for local application . the required therapeutic dose of gentamicin or other drugs can be easily obtained by adjusting the thickness ( basis weight ) of the middle drug - loaded layer or by adjusting the drug s concentration in the electrospun mixture . nevertheless , the thickness of the layers needs to be further optimized to control the drug release rate and to maintain the mechanical properties or other features of multi - layer nanofibrous mats . hplc / ms analysis , as well as inhibition of bacterial growth , proved the stability of the gentamicin after its incorporation into the nanofibers during electrospinning .

preoperative anxiety , a common phenomenon in preoperative patient evaluation , is a process that starts from the date of planning a given operation and progressively intensifies up to the moment of the operation itself . the symptoms of preoperative anxiety are stress and discomfort and the sympathetic , parasympathetic , and endocrine systems are known to play a role . because preoperative anxiety causes a decrease in patient comfort and quality of life , difficulties in making rational preferences between treatment choices , a decrease in various cognitive functions , and even difficulties in handling postoperative pain during the postoperative period , it is important to thoroughly analyze the possible risk factors associated with preoperative anxiety and to take the relevant precautions . in this study , we investigated the anxiety status of the patient before elective cholecystectomy and analyzed the relation between the level of anxiety for a given operation type ( laparoscopic and open cholecystectomy ) and the corresponding demographic and social data . a total of 333 patients undergoing cholecystectomy due to cholelithiasis ( gallstone disease ) between may 2005 and april 2011 were included in the study . a total of 218 patients ( 66.1% ) received laparoscopic cholecystectomy , and 115 patients ( 33.9% ) were treated with open cholecystectomy . all patients who underwent surgery due to cholecystectomy in this period were included in the study . however , to get reliable results from the study , the psychiatric histories of all patients were questioned in detail . whether associated with anxiety disorder or not in the past , the patients with any psychiatric diagnosis , the patients who have been using drugs for any reason , or the patients who had used drugs but stopped , especially in patients with disease associated with anxiety disorders , were excluded from the study . additionally , some other systemic diseases that can cause a defect on cognitive functions , such as hypertension , diabetes mellitus , atherosclerotic cardiovascular disease ; patients presenting with chronic diseases that necessitate continuous drug use ; and those having had a surgical operation prior to the present intervention were not included into the study . all patients underwent preoperative systematic physical examination , complete blood cell count , and routine biochemical examination . moreover , following consultation with the department of anesthesia , only patients who were classified as being in the american society of anesthesiologists ' categories i and ii were included in the study . furthermore , all patients were informed about the possible risks and complications associated with anesthesia by the anesthetist , and an informed consent form was signed by the patient 's relatives . in terms of standardization , patients were hospitalized 1 day prior to the operation , and their vital signs were monitored once every 6 hours . patients presenting with abnormal fever , pulse , or tension values were excluded from the study . all patients were informed in detail about the operation and the study by the same operator . informed consent forms for the operation and consent forms for the study were signed by all patients . our study was conducted in accordance with the world medical association declaration of helsinki and received approval by the institutional ethics committee . the beck anxiety inventory measures the strength of the anxiety indications that an individual experiences . the inventory consists of 21 items and has a likert - type self - evaluation scale pointing between 0 and 3 ; the high total point indicates the high level of anxiety that an individual experiences . however , according to the points scored in the beck anxiety inventory , the level of anxiety of the patient is classified as follows : 0 to 7 , minimal level of anxiety ; 8 to 15 , mild anxiety ; 16 to 25 , moderate anxiety ; and 26 to 63 , severe anxiety . in this study , we evaluated levels of anxiety in 3 groups as follows : 0 to 15 , low to mild anxiety ; 16 to 25 , moderate anxiety ; 26 to 63 , severe anxiety . for illiterate patients , the beck anxiety inventory form was completed by their chosen relatives , by reading and simultaneously completing the inventory in accordance with the answers provided by the patients . demographical data and applied surgical procedures from all patients were recorded into the work file database . the following patient information remained confidential and was recorded : age and sex ( female [ f ] or male [ m ] ) ; associated disease : have / do not have ; civil status : ( 1 ) single , ( 2 ) married , or ( 3 ) divorced ; educational status : ( 1 ) illiterate , ( 2 ) primary education , or ( 3 ) high school and university ; beck anxiety inventory score ; having open / laparoscopic cholecystectomy ; previous knowledge of the operation : ( 1 ) health personnel , ( 2 ) family doctor and/or house nurse , etc . , ( 3 ) other , such as friend and/or neighbor , etc . , or ( 4 ) not informed ; job status : ( 1 ) unemployed , ( 2 ) self - employed , ( 3 ) employed , or ( 4 ) homemaker ; economic status : ( 1 ) good , ( 2 ) average , or ( 3 ) bad ; health insurance : have / do not have ; and child in need of care : have / do not have . data analysis was performed with spss for windows 11.5 package software ( spss inc , chicago , illinois ) . multinomial logistic regression analysis was used to identify the factors that affect distinguishing the group with a 0 to 15 score in beck anxiety inventory from the groups with 16 to 25 and 26 to 53 scores . every variable 's odds ratio , 95% confidence interval , and wald statistics were calculated . a total of 333 patients undergoing cholecystectomy due to cholelithiasis ( gallstone disease ) between may 2005 and april 2011 were included in the study . a total of 218 patients ( 66.1% ) received laparoscopic cholecystectomy , and 115 patients ( 33.9% ) were treated with open cholecystectomy . all patients who underwent surgery due to cholecystectomy in this period were included in the study . however , to get reliable results from the study , the psychiatric histories of all patients were questioned in detail . whether associated with anxiety disorder or not in the past , the patients with any psychiatric diagnosis , the patients who have been using drugs for any reason , or the patients who had used drugs but stopped , especially in patients with disease associated with anxiety disorders , were excluded from the study . additionally , some other systemic diseases that can cause a defect on cognitive functions , such as hypertension , diabetes mellitus , atherosclerotic cardiovascular disease ; patients presenting with chronic diseases that necessitate continuous drug use ; and those having had a surgical operation prior to the present intervention were not included into the study . all patients underwent preoperative systematic physical examination , complete blood cell count , and routine biochemical examination . moreover , following consultation with the department of anesthesia , only patients who were classified as being in the american society of anesthesiologists ' categories i and ii were included in the study . furthermore , all patients were informed about the possible risks and complications associated with anesthesia by the anesthetist , and an informed consent form was signed by the patient 's relatives . in terms of standardization , patients were hospitalized 1 day prior to the operation , and their vital signs were monitored once every 6 hours . patients presenting with abnormal fever , pulse , or tension values were excluded from the study . all patients were informed in detail about the operation and the study by the same operator . informed consent forms for the operation and consent forms for the study were signed by all patients . our study was conducted in accordance with the world medical association declaration of helsinki and received approval by the institutional ethics committee . the beck anxiety inventory measures the strength of the anxiety indications that an individual experiences . the inventory consists of 21 items and has a likert - type self - evaluation scale pointing between 0 and 3 ; the high total point indicates the high level of anxiety that an individual experiences . however , according to the points scored in the beck anxiety inventory , the level of anxiety of the patient is classified as follows : 0 to 7 , minimal level of anxiety ; 8 to 15 , mild anxiety ; 16 to 25 , moderate anxiety ; and 26 to 63 , severe anxiety . in this study , we evaluated levels of anxiety in 3 groups as follows : 0 to 15 , low to mild anxiety ; 16 to 25 , moderate anxiety ; 26 to 63 , severe anxiety . for illiterate patients , the beck anxiety inventory form was completed by their chosen relatives , by reading and simultaneously completing the inventory in accordance with the answers provided by the patients . demographical data and applied surgical procedures from all patients were recorded into the work file database . the following patient information remained confidential and was recorded : age and sex ( female [ f ] or male [ m ] ) ; associated disease : have / do not have ; civil status : ( 1 ) single , ( 2 ) married , or ( 3 ) divorced ; educational status : ( 1 ) illiterate , ( 2 ) primary education , or ( 3 ) high school and university ; beck anxiety inventory score ; having open / laparoscopic cholecystectomy ; previous knowledge of the operation : ( 1 ) health personnel , ( 2 ) family doctor and/or house nurse , etc . , ( 3 ) other , such as friend and/or neighbor , etc . , or ( 4 ) not informed ; job status : ( 1 ) unemployed , ( 2 ) self - employed , ( 3 ) employed , or ( 4 ) homemaker ; economic status : ( 1 ) good , ( 2 ) average , or ( 3 ) bad ; health insurance : have / do not have ; and child in need of care : have / do not have . data analysis was performed with spss for windows 11.5 package software ( spss inc , chicago , illinois ) . multinomial logistic regression analysis was used to identify the factors that affect distinguishing the group with a 0 to 15 score in beck anxiety inventory from the groups with 16 to 25 and 26 to 53 scores . every variable 's odds ratio , 95% confidence interval , and wald statistics were calculated . a total of 333 patients , 300 ( 90.1% ) of whom were women and 33 ( 9.9% ) of whom were men , were included in our study . laparoscopic cholecystectomy ( lap ) was performed on 220 patients ( 66.1% ) and open cholecystectomy ( open ) was performed on 113 patients ( 33.9% ) . systematic evaluation of the patient demographical data within lap and open groups determined the following : there was a significant difference between lap and open groups in terms of age distribution : the 25- to 50-year age - group was predominant in the lap group , and the 50- to 89-year age - group was predominant in the open group ( p < .001 ) . regarding sex distribution , women were more predominant in the lap group than they were in the open group ( p < .001 ) . the frequency of associated disease was significantly higher in the lap group than in the open group ( p < .001 ) . regarding civil status , the frequency of single or divorced patients was significantly higher in the lap group than in the open group ( p < .001 and p = .003 for single and divorced patients , respectively ) , whereas the frequency of married patients was significantly lower in the lap group than in the open group ( p < .001 ) . regarding educational status , the frequency of illiterate patients was statistically higher in the open group than in the lap group ( p < .001 ) . the frequency of primary school graduates was significantly lower in the open group than in the lap group ( p < .001 ) . regarding job status , the frequency of unemployed patients was similar between the 2 groups ( p = .101 ) ; by contrast , the frequencies of self - employed patients and housewife patients were significantly higher in the open group than they were in the lap group ( p < .001 and p = .005 , respectively ) , and the frequency of employed patients was significantly higher in the lap group than in the open group ( p < .001 ) . regarding economic status , the frequency of patients with good economic status was similar between the 2 groups ( p = .606 ) , and while patients with average economic status were predominant in the open group , patients with weak economic status were predominant in the lap group ( p < .001 ) . the frequencies of patients without health insurance and having children were significantly higher in the lap group and the open group , respectively ( p = .024 and p < .001 ) . the frequencies of patients informed about the operation prior to applying to the hospital by health personnel or by others was significantly higher in the lap group than in the open group ( p < .001 and p = .002 , respectively ) , whereas the frequency of patients not informed about the operation was significantly higher in open group ( p < .001 ) ( table 1 ) . distribution of the demographical and clinical features of the patients according to surgical groups lap , laparoscopic cholecystectomy . when the 2 groups were evaluated according to their beck anxiety inventory scores , it was determined that the frequency of patients having 0 to 15 scores was statistically similar between the groups ( p = .538 ) , the frequency of patients having 16 to 25 scores was significantly lower ( p < .001 ) , and the frequency of patients having 26 to 53 scores was significantly higher ( p < .001 ) in the lap group than in the open group ( table 2 ) . further investigation was conducted to assess the determinant factors in differentiating between patients having 0 to 15 scores and those having 16 to 25 or 26 to 53 scores . according to the results of this investigation , the following criteria were determined : the most determinant factors in differentiating between the score groups were having a low level of education , being of the female sex , being single , and having laparoscopic operation ; the factors of being a homemaker and over the age of 25 years were determined to have significant effects . finally , no significant effect was determined in terms of being in a bad economic status ( table 3 ) . distribution of the demographical and clinical features of the patients according to their scores in the beck anxiety inventory the difference between the 0 to 15 and 16 to 25 score groups is statistically significant ( p < .05 ) . the difference between the 0 to 15 and 16 to 25 score groups is statistically significant ( p < .001 ) . the difference between the 0 to 15 and 16 to 25 score groups is statistically significant ( p < .01 ) . the difference between the 0 to 15 and 26 to 53 score groups is statistically significant ( p < .05 ) . the difference between the 0 to 15 and 26 to 53 score groups is statistically significant ( p < .01 ) . the difference between the 0 to 15 and 26 to 53 score groups is statistically significant ( p < .001 ) . the difference between the 16 to 25 and 26 to 53 score groups is statistically significant ( p < .001 ) . the difference between the 16 to 25 and 26 to 53 score group is statistically significant ( p < .01 ) . the difference between the 16 to 25 and 26 to 53 score groups is statistically significant ( p < .05 ) . determining the factors playing a role in distinguishing between the 0 to 15 score group and , respectively , the 16 to 25 and 26 to 53 score groups with multinomial logistic regression analysis lap , laparoscopic cholecystectomy . anxiety is a mood disorder emerging by a trigger or an acute situation and manifesting itself with autonomic nervous system components , such as stress , discomfort , nervosity and anxiety , and individuals having this disorder are mostly nervous , more reactive , and alert to all kinds of stimuli . in this study , we investigated patients who had preoperative state anxiety , which is characterized by the fear of obscurity , the feeling of being in a strange situation , and the feeling of loss of control . when patients are asked what the causes of their anxiety are so that health personnel can perceive the different components of preoperative anxiety , many different answers are provided . for instance , most patients expressed that remaining separated from their family , and most importantly from their children , causes them anxiety . other patients expressed that waiting for an operation causes them anxiety ; contrary to expectations , being awake during the operation is not a common reason for anxiety . according to jawaid et al the most common reasons for anxiety are postoperative pain , waiting a very long time for the operation , nausea and vomiting , inability to wake from anesthesia , and fear of injection . moreover , other studies demonstrate that patients develop anxiety because of the environment they are in . the most common reasons that patients give for anxiety due to the surrounding environment are feeling uncomfortable about the environment , having difficulty in reaching for personal belongings , sleeping in a foreign bed , and interrupted lifestyle . nowadays , patients to be hospitalized for an operation are advised by most health care organizations to bring their own belongings ( such as a pillow ) with them . a common conclusion drawn by most studies conducted to understand the reasons for preoperative anxiety is that the anxiety of the patient is such a complex process that it is not only relevant to surgery or anesthesia , but it is also relevant to the postoperative phase and to being separated from the family . even in this complex and multifactorial process , there are some predictable risk factors . for example , a recent study ( 8) revealed that the preoperative anxiety risk of patients having future anxiety is relatively high ; therefore , patients having a known psychiatric disease were excluded from our study . furthermore , females constitute another high - risk group for preoperative anxiety , and the results of our study support this finding . when being a housewife is considered as an anxiety - promoting factor , it can be suggested that being a female and having a low level of education are the 2 factors affecting this parameter ; accordingly , the level of education is considered to be a risk factor from various aspects in previous studies . in our study , a low level of education is determined to be one of the predominant and most effective factors responsible for increasing preoperative anxiety . it is considered that this situation is especially relevant to having difficulty in reaching accurate information because of the level of education , the lack of self - confidence in understanding what is being told and making the right decision after evaluating the information . recent operation history can lessen the feeling of obscurity , and there are arguments that the preoperative anxiety level increases or decreases in accordance with the quality of the former operation . patients who had an operation before the present intervention were excluded from our study in terms of standardization . in this context , some studies claim that religious belief is another factor . in the study of jawaid et al , the investigators indicate , as an interesting note , that a patient presenting with a low anxiety score attributes this to believing and trusting in allah . from the results of our study this finding begs the question to what extent we , as doctors , can be successful in optimizing the patient 's adaptation , despite the rapid and excellent technical developments in endoscopic and laparoscopic surgeries . we think that it should be discussed whether we should take into account the emotional and psychological state of the patient in its entirety while we are making efforts to be up to date and to apply surgical techniques that are constantly being developed and improved . although the clinic in which this study was conducted reaches out to a wide population , has been performing laparoscopic operations for many years , and closely tracks the developments in the field , only 14.7% of patients were informed by health personnel about the laparoscopic operation , 66.6% of patients had speculative information by their relatives or neighbors who underwent the operation before , and 18.6% of patients were never informed about it , and especially the last 2 patient groups were informed about the technique of the surgery by the operator just prior to surgery in the clinic . from bilateral discussions the following quotation demonstrates this concept from one patient 's perspective : an operation can not be properly done through two holes ; i want to have an open surgery so that you see clearly what you do . in this situation , the problem to deal with is the preoperative anxiety of a patient who is going to have an operation with a technique that the patient does not have confidence in . when analyzing the results that may appear during the intraoperative and postoperative periods , to understand preoperative anxiety , to analyze the risk factors in depth , and to take the necessary precautions are all considerations that need to be the primary objectives of the surgeons who are involved with laparoscopic , endoscopic , and robotic surgeries . preoperative anxiety requires a higher dosage of agent for induction of anesthesia during the intraoperative period , and the studies of carr et al demonstrated that the patients who have preoperative anxiety are more likely to show nausea and vomiting in the postoperative period , need a higher dosage of agent for pain alleviation , and stay for a longer period at the hospital in the postoperative period . some publications even assert that preoperative anxiety causes symptoms such as delirium , cognitive disorders , and some behavioral pathologies in the postoperative period . furthermore , preoperative anxiety is seen as a factor that decreases the patient 's reassurance in terms of the surgery itself . for all of these reasons , preoperative anxiety needs to be identified at the right time to take the necessary precautions . although there are various tests to diagnose and assess preoperative anxiety , talking to the patient during the preoperative period is a good first step in ensuring fruitful results . prior duty in coping with preoperative anxiety belongs to the health personnel , doctors or other relevant health personnel who perform field studies on public health shall thoroughly analyze the symptoms and findings , shall have information about the medical applications that the patient may encounter especially in next phases , and shall adequately inform the patient about these applications . then , the operator who plans the operation shall inform the patient about the operation in accordance with the sociocultural level of the patient , shall listen to the anxiety reasons and answer his / her questions , and shall maintain a reassuring attitude in order to relieve the anxiety of the patient . lastly , we believe in the importance of informing the patient with a patient visit by the anesthetist , ideally 1 day before the operation , thereby attempting to minimize the preoperative anxiety of the patient with a gradual informing strategy processing from simple to complicated . for training and research hospitals , both the surgical and anesthesia assistants shall have the responsibility of keeping in touch with the patient in accordance with the operators and specialists . the greatest responsibilities of the surgeon are to allocate sufficient time for the questions of the patients and to give responses to them by encouraging them to ask questions and to enable illumination on the issue for the patient to a satisfactory extent . however , to ensure effective communication between the patient and the anesthesia department , the health care staff should observe the patient and , if anxiety symptoms are noticed , refer the patient to any relevant branches . at this stage , after all these observations , if the patient is perceived to present with anxiety symptoms , it is recommended to apply the necessary tests and to ask for a consultation with the psychiatric clinic , and to take steps in accordance with the recommendations of the psychiatric clinic to avoid intra- and postoperative results . for the reasons explained in our study , it is highly important in today 's surgical practice to observe patients carefully and to take the necessary precautions to avoid the bothersome intra- and postoperative results of preoperative anxiety .

background and objectives : our aim is to investigate the anxiety status of the patient before elective cholecystectomy and to analyze the relation between the level of anxiety for a given operation type ( laparoscopic and open cholecystectomy ) and the corresponding demographic and social data.methods:a total of 333 patients undergoing cholecystectomy due to cholelithiasis were included in the study ; 218 patients ( 66.1% ) received laparoscopic cholecystectomy and 115 patients ( 33.9% ) were treated with open cholecystectomy . the beck anxiety inventory was given to all patients to be completed . we evaluated levels of anxiety in 3 groups as follows : 0 to 15 , low to mild anxiety ; 16 to 25 , moderate anxiety ; 26 to 63 , severe anxiety . the following patient information remained confidential and was recorded : age and sex , associated disease , civil status , educational status , having open / laparoscopic cholecystectomy , previous knowledge of the operation , job status , economic status , health insurance , and having a child in need of care.results:the following criteria were determined : the most determinant factors in differentiating between the score groups were having a low level of education , being of the female sex , being single , and having laparoscopic operation ; the factors of being a homemaker and over the age of 25 years were determined to have significant effects.conclusions:when analyzing the results that may appear during the intraoperative and postoperative period , understanding preoperative anxiety , analyzing the risk factors in depth , and taking the necessary precautions are all considerations that need to be the primary objectives of operators who are involved with laparoscopic , endoscopic , and robotic surgery .

INTRODUCTION PATIENTS AND METHODS Patients Beck Anxiety Inventory Evaluated Parameters Data Evaluation RESULTS DISCUSSION CONCLUSIONS

preoperative anxiety , a common phenomenon in preoperative patient evaluation , is a process that starts from the date of planning a given operation and progressively intensifies up to the moment of the operation itself . the symptoms of preoperative anxiety are stress and discomfort and the sympathetic , parasympathetic , and endocrine systems are known to play a role . because preoperative anxiety causes a decrease in patient comfort and quality of life , difficulties in making rational preferences between treatment choices , a decrease in various cognitive functions , and even difficulties in handling postoperative pain during the postoperative period , it is important to thoroughly analyze the possible risk factors associated with preoperative anxiety and to take the relevant precautions . in this study , we investigated the anxiety status of the patient before elective cholecystectomy and analyzed the relation between the level of anxiety for a given operation type ( laparoscopic and open cholecystectomy ) and the corresponding demographic and social data . a total of 333 patients undergoing cholecystectomy due to cholelithiasis ( gallstone disease ) between may 2005 and april 2011 were included in the study . a total of 218 patients ( 66.1% ) received laparoscopic cholecystectomy , and 115 patients ( 33.9% ) were treated with open cholecystectomy . all patients who underwent surgery due to cholecystectomy in this period were included in the study . however , to get reliable results from the study , the psychiatric histories of all patients were questioned in detail . all patients underwent preoperative systematic physical examination , complete blood cell count , and routine biochemical examination . moreover , following consultation with the department of anesthesia , only patients who were classified as being in the american society of anesthesiologists ' categories i and ii were included in the study . furthermore , all patients were informed about the possible risks and complications associated with anesthesia by the anesthetist , and an informed consent form was signed by the patient 's relatives . in terms of standardization , patients were hospitalized 1 day prior to the operation , and their vital signs were monitored once every 6 hours . all patients were informed in detail about the operation and the study by the same operator . informed consent forms for the operation and consent forms for the study were signed by all patients . the beck anxiety inventory measures the strength of the anxiety indications that an individual experiences . the inventory consists of 21 items and has a likert - type self - evaluation scale pointing between 0 and 3 ; the high total point indicates the high level of anxiety that an individual experiences . however , according to the points scored in the beck anxiety inventory , the level of anxiety of the patient is classified as follows : 0 to 7 , minimal level of anxiety ; 8 to 15 , mild anxiety ; 16 to 25 , moderate anxiety ; and 26 to 63 , severe anxiety . in this study , we evaluated levels of anxiety in 3 groups as follows : 0 to 15 , low to mild anxiety ; 16 to 25 , moderate anxiety ; 26 to 63 , severe anxiety . for illiterate patients , the beck anxiety inventory form was completed by their chosen relatives , by reading and simultaneously completing the inventory in accordance with the answers provided by the patients . the following patient information remained confidential and was recorded : age and sex ( female [ f ] or male [ m ] ) ; associated disease : have / do not have ; civil status : ( 1 ) single , ( 2 ) married , or ( 3 ) divorced ; educational status : ( 1 ) illiterate , ( 2 ) primary education , or ( 3 ) high school and university ; beck anxiety inventory score ; having open / laparoscopic cholecystectomy ; previous knowledge of the operation : ( 1 ) health personnel , ( 2 ) family doctor and/or house nurse , etc . , or ( 4 ) not informed ; job status : ( 1 ) unemployed , ( 2 ) self - employed , ( 3 ) employed , or ( 4 ) homemaker ; economic status : ( 1 ) good , ( 2 ) average , or ( 3 ) bad ; health insurance : have / do not have ; and child in need of care : have / do not have . multinomial logistic regression analysis was used to identify the factors that affect distinguishing the group with a 0 to 15 score in beck anxiety inventory from the groups with 16 to 25 and 26 to 53 scores . a total of 333 patients undergoing cholecystectomy due to cholelithiasis ( gallstone disease ) between may 2005 and april 2011 were included in the study . a total of 218 patients ( 66.1% ) received laparoscopic cholecystectomy , and 115 patients ( 33.9% ) were treated with open cholecystectomy . all patients who underwent surgery due to cholecystectomy in this period were included in the study . however , to get reliable results from the study , the psychiatric histories of all patients were questioned in detail . whether associated with anxiety disorder or not in the past , the patients with any psychiatric diagnosis , the patients who have been using drugs for any reason , or the patients who had used drugs but stopped , especially in patients with disease associated with anxiety disorders , were excluded from the study . all patients underwent preoperative systematic physical examination , complete blood cell count , and routine biochemical examination . moreover , following consultation with the department of anesthesia , only patients who were classified as being in the american society of anesthesiologists ' categories i and ii were included in the study . furthermore , all patients were informed about the possible risks and complications associated with anesthesia by the anesthetist , and an informed consent form was signed by the patient 's relatives . in terms of standardization , patients were hospitalized 1 day prior to the operation , and their vital signs were monitored once every 6 hours . all patients were informed in detail about the operation and the study by the same operator . informed consent forms for the operation and consent forms for the study were signed by all patients . the beck anxiety inventory measures the strength of the anxiety indications that an individual experiences . the inventory consists of 21 items and has a likert - type self - evaluation scale pointing between 0 and 3 ; the high total point indicates the high level of anxiety that an individual experiences . however , according to the points scored in the beck anxiety inventory , the level of anxiety of the patient is classified as follows : 0 to 7 , minimal level of anxiety ; 8 to 15 , mild anxiety ; 16 to 25 , moderate anxiety ; and 26 to 63 , severe anxiety . in this study , we evaluated levels of anxiety in 3 groups as follows : 0 to 15 , low to mild anxiety ; 16 to 25 , moderate anxiety ; 26 to 63 , severe anxiety . for illiterate patients , the beck anxiety inventory form was completed by their chosen relatives , by reading and simultaneously completing the inventory in accordance with the answers provided by the patients . the following patient information remained confidential and was recorded : age and sex ( female [ f ] or male [ m ] ) ; associated disease : have / do not have ; civil status : ( 1 ) single , ( 2 ) married , or ( 3 ) divorced ; educational status : ( 1 ) illiterate , ( 2 ) primary education , or ( 3 ) high school and university ; beck anxiety inventory score ; having open / laparoscopic cholecystectomy ; previous knowledge of the operation : ( 1 ) health personnel , ( 2 ) family doctor and/or house nurse , etc . , or ( 4 ) not informed ; job status : ( 1 ) unemployed , ( 2 ) self - employed , ( 3 ) employed , or ( 4 ) homemaker ; economic status : ( 1 ) good , ( 2 ) average , or ( 3 ) bad ; health insurance : have / do not have ; and child in need of care : have / do not have . multinomial logistic regression analysis was used to identify the factors that affect distinguishing the group with a 0 to 15 score in beck anxiety inventory from the groups with 16 to 25 and 26 to 53 scores . a total of 333 patients , 300 ( 90.1% ) of whom were women and 33 ( 9.9% ) of whom were men , were included in our study . laparoscopic cholecystectomy ( lap ) was performed on 220 patients ( 66.1% ) and open cholecystectomy ( open ) was performed on 113 patients ( 33.9% ) . systematic evaluation of the patient demographical data within lap and open groups determined the following : there was a significant difference between lap and open groups in terms of age distribution : the 25- to 50-year age - group was predominant in the lap group , and the 50- to 89-year age - group was predominant in the open group ( p < .001 ) . regarding civil status , the frequency of single or divorced patients was significantly higher in the lap group than in the open group ( p < .001 and p = .003 for single and divorced patients , respectively ) , whereas the frequency of married patients was significantly lower in the lap group than in the open group ( p < .001 ) . regarding educational status , the frequency of illiterate patients was statistically higher in the open group than in the lap group ( p < .001 ) . regarding job status , the frequency of unemployed patients was similar between the 2 groups ( p = .101 ) ; by contrast , the frequencies of self - employed patients and housewife patients were significantly higher in the open group than they were in the lap group ( p < .001 and p = .005 , respectively ) , and the frequency of employed patients was significantly higher in the lap group than in the open group ( p < .001 ) . regarding economic status , the frequency of patients with good economic status was similar between the 2 groups ( p = .606 ) , and while patients with average economic status were predominant in the open group , patients with weak economic status were predominant in the lap group ( p < .001 ) . the frequencies of patients without health insurance and having children were significantly higher in the lap group and the open group , respectively ( p = .024 and p < .001 ) . the frequencies of patients informed about the operation prior to applying to the hospital by health personnel or by others was significantly higher in the lap group than in the open group ( p < .001 and p = .002 , respectively ) , whereas the frequency of patients not informed about the operation was significantly higher in open group ( p < .001 ) ( table 1 ) . distribution of the demographical and clinical features of the patients according to surgical groups lap , laparoscopic cholecystectomy . when the 2 groups were evaluated according to their beck anxiety inventory scores , it was determined that the frequency of patients having 0 to 15 scores was statistically similar between the groups ( p = .538 ) , the frequency of patients having 16 to 25 scores was significantly lower ( p < .001 ) , and the frequency of patients having 26 to 53 scores was significantly higher ( p < .001 ) in the lap group than in the open group ( table 2 ) . further investigation was conducted to assess the determinant factors in differentiating between patients having 0 to 15 scores and those having 16 to 25 or 26 to 53 scores . according to the results of this investigation , the following criteria were determined : the most determinant factors in differentiating between the score groups were having a low level of education , being of the female sex , being single , and having laparoscopic operation ; the factors of being a homemaker and over the age of 25 years were determined to have significant effects . finally , no significant effect was determined in terms of being in a bad economic status ( table 3 ) . distribution of the demographical and clinical features of the patients according to their scores in the beck anxiety inventory the difference between the 0 to 15 and 16 to 25 score groups is statistically significant ( p < .05 ) . the difference between the 0 to 15 and 16 to 25 score groups is statistically significant ( p < .001 ) . the difference between the 0 to 15 and 16 to 25 score groups is statistically significant ( p < .01 ) . the difference between the 0 to 15 and 26 to 53 score groups is statistically significant ( p < .05 ) . the difference between the 0 to 15 and 26 to 53 score groups is statistically significant ( p < .01 ) . the difference between the 0 to 15 and 26 to 53 score groups is statistically significant ( p < .001 ) . the difference between the 16 to 25 and 26 to 53 score groups is statistically significant ( p < .001 ) . the difference between the 16 to 25 and 26 to 53 score group is statistically significant ( p < .01 ) . the difference between the 16 to 25 and 26 to 53 score groups is statistically significant ( p < .05 ) . determining the factors playing a role in distinguishing between the 0 to 15 score group and , respectively , the 16 to 25 and 26 to 53 score groups with multinomial logistic regression analysis lap , laparoscopic cholecystectomy . anxiety is a mood disorder emerging by a trigger or an acute situation and manifesting itself with autonomic nervous system components , such as stress , discomfort , nervosity and anxiety , and individuals having this disorder are mostly nervous , more reactive , and alert to all kinds of stimuli . in this study , we investigated patients who had preoperative state anxiety , which is characterized by the fear of obscurity , the feeling of being in a strange situation , and the feeling of loss of control . when patients are asked what the causes of their anxiety are so that health personnel can perceive the different components of preoperative anxiety , many different answers are provided . other patients expressed that waiting for an operation causes them anxiety ; contrary to expectations , being awake during the operation is not a common reason for anxiety . according to jawaid et al the most common reasons for anxiety are postoperative pain , waiting a very long time for the operation , nausea and vomiting , inability to wake from anesthesia , and fear of injection . the most common reasons that patients give for anxiety due to the surrounding environment are feeling uncomfortable about the environment , having difficulty in reaching for personal belongings , sleeping in a foreign bed , and interrupted lifestyle . a common conclusion drawn by most studies conducted to understand the reasons for preoperative anxiety is that the anxiety of the patient is such a complex process that it is not only relevant to surgery or anesthesia , but it is also relevant to the postoperative phase and to being separated from the family . for example , a recent study ( 8) revealed that the preoperative anxiety risk of patients having future anxiety is relatively high ; therefore , patients having a known psychiatric disease were excluded from our study . furthermore , females constitute another high - risk group for preoperative anxiety , and the results of our study support this finding . when being a housewife is considered as an anxiety - promoting factor , it can be suggested that being a female and having a low level of education are the 2 factors affecting this parameter ; accordingly , the level of education is considered to be a risk factor from various aspects in previous studies . in our study , a low level of education is determined to be one of the predominant and most effective factors responsible for increasing preoperative anxiety . it is considered that this situation is especially relevant to having difficulty in reaching accurate information because of the level of education , the lack of self - confidence in understanding what is being told and making the right decision after evaluating the information . recent operation history can lessen the feeling of obscurity , and there are arguments that the preoperative anxiety level increases or decreases in accordance with the quality of the former operation . in the study of jawaid et al , the investigators indicate , as an interesting note , that a patient presenting with a low anxiety score attributes this to believing and trusting in allah . from the results of our study this finding begs the question to what extent we , as doctors , can be successful in optimizing the patient 's adaptation , despite the rapid and excellent technical developments in endoscopic and laparoscopic surgeries . we think that it should be discussed whether we should take into account the emotional and psychological state of the patient in its entirety while we are making efforts to be up to date and to apply surgical techniques that are constantly being developed and improved . although the clinic in which this study was conducted reaches out to a wide population , has been performing laparoscopic operations for many years , and closely tracks the developments in the field , only 14.7% of patients were informed by health personnel about the laparoscopic operation , 66.6% of patients had speculative information by their relatives or neighbors who underwent the operation before , and 18.6% of patients were never informed about it , and especially the last 2 patient groups were informed about the technique of the surgery by the operator just prior to surgery in the clinic . from bilateral discussions the following quotation demonstrates this concept from one patient 's perspective : an operation can not be properly done through two holes ; i want to have an open surgery so that you see clearly what you do . in this situation , the problem to deal with is the preoperative anxiety of a patient who is going to have an operation with a technique that the patient does not have confidence in . when analyzing the results that may appear during the intraoperative and postoperative periods , to understand preoperative anxiety , to analyze the risk factors in depth , and to take the necessary precautions are all considerations that need to be the primary objectives of the surgeons who are involved with laparoscopic , endoscopic , and robotic surgeries . preoperative anxiety requires a higher dosage of agent for induction of anesthesia during the intraoperative period , and the studies of carr et al demonstrated that the patients who have preoperative anxiety are more likely to show nausea and vomiting in the postoperative period , need a higher dosage of agent for pain alleviation , and stay for a longer period at the hospital in the postoperative period . some publications even assert that preoperative anxiety causes symptoms such as delirium , cognitive disorders , and some behavioral pathologies in the postoperative period . furthermore , preoperative anxiety is seen as a factor that decreases the patient 's reassurance in terms of the surgery itself . for all of these reasons , preoperative anxiety needs to be identified at the right time to take the necessary precautions . although there are various tests to diagnose and assess preoperative anxiety , talking to the patient during the preoperative period is a good first step in ensuring fruitful results . prior duty in coping with preoperative anxiety belongs to the health personnel , doctors or other relevant health personnel who perform field studies on public health shall thoroughly analyze the symptoms and findings , shall have information about the medical applications that the patient may encounter especially in next phases , and shall adequately inform the patient about these applications . then , the operator who plans the operation shall inform the patient about the operation in accordance with the sociocultural level of the patient , shall listen to the anxiety reasons and answer his / her questions , and shall maintain a reassuring attitude in order to relieve the anxiety of the patient . lastly , we believe in the importance of informing the patient with a patient visit by the anesthetist , ideally 1 day before the operation , thereby attempting to minimize the preoperative anxiety of the patient with a gradual informing strategy processing from simple to complicated . the greatest responsibilities of the surgeon are to allocate sufficient time for the questions of the patients and to give responses to them by encouraging them to ask questions and to enable illumination on the issue for the patient to a satisfactory extent . however , to ensure effective communication between the patient and the anesthesia department , the health care staff should observe the patient and , if anxiety symptoms are noticed , refer the patient to any relevant branches . at this stage , after all these observations , if the patient is perceived to present with anxiety symptoms , it is recommended to apply the necessary tests and to ask for a consultation with the psychiatric clinic , and to take steps in accordance with the recommendations of the psychiatric clinic to avoid intra- and postoperative results . for the reasons explained in our study , it is highly important in today 's surgical practice to observe patients carefully and to take the necessary precautions to avoid the bothersome intra- and postoperative results of preoperative anxiety .

the online version of this article ( doi:10.1007/s00134 - 014 - 3381-x ) contains supplementary material , which is available to authorized users . extremes of weight - for - age are common in children requiring intensive care and are likely to impact upon morbidity and mortality [ 1 , 2 ] . being underweight may indicate malnutrition , be associated with genetic syndromes , chronic conditions , or represent increased energy consumption [ 3 , 4 ] . protein - energy malnutrition is associated with increased mortality , can be complex to diagnose , and is often under recognised and underestimated [ 6 , 7 ] . being overweight may reflect underlying disease or syndromes , limited mobility , or may simply represent increasing prevalence of obesity in the general population . being significantly underweight is associated with prolonged icu stay and increased mortality [ 1 , 9 , 10 ] . however , published data on outcome in overweight children and adults is conflicting [ 1114 ] . broadly , adult intensive care unit ( aicu ) data suggest increased duration of mechanical ventilation and icu stay but no clear association with mortality [ 1517 ] . weight - for - age may be an independent risk factor for mortality in critically ill neonates and children . in this study we tested the null hypotheses that the paediatric intensive care ( pic ) population weight distribution does not differ from that of the general population and that weight - for - age at admission does not influence risk - adjusted mortality . we queried the prospectively collected great ormond street hospital ( gosh , london , uk ) paediatric intensive care audit network ( picanet ) database to identify all children aged 018.0 years admitted to the paediatric , cardiac and neonatal intensive care units between 1st march 2003 and 30th december 2011 . gosh is a tertiary and quaternary referral hospital for children with approximately 1,5001,800 pic admissions per year . demographic , admission and outcome information were recorded , including the paediatric index of mortality-2 ( pim2 ) score . children were recorded as dead if they died during the primary admission or within 30 days of discharge from picu ( including readmissions ) . up to chronological age 2 years , children born premature ( < 37/40 gestation ) had their age corrected . admission weight for each child was compared to uk population reference data by calculation of a weight - for - age standard deviation score ( sds ) ; sds is the number of standard deviations by which a weight value differs from the population mean . standardising all weight values in this way allows comparison of children of differing age and gender . lmsgrowth software for microsoft excel was used , with uk growth reference data as per current royal college of paediatrics and child health guidelines [ 2022 ] . medical records of outliers were reviewed ; children with anomalous or absent data were excluded . patients were categorised into nine groups according to their weight - for - age sds ( table 1 ) . four ethnic subgroups were chosen to avoid over - dividing the study cohort : white , black , south asian and other. the south asian ethnic group was chosen since recent picanet data has indicated poor outcomes across uk picus for this group of children [ 23 , 24 ] . age groups were chosen pragmatically : neonates including premature babies ( group a ) , preschool infants and children ( 1 month4.9 years , group b ) and school - age ( 518 years , group c).table 1study population characteristics and primary outcome data including details of distribution across demographic indices and weight - for - age at admission groupstotal study records , n 14,307 excluded : absent / spurious data102 excluded : readmission < 30 days1,747records analysed12,458gender m : f ( % ) 6,851:5,607 ( 55:45 ) n ( % ) age distribution a ( < 1 months)3,562 ( 29)b ( 1 months4.9 years)5,972 ( 48)c ( 518 years)2,924 ( 23 ) admission type planned : post op4,511 ( 36)planned : other913 ( 7)unplanned : post op311 ( 2)unplanned : other6,723 ( 54)mechanical ventilation in first hour9,544 ( 77 ) ethnicity white origin6,515 ( 52)black origin1,369 ( 11)south asian1,323 ( 11)other ( see supplementary appendix 1)3,251 ( 26 ) diagnostic group cardiovascular 5,137 ( 41)respiratory2,678 ( 21)neurological1,067 ( 9)gastrointestinal827 ( 7)trauma405 ( 3)infection392 ( 3)other1,952 ( 16)distribution by weight - for - age sdsweight - for - age ( sds ) n ( % ) deathssmr ( 95 % ci)logistic regressionor ( 95 % ci)<3.51,004 ( 8)841.19 ( 0.97,1.46)1.46 ( 1.101.94)3.5 to 2.511,210 ( 10)981.26 ( 1.041.53)1.46 ( 1.121.92)2.5 to 1.512,281 ( 18)1941.02 ( 0.891.16)1.18 ( 0.951.48)1.5 to 0.513,129 ( 25)2220.87 ( 0.760.98)0.98 ( 0.791.21)0.5 to 0.492,646 ( 21)2000.88 ( 0.771.00)reference0.51.491,509 ( 12)1010.82 ( 0.670.99)0.88 ( 0.681.15)1.52.49504 ( 4)280.71 ( 0.501.01)0.75 ( 0.481.16)2.53.49144 ( 1)131.14 ( 0.681.89)1.31 ( 0.712.43)>3.531 ( 0.2)20.95 ( 0.273.05)1.15 ( 0.255.2)study population weight distribution compared to uk reference populationmean weight - for - age sds ( 95 % ci)1.04 ( 1.07 to 1.01 ) p < 0.0001*standard deviation1.7median z - score weight - for - age0.93study population summary of deathstotal deaths during study period942 ( 7.5)in picu895<30 days post discharge47smr for whole study population0.94distribution of deaths , standardised mortality ratios ( smr ) and odds ratio ( or ) risk of death determined by logistic regression are provided by weight - for - age*one - sample t test comparison with population mean post cardiac bypass n ( % ) : 2,859 ( 23 ) study population characteristics and primary outcome data including details of distribution across demographic indices and weight - for - age at admission groups distribution of deaths , standardised mortality ratios ( smr ) and odds ratio ( or ) risk of death determined by logistic regression are provided by weight - for - age * one - sample t test comparison with population mean post cardiac bypass n ( % ) : 2,859 ( 23 ) the study population frequency distribution was compared to the uk reference population distribution using a one - sample t test . a histogram of the study data was plotted against a representative distribution of the uk reference population.fig . 1stacked histogram of study population admission weight - for - age standard deviation score compared to uk reference population . the mean weight - for - age at admission of the study population was 1.04 standard deviations less than that of the uk reference population ( 95 % ci 1.07 to 1.01 , p < 0.0001 ) , with an over - representation of outliers , especially extreme underweight . sds standard deviation score , sd standard deviation stacked histogram of study population admission weight - for - age standard deviation score compared to uk reference population . survivors and deaths are differentiated within the histogram . the mean weight - for - age at admission of the study population was 1.04 standard deviations less than that of the uk reference population ( 95 % ci 1.07 to 1.01 , p < 0.0001 ) , with an over - representation of outliers , especially extreme underweight . sds standard deviation score , sd standard deviation the primary outcome was risk - adjusted mortality during picu admission or within 30 days of discharge . standardised mortality ratios ( smr ) and 95 % confidence interval ( 95 % ci ) for each of the nine sds groups were calculated using pim-2 scores . multivariable statistical analysis was undertaken using logistic regression ( stata 12 , statacorp lp , texas , usa ) to assess the relationship between the main exposure variable ( weight - for - age at admission ) , three covariates ( age , gender , ethnicity ) and the dependent variable ( death ) . the logit form of pim2 was used for mortality risk - adjustment and models were sequentially adjusted for each of the covariates . likelihood ratio testing was used to select the best - fit model ( supplementary appendix 2 ) . a significance level of 5 % ( two - sided p value 0.05 ) was used throughout . results were expressed as odds ratios ( or ) and 95 % confidence intervals for risk of mortality.fig . 2plot of standardised mortality ratios ( smrs ) vs. weight - for - age at admission standard deviation scores , with 95 % confidence intervals . expected deaths are estimated by the sum of pim2 scores for all patients in a group . a u - shaped association is indicated : highest risk of mortality occurs with extreme under- or overweight , which this study has shown to be over - represented in children admitted to picu . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age . asterisk ( * ) indicates the overall smr for the whole study cohort is displayed on the chart at 0.94 plot of standardised mortality ratios ( smrs ) vs. weight - for - age at admission standard deviation scores , with 95 % confidence intervals . expected deaths are estimated by the sum of pim2 scores for all patients in a group . a u - shaped association is indicated : highest risk of mortality occurs with extreme under- or overweight , which this study has shown to be over - represented in children admitted to picu . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age . asterisk ( * ) indicates the overall smr for the whole study cohort is displayed on the chart at 0.94 up to chronological age 2 years , children born premature ( < 37/40 gestation ) had their age corrected . admission weight for each child was compared to uk population reference data by calculation of a weight - for - age standard deviation score ( sds ) ; sds is the number of standard deviations by which a weight value differs from the population mean . standardising all weight values in this way allows comparison of children of differing age and gender . lmsgrowth software for microsoft excel was used , with uk growth reference data as per current royal college of paediatrics and child health guidelines [ 2022 ] . medical records of outliers were reviewed ; children with anomalous or absent data were excluded . patients were categorised into nine groups according to their weight - for - age sds ( table 1 ) . four ethnic subgroups were chosen to avoid over - dividing the study cohort : white , black , south asian and other. the south asian ethnic group was chosen since recent picanet data has indicated poor outcomes across uk picus for this group of children [ 23 , 24 ] . age groups were chosen pragmatically : neonates including premature babies ( group a ) , preschool infants and children ( 1 month4.9 years , group b ) and school - age ( 518 years , group c).table 1study population characteristics and primary outcome data including details of distribution across demographic indices and weight - for - age at admission groupstotal study records , n 14,307 excluded : absent / spurious data102 excluded : readmission < 30 days1,747records analysed12,458gender m : f ( % ) 6,851:5,607 ( 55:45 ) n ( % ) age distribution a ( < 1 months)3,562 ( 29)b ( 1 months4.9 years)5,972 ( 48)c ( 518 years)2,924 ( 23 ) admission type planned : post op4,511 ( 36)planned : other913 ( 7)unplanned : post op311 ( 2)unplanned : other6,723 ( 54)mechanical ventilation in first hour9,544 ( 77 ) ethnicity white origin6,515 ( 52)black origin1,369 ( 11)south asian1,323 ( 11)other ( see supplementary appendix 1)3,251 ( 26 ) diagnostic group cardiovascular 5,137 ( 41)respiratory2,678 ( 21)neurological1,067 ( 9)gastrointestinal827 ( 7)trauma405 ( 3)infection392 ( 3)other1,952 ( 16)distribution by weight - for - age sdsweight - for - age ( sds ) n ( % ) deathssmr ( 95 % ci)logistic regressionor ( 95 % ci)<3.51,004 ( 8)841.19 ( 0.97,1.46)1.46 ( 1.101.94)3.5 to 2.511,210 ( 10)981.26 ( 1.041.53)1.46 ( 1.121.92)2.5 to 1.512,281 ( 18)1941.02 ( 0.891.16)1.18 ( 0.951.48)1.5 to 0.513,129 ( 25)2220.87 ( 0.760.98)0.98 ( 0.791.21)0.5 to 0.492,646 ( 21)2000.88 ( 0.771.00)reference0.51.491,509 ( 12)1010.82 ( 0.670.99)0.88 ( 0.681.15)1.52.49504 ( 4)280.71 ( 0.501.01)0.75 ( 0.481.16)2.53.49144 ( 1)131.14 ( 0.681.89)1.31 ( 0.712.43)>3.531 ( 0.2)20.95 ( 0.273.05)1.15 ( 0.255.2)study population weight distribution compared to uk reference populationmean weight - for - age sds ( 95 % ci)1.04 ( 1.07 to 1.01 ) p < 0.0001*standard deviation1.7median z - score weight - for - age0.93study population summary of deathstotal deaths during study period942 ( 7.5)in picu895<30 days post discharge47smr for whole study population0.94distribution of deaths , standardised mortality ratios ( smr ) and odds ratio ( or ) risk of death determined by logistic regression are provided by weight - for - age*one - sample t test comparison with population mean post cardiac bypass n ( % ) : 2,859 ( 23 ) study population characteristics and primary outcome data including details of distribution across demographic indices and weight - for - age at admission groups distribution of deaths , standardised mortality ratios ( smr ) and odds ratio ( or ) risk of death determined by logistic regression are provided by weight - for - age * one - sample t test comparison with population mean post cardiac bypass n ( % ) : 2,859 ( 23 ) the study population frequency distribution was compared to the uk reference population distribution using a one - sample t test . a histogram of the study data was plotted against a representative distribution of the uk reference population.fig . 1stacked histogram of study population admission weight - for - age standard deviation score compared to uk reference population . the mean weight - for - age at admission of the study population was 1.04 standard deviations less than that of the uk reference population ( 95 % ci 1.07 to 1.01 , p < 0.0001 ) , with an over - representation of outliers , especially extreme underweight . sds standard deviation score , sd standard deviation stacked histogram of study population admission weight - for - age standard deviation score compared to uk reference population . survivors and deaths are differentiated within the histogram . the mean weight - for - age at admission of the study population was 1.04 standard deviations less than that of the uk reference population ( 95 % ci 1.07 to 1.01 , p < 0.0001 ) , with an over - representation of outliers , especially extreme underweight . sds standard deviation score , sd standard deviation the primary outcome was risk - adjusted mortality during picu admission or within 30 days of discharge . standardised mortality ratios ( smr ) and 95 % confidence interval ( 95 % ci ) for each of the nine sds groups were calculated using pim-2 scores . multivariable statistical analysis was undertaken using logistic regression ( stata 12 , statacorp lp , texas , usa ) to assess the relationship between the main exposure variable ( weight - for - age at admission ) , three covariates ( age , gender , ethnicity ) and the dependent variable ( death ) . the logit form of pim2 was used for mortality risk - adjustment and models were sequentially adjusted for each of the covariates . likelihood ratio testing was used to select the best - fit model ( supplementary appendix 2 ) . a significance level of 5 % ( two - sided p value 0.05 ) was used throughout . results were expressed as odds ratios ( or ) and 95 % confidence intervals for risk of mortality.fig . 2plot of standardised mortality ratios ( smrs ) vs. weight - for - age at admission standard deviation scores , with 95 % confidence intervals . expected deaths are estimated by the sum of pim2 scores for all patients in a group . a u - shaped association is indicated : highest risk of mortality occurs with extreme under- or overweight , which this study has shown to be over - represented in children admitted to picu . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age . asterisk ( * ) indicates the overall smr for the whole study cohort is displayed on the chart at 0.94 plot of standardised mortality ratios ( smrs ) vs. weight - for - age at admission standard deviation scores , with 95 % confidence intervals . expected deaths are estimated by the sum of pim2 scores for all patients in a group . a u - shaped association is indicated : highest risk of mortality occurs with extreme under- or overweight , which this study has shown to be over - represented in children admitted to picu . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age . asterisk ( * ) indicates the overall smr for the whole study cohort is displayed on the chart at 0.94 a total of 14,307 individual records were identified and 12,458 analysed : we excluded 102 records with absent or spurious weight data and 1,747 readmissions within 30 days . fifty - five per cent were male , 52 % were children of white origin , 11 % black origin and 11 % south asian ; 26 % of children were therefore grouped in the other ethnicity category which included 4.9 % middle eastern , 4 % asian , 3 % mixed race , 7.5 % ethnicity not provided and less than 1 % each for all other ethnicities ( supplementary appendix 1 ) . the mean weight - for - age at admission of the study population was 1.04 standard deviations less than that of the uk reference population ( 95 % ci 1.07 to 1.01 , p < 0.0001 ) . 18 % and 1.4 % of the study population children were extremely under- or overweight ( more than 2.5 standard deviations from uk mean weight - for - age ) , respectively ( fig . a total of 942 patients ( 7.5 % ) died during the study period . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age ( table 1 , fig . the highest risk of mortality occurred in children with extreme under- or overweight ( more than 2.5 standard deviations from the uk reference mean ) . confidence intervals are wide for extreme overweight children because a relatively small proportion of the study population falls within these groups . trends in ors for risk of death by weight - for - age at admission , determined by logistic regression , are comparable to smr trends ( supplementary appendix 2 ) . the full logistic regression model , adjusted for covariates age , gender and ethnicity , was the best fit for the data ; therefore the main exposure variable , weight - for - age at admission , and the covariates are all independent risk factors for mortality in children admitted to picu . age , gender and ethnic subgroups ( supplementary appendix 3 ) have weight - for - age distributions which are comparable to the whole study cohort with two exceptions . first , young children ( < 5 years ) have markedly lower mean weight - for - age at admission than older children ( < 1 months : 1.0 , 1 months5 years : 1.2 and 518 years : 0.6 standard deviations below the uk reference population mean , respectively ) . second , south asian children have a markedly lower mean weight - for - age compared to non - south asians : 1.4 standard deviations below the uk mean vs. 1.0 , 1.0 and 1.1 for children of white , black and other ethnic origin , respectively . exclusion of neonates did not substantially alter trends ( supplementary appendix 5 , fig . odds ratios for risk of death within subgroups are presented in fig . 3 and supplementary appendix 3 . children 1 months4.9 years have the lowest risk of dying whilst children of south asian and other ethnicity have significantly increased risk of death compared to those of white and black origin.fig . 3plot of odds ratio ( or ) risk of death comparing categories within subgroups , determined by multivariable logistic regression , with 95 % confidence intervals . the logit form of pim2 was used for mortality risk adjustment with further sequential adjustment of the model for each covariate ( gender , age and ethnicity ) . one category in each subgroup is used as the reference ( or = 1 ) ( see also supplementary appendix 3 , table 2 ) . age group : a 1 month , b = 1 month4.9 years , c = 516 years . wh white , bl black , sa south asian , o other plot of odds ratio ( or ) risk of death comparing categories within subgroups , determined by multivariable logistic regression , with 95 % confidence intervals . the logit form of pim2 was used for mortality risk adjustment with further sequential adjustment of the model for each covariate ( gender , age and ethnicity ) . one category in each subgroup is used as the reference ( or = 1 ) ( see also supplementary appendix 3 , table 2 ) . age group : a 1 month , b = 1 month4.9 years , c = 516 years . wh white , bl black , sa south asian , o other subgroups have weight - for - age risk - adjusted mortality trends ( supplementary appendices 36 ) comparable to the whole study cohort with two exceptions . first , in children older than 5 years of age , extremes of weight - for - age are not associated with increased risk of death ( supplementary appendix 5 ) . second , in children of south asian origin , moderate overweight does not appear to be associated with the lowest risk of mortality , though wide confidence intervals limit interpretation . a total of 942 patients ( 7.5 % ) died during the study period . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age ( table 1 , fig . the highest risk of mortality occurred in children with extreme under- or overweight ( more than 2.5 standard deviations from the uk reference mean ) . confidence intervals are wide for extreme overweight children because a relatively small proportion of the study population falls within these groups . trends in ors for risk of death by weight - for - age at admission , determined by logistic regression , are comparable to smr trends ( supplementary appendix 2 ) . the full logistic regression model , adjusted for covariates age , gender and ethnicity , was the best fit for the data ; therefore the main exposure variable , weight - for - age at admission , and the covariates are all independent risk factors for mortality in children admitted to picu . age , gender and ethnic subgroups ( supplementary appendix 3 ) have weight - for - age distributions which are comparable to the whole study cohort with two exceptions . first , young children ( < 5 years ) have markedly lower mean weight - for - age at admission than older children ( < 1 months : 1.0 , 1 months5 years : 1.2 and 518 years : 0.6 standard deviations below the uk reference population mean , respectively ) . second , south asian children have a markedly lower mean weight - for - age compared to non - south asians : 1.4 standard deviations below the uk mean vs. 1.0 , 1.0 and 1.1 for children of white , black and other ethnic origin , respectively . exclusion of neonates did not substantially alter trends ( supplementary appendix 5 , fig . odds ratios for risk of death within subgroups are presented in fig . 3 and supplementary appendix 3 . children 1 months4.9 years have the lowest risk of dying whilst children of south asian and other ethnicity have significantly increased risk of death compared to those of white and black origin.fig . 3plot of odds ratio ( or ) risk of death comparing categories within subgroups , determined by multivariable logistic regression , with 95 % confidence intervals . the logit form of pim2 was used for mortality risk adjustment with further sequential adjustment of the model for each covariate ( gender , age and ethnicity ) . one category in each subgroup is used as the reference ( or = 1 ) ( see also supplementary appendix 3 , table 2 ) . age group : a 1 month , b = 1 month4.9 years , c = 516 years . wh white , bl black , sa south asian , o other plot of odds ratio ( or ) risk of death comparing categories within subgroups , determined by multivariable logistic regression , with 95 % confidence intervals . the logit form of pim2 was used for mortality risk adjustment with further sequential adjustment of the model for each covariate ( gender , age and ethnicity ) . one category in each subgroup is used as the reference ( or = 1 ) ( see also supplementary appendix 3 , table 2 ) . age group : a 1 month , b = 1 month4.9 years , c = 516 years . wh white , bl black , sa south asian , o other subgroups have weight - for - age risk - adjusted mortality trends ( supplementary appendices 36 ) comparable to the whole study cohort with two exceptions . first , in children older than 5 years of age , extremes of weight - for - age are not associated with increased risk of death ( supplementary appendix 5 ) . second , in children of south asian origin , moderate overweight does not appear to be associated with the lowest risk of mortality , though wide confidence intervals limit interpretation . we have demonstrated that the pic population weight distribution at admission differs significantly from that of the uk reference population . critically ill children have significantly lower weight - for - age than the uk average and also have an increased proportion at the extremes of weight - for - age , especially extreme underweight : 1.4 and 18 % of the study population have a weight - for - age at admission more than 2.5 standard deviations above and below the uk population mean , respectively , rather than the expected 0.6 % for each . multivariable logistic regression analysis demonstrated that weight - for - age at admission is an independent risk factor for mortality , as are age , gender and ethnicity . we observed a u - shaped association between weight - for - age at admission and risk - adjusted mortality . u - shaped interactions occur between many biological risk factors and outcome [ 26 , 27 ] , but it is of particular interest that the lowest risk of death is associated with mild - to - moderately raised , rather than mean , weight - for - age . our data demonstrates that extreme under- and overweight is strongly associated with increased risk of death ( supplementary appendix 5 ) and that these high - risk groups of children are markedly over - represented in the pic population . the associations we have observed could be unique to gosh ; our picu differs from many others by providing very specialist services with a larger proportion of high - risk cases . the study mortality rate ( 7.5 % ) is markedly higher than the uk average during the same period ( 4.6 % ) whilst the risk - adjusted smr is similar to other centres . however , comparable published data suggest that the results of this study may be generalizable to other centres . our findings are consistent with numa et al . who analysed 6,316 consecutive picu admissions in australia : a u - shaped relationship was seen with the lowest risk of death occurring at the 75th centile weight - for - age ( equivalent to sds + 1.15 ) . a similar relationship ( the obesity paradox ) has been reported in adults with heart failure : lowest risk of mortality associated with mild - to - moderately overweight patients [ 2830 ] . the proportion of underweight children in our cohort is very similar to two previous studies spanning 30 years . in the netherlands , admission weight - for - age has been shown to be 0.30.9 sds lower than the national average and 924 % of patients are acutely malnourished at admission , dependent on age . a us study in 1982 found 19 % of critically ill children had acute protein - energy malnutrition at admission . given the specialist services provided at gosh , these results should be replicated at other uk centres to confirm whether they are indeed generalizable . in this study height measurements were not available and the study was not designed to take into account change in weight or nutritional status by picu discharge or death . a recent study of admission , discharge and follow - up weight - for - age alongside anthropometric measurements found 24 % of children were undernourished at admission ; this increased by discharge but recovered to baseline at 6 months . their proportion of underweight children at admission is comparable both to historical data and our own . syndromes may be associated with under- or overweight and several specific growth charts have been developed . we were unable to extract information regarding genetic syndromes but consider the effect on our results to be small . trisomy 21 ( t21 ) is the most frequent chromosomal abnormality and accounts for just 3.8 % of picu admissions . specific t21 growth chart data excluded children with known additional pathology and therefore may not be representative of children admitted to picu . we found a significantly increased risk of mortality in children of south asian and other ethnicity . a south asian phenotype of central adiposity , insulin resistance and larger , possibly dysfunctional , adipocytes has been described . published data suggests this may be present from childhood or even in utero : cord leptin , a biomarker of body fat mass at birth , has been found to be higher in south asian babies compared to white europeans . why and how ethnicity is associated with differing risk - adjusted pic mortality warrants further investigation . aetiology is likely to be complex including , but not limited to , biological polymorphisms , socio - economic status , access to and equity of healthcare provision , lifestyle , diet and varying end - of - life care decisions according to ethnicity and culture [ 36 , 37 ] . uk reference population anthropometric data largely excludes children of non - white ethnic origin due to known difference in growth , limiting our ability to interpret weight - for - age in children of non - white ethnicity ( supplementary appendix 3 ) . this study benefits from a large data set , which was collected prospectively and is externally validated . all admissions were included with only two pragmatic exclusions : to remove spurious data and to reduce bias from short - timescale readmissions of the same patient whilst including deaths within 30 days of discharge . picu admission weight often relies on weight measured at admission to the referring hospital or ward where most children are weighed as a routine . we acknowledge that some recorded values may be estimates and this reflects the reality of picu practice ; consistent with the approach taken in comparable recent studies , we believe any resultant error will be random and offset by our large data set . subgroup analysis of elective admission , which might be expected to all have accurate admission weight , demonstrates similar trends to that shown in the whole study ( supplementary appendix 7 ) . uk reference anthropometric data will become increasingly outdated with time , especially with trends to increasing obesity . exclusion of non - white children means the data will not be representative of an increasingly diversified uk population . it defines the uk pic population admission weight - for - age distribution , demonstrates weight to be a prognostic factor which should be considered at the bedside when assessing critically ill children and outlines variation with ethnicity . these results indicate that further work to fully understand the impact of weight - for - age and nutrition on outcome would be valuable . weight was not considered during the development of pim and pim2 ; further work here may have some merit . one author has shown that including a weight variable in the pim2 model leads to a small but statistically significant increase in the area under a receiver operating characteristic curve and considers how this may impact upon discriminatory predictive performance . however , the benefits of adding variables must be balanced against the risk of complicating the tool and reducing accessibility and ease of use . the mean is significantly lower and there is an over - representation of patients at the extremes of weight - for - age ; especially extreme underweight . this study has shown that weight - for - age at admission is an independent risk factor for mortality . there is a u - shaped association between weight - for - age and risk - adjusted mortality , with the lowest risk of death in children who are mild - to - moderately overweight . the over - represented extremes of weight - for - age are associated with the highest risk - adjusted mortality . future studies should determine the impact of malnutrition on risk - adjusted mortality and investigate the aetiology of risk disparities with ethnicity .

aimsto determine whether the paediatric intensive care ( pic ) population weight distribution differs from the uk reference population and whether weight - for - age at admission is an independent risk factor for mortality.methodsadmission weight - for - age standard deviation scores ( sds ) were calculated for all pic admissions ( march 2003december 2011 ) to great ormond street hospital : this is the number of standard deviations ( sd ) between a child s weight and the uk mean weight - for - age . categorised into nine sds groups , standardised mortality ratios ( smr ) and logistic regression were used to assess the relationship between weight - for - age at admission and risk - adjusted mortality.resultsout of 12,458 admissions , mean weight - for - age was 1.04 sd below the uk reference population mean ( p < 0.0001 ) . based on 942 deaths , risk - adjusted mortality was lowest in those with mild - to - moderately raised weight - for - age ( sds 0.52.5 ) and highest in children with extreme under- or overweight ( sds < 3.5 and sds > + 3.5 ) . logistic regression indicated that age , gender , ethnicity and weight - for - age are independent risk factors for mortality . south asian and other ethnicities had significantly increased risk of death compared to children of white and black ethnic origin.conclusionthe pic population mean weight - for - age is significantly lower than the uk reference mean . the extremes of weight - for - age are over - represented , especially underweight . weight - for - age at admission is an independent risk factor for mortality . a u - shaped association between weight and risk - adjusted mortality exists , with the lowest risk of death in children who are mild - to - moderately overweight . future studies should determine the impact of malnutrition on risk - adjusted mortality and investigate the aetiology of risk disparities with ethnicity.electronic supplementary materialthe online version of this article ( doi:10.1007/s00134 - 014 - 3381-x ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Methods Data preparation Data analysis Results Risk-adjusted mortality Subgroup analysis Discussion Conclusion Electronic supplementary material Conflicts of interest

the online version of this article ( doi:10.1007/s00134 - 014 - 3381-x ) contains supplementary material , which is available to authorized users . in this study we tested the null hypotheses that the paediatric intensive care ( pic ) population weight distribution does not differ from that of the general population and that weight - for - age at admission does not influence risk - adjusted mortality . admission weight for each child was compared to uk population reference data by calculation of a weight - for - age standard deviation score ( sds ) ; sds is the number of standard deviations by which a weight value differs from the population mean . age groups were chosen pragmatically : neonates including premature babies ( group a ) , preschool infants and children ( 1 month4.9 years , group b ) and school - age ( 518 years , group c).table 1study population characteristics and primary outcome data including details of distribution across demographic indices and weight - for - age at admission groupstotal study records , n 14,307 excluded : absent / spurious data102 excluded : readmission < 30 days1,747records analysed12,458gender m : f ( % ) 6,851:5,607 ( 55:45 ) n ( % ) age distribution a ( < 1 months)3,562 ( 29)b ( 1 months4.9 years)5,972 ( 48)c ( 518 years)2,924 ( 23 ) admission type planned : post op4,511 ( 36)planned : other913 ( 7)unplanned : post op311 ( 2)unplanned : other6,723 ( 54)mechanical ventilation in first hour9,544 ( 77 ) ethnicity white origin6,515 ( 52)black origin1,369 ( 11)south asian1,323 ( 11)other ( see supplementary appendix 1)3,251 ( 26 ) diagnostic group cardiovascular 5,137 ( 41)respiratory2,678 ( 21)neurological1,067 ( 9)gastrointestinal827 ( 7)trauma405 ( 3)infection392 ( 3)other1,952 ( 16)distribution by weight - for - age sdsweight - for - age ( sds ) n ( % ) deathssmr ( 95 % ci)logistic regressionor ( 95 % ci)<3.51,004 ( 8)841.19 ( 0.97,1.46)1.46 ( 1.101.94)3.5 to 2.511,210 ( 10)981.26 ( 1.041.53)1.46 ( 1.121.92)2.5 to 1.512,281 ( 18)1941.02 ( 0.891.16)1.18 ( 0.951.48)1.5 to 0.513,129 ( 25)2220.87 ( 0.760.98)0.98 ( 0.791.21)0.5 to 0.492,646 ( 21)2000.88 ( 0.771.00)reference0.51.491,509 ( 12)1010.82 ( 0.670.99)0.88 ( 0.681.15)1.52.49504 ( 4)280.71 ( 0.501.01)0.75 ( 0.481.16)2.53.49144 ( 1)131.14 ( 0.681.89)1.31 ( 0.712.43)>3.531 ( 0.2)20.95 ( 0.273.05)1.15 ( 0.255.2)study population weight distribution compared to uk reference populationmean weight - for - age sds ( 95 % ci)1.04 ( 1.07 to 1.01 ) p < 0.0001*standard deviation1.7median z - score weight - for - age0.93study population summary of deathstotal deaths during study period942 ( 7.5)in picu895<30 days post discharge47smr for whole study population0.94distribution of deaths , standardised mortality ratios ( smr ) and odds ratio ( or ) risk of death determined by logistic regression are provided by weight - for - age*one - sample t test comparison with population mean post cardiac bypass n ( % ) : 2,859 ( 23 ) study population characteristics and primary outcome data including details of distribution across demographic indices and weight - for - age at admission groups distribution of deaths , standardised mortality ratios ( smr ) and odds ratio ( or ) risk of death determined by logistic regression are provided by weight - for - age * one - sample t test comparison with population mean post cardiac bypass n ( % ) : 2,859 ( 23 ) the study population frequency distribution was compared to the uk reference population distribution using a one - sample t test . 1stacked histogram of study population admission weight - for - age standard deviation score compared to uk reference population . the mean weight - for - age at admission of the study population was 1.04 standard deviations less than that of the uk reference population ( 95 % ci 1.07 to 1.01 , p < 0.0001 ) , with an over - representation of outliers , especially extreme underweight . the mean weight - for - age at admission of the study population was 1.04 standard deviations less than that of the uk reference population ( 95 % ci 1.07 to 1.01 , p < 0.0001 ) , with an over - representation of outliers , especially extreme underweight . multivariable statistical analysis was undertaken using logistic regression ( stata 12 , statacorp lp , texas , usa ) to assess the relationship between the main exposure variable ( weight - for - age at admission ) , three covariates ( age , gender , ethnicity ) and the dependent variable ( death ) . 2plot of standardised mortality ratios ( smrs ) vs. weight - for - age at admission standard deviation scores , with 95 % confidence intervals . a u - shaped association is indicated : highest risk of mortality occurs with extreme under- or overweight , which this study has shown to be over - represented in children admitted to picu . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age . asterisk ( * ) indicates the overall smr for the whole study cohort is displayed on the chart at 0.94 plot of standardised mortality ratios ( smrs ) vs. weight - for - age at admission standard deviation scores , with 95 % confidence intervals . a u - shaped association is indicated : highest risk of mortality occurs with extreme under- or overweight , which this study has shown to be over - represented in children admitted to picu . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age . admission weight for each child was compared to uk population reference data by calculation of a weight - for - age standard deviation score ( sds ) ; sds is the number of standard deviations by which a weight value differs from the population mean . age groups were chosen pragmatically : neonates including premature babies ( group a ) , preschool infants and children ( 1 month4.9 years , group b ) and school - age ( 518 years , group c).table 1study population characteristics and primary outcome data including details of distribution across demographic indices and weight - for - age at admission groupstotal study records , n 14,307 excluded : absent / spurious data102 excluded : readmission < 30 days1,747records analysed12,458gender m : f ( % ) 6,851:5,607 ( 55:45 ) n ( % ) age distribution a ( < 1 months)3,562 ( 29)b ( 1 months4.9 years)5,972 ( 48)c ( 518 years)2,924 ( 23 ) admission type planned : post op4,511 ( 36)planned : other913 ( 7)unplanned : post op311 ( 2)unplanned : other6,723 ( 54)mechanical ventilation in first hour9,544 ( 77 ) ethnicity white origin6,515 ( 52)black origin1,369 ( 11)south asian1,323 ( 11)other ( see supplementary appendix 1)3,251 ( 26 ) diagnostic group cardiovascular 5,137 ( 41)respiratory2,678 ( 21)neurological1,067 ( 9)gastrointestinal827 ( 7)trauma405 ( 3)infection392 ( 3)other1,952 ( 16)distribution by weight - for - age sdsweight - for - age ( sds ) n ( % ) deathssmr ( 95 % ci)logistic regressionor ( 95 % ci)<3.51,004 ( 8)841.19 ( 0.97,1.46)1.46 ( 1.101.94)3.5 to 2.511,210 ( 10)981.26 ( 1.041.53)1.46 ( 1.121.92)2.5 to 1.512,281 ( 18)1941.02 ( 0.891.16)1.18 ( 0.951.48)1.5 to 0.513,129 ( 25)2220.87 ( 0.760.98)0.98 ( 0.791.21)0.5 to 0.492,646 ( 21)2000.88 ( 0.771.00)reference0.51.491,509 ( 12)1010.82 ( 0.670.99)0.88 ( 0.681.15)1.52.49504 ( 4)280.71 ( 0.501.01)0.75 ( 0.481.16)2.53.49144 ( 1)131.14 ( 0.681.89)1.31 ( 0.712.43)>3.531 ( 0.2)20.95 ( 0.273.05)1.15 ( 0.255.2)study population weight distribution compared to uk reference populationmean weight - for - age sds ( 95 % ci)1.04 ( 1.07 to 1.01 ) p < 0.0001*standard deviation1.7median z - score weight - for - age0.93study population summary of deathstotal deaths during study period942 ( 7.5)in picu895<30 days post discharge47smr for whole study population0.94distribution of deaths , standardised mortality ratios ( smr ) and odds ratio ( or ) risk of death determined by logistic regression are provided by weight - for - age*one - sample t test comparison with population mean post cardiac bypass n ( % ) : 2,859 ( 23 ) study population characteristics and primary outcome data including details of distribution across demographic indices and weight - for - age at admission groups distribution of deaths , standardised mortality ratios ( smr ) and odds ratio ( or ) risk of death determined by logistic regression are provided by weight - for - age * one - sample t test comparison with population mean post cardiac bypass n ( % ) : 2,859 ( 23 ) the study population frequency distribution was compared to the uk reference population distribution using a one - sample t test . the mean weight - for - age at admission of the study population was 1.04 standard deviations less than that of the uk reference population ( 95 % ci 1.07 to 1.01 , p < 0.0001 ) , with an over - representation of outliers , especially extreme underweight . the mean weight - for - age at admission of the study population was 1.04 standard deviations less than that of the uk reference population ( 95 % ci 1.07 to 1.01 , p < 0.0001 ) , with an over - representation of outliers , especially extreme underweight . multivariable statistical analysis was undertaken using logistic regression ( stata 12 , statacorp lp , texas , usa ) to assess the relationship between the main exposure variable ( weight - for - age at admission ) , three covariates ( age , gender , ethnicity ) and the dependent variable ( death ) . 2plot of standardised mortality ratios ( smrs ) vs. weight - for - age at admission standard deviation scores , with 95 % confidence intervals . a u - shaped association is indicated : highest risk of mortality occurs with extreme under- or overweight , which this study has shown to be over - represented in children admitted to picu . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age . asterisk ( * ) indicates the overall smr for the whole study cohort is displayed on the chart at 0.94 plot of standardised mortality ratios ( smrs ) vs. weight - for - age at admission standard deviation scores , with 95 % confidence intervals . a u - shaped association is indicated : highest risk of mortality occurs with extreme under- or overweight , which this study has shown to be over - represented in children admitted to picu . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age . the mean weight - for - age at admission of the study population was 1.04 standard deviations less than that of the uk reference population ( 95 % ci 1.07 to 1.01 , p < 0.0001 ) . 18 % and 1.4 % of the study population children were extremely under- or overweight ( more than 2.5 standard deviations from uk mean weight - for - age ) , respectively ( fig . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age ( table 1 , fig . the highest risk of mortality occurred in children with extreme under- or overweight ( more than 2.5 standard deviations from the uk reference mean ) . the full logistic regression model , adjusted for covariates age , gender and ethnicity , was the best fit for the data ; therefore the main exposure variable , weight - for - age at admission , and the covariates are all independent risk factors for mortality in children admitted to picu . first , young children ( < 5 years ) have markedly lower mean weight - for - age at admission than older children ( < 1 months : 1.0 , 1 months5 years : 1.2 and 518 years : 0.6 standard deviations below the uk reference population mean , respectively ) . second , south asian children have a markedly lower mean weight - for - age compared to non - south asians : 1.4 standard deviations below the uk mean vs. 1.0 , 1.0 and 1.1 for children of white , black and other ethnic origin , respectively . children 1 months4.9 years have the lowest risk of dying whilst children of south asian and other ethnicity have significantly increased risk of death compared to those of white and black origin.fig . first , in children older than 5 years of age , extremes of weight - for - age are not associated with increased risk of death ( supplementary appendix 5 ) . the lowest risk of mortality occurred in children with a weight - for - age at admission between 0.5 and 2.5 standard deviations above the uk mean weight - for - age ( table 1 , fig . the highest risk of mortality occurred in children with extreme under- or overweight ( more than 2.5 standard deviations from the uk reference mean ) . the full logistic regression model , adjusted for covariates age , gender and ethnicity , was the best fit for the data ; therefore the main exposure variable , weight - for - age at admission , and the covariates are all independent risk factors for mortality in children admitted to picu . first , young children ( < 5 years ) have markedly lower mean weight - for - age at admission than older children ( < 1 months : 1.0 , 1 months5 years : 1.2 and 518 years : 0.6 standard deviations below the uk reference population mean , respectively ) . second , south asian children have a markedly lower mean weight - for - age compared to non - south asians : 1.4 standard deviations below the uk mean vs. 1.0 , 1.0 and 1.1 for children of white , black and other ethnic origin , respectively . children 1 months4.9 years have the lowest risk of dying whilst children of south asian and other ethnicity have significantly increased risk of death compared to those of white and black origin.fig . first , in children older than 5 years of age , extremes of weight - for - age are not associated with increased risk of death ( supplementary appendix 5 ) . critically ill children have significantly lower weight - for - age than the uk average and also have an increased proportion at the extremes of weight - for - age , especially extreme underweight : 1.4 and 18 % of the study population have a weight - for - age at admission more than 2.5 standard deviations above and below the uk population mean , respectively , rather than the expected 0.6 % for each . multivariable logistic regression analysis demonstrated that weight - for - age at admission is an independent risk factor for mortality , as are age , gender and ethnicity . we observed a u - shaped association between weight - for - age at admission and risk - adjusted mortality . u - shaped interactions occur between many biological risk factors and outcome [ 26 , 27 ] , but it is of particular interest that the lowest risk of death is associated with mild - to - moderately raised , rather than mean , weight - for - age . who analysed 6,316 consecutive picu admissions in australia : a u - shaped relationship was seen with the lowest risk of death occurring at the 75th centile weight - for - age ( equivalent to sds + 1.15 ) . the mean is significantly lower and there is an over - representation of patients at the extremes of weight - for - age ; especially extreme underweight . this study has shown that weight - for - age at admission is an independent risk factor for mortality . there is a u - shaped association between weight - for - age and risk - adjusted mortality , with the lowest risk of death in children who are mild - to - moderately overweight . the over - represented extremes of weight - for - age are associated with the highest risk - adjusted mortality . future studies should determine the impact of malnutrition on risk - adjusted mortality and investigate the aetiology of risk disparities with ethnicity .

determining and characterizing past exposures to chemicals and physical agents is a vexing but essential challenge in industrial hygiene , pathology , occupational medicine and epidemiologic research . in particular , cumulative retrospective exposure assessment ( rea ) of asbestos has allowed dose response characterization of cohorts and individuals with respect to asbestosis , lung and other cancers , as well as mesothelioma . the determination of reliability and linearity of either industrial hygiene exposure estimation or asbestos lung burden analysis ( lba ) to characterize past asbestos exposures is necessary to effectively perform either cohort or case control studies and risk assessment of exposed individuals . determination of disease risk and causation for individuals also often requires reliable characterization of past exposures . exposure reconstruction is also essential in determining cumulative population background levels so that occupational exposure limits may be properly determined . risk characterization and risk assessment associated for both chrysotile and amphibole asbestos exposures remain important in risk - based decision making for contemporaneous asbestos exposures in both the community and in the workplace . even in the us and western world , low - level asbestos exposures are evidenced in ambient air , mining , quarrying , earth - moving construction , general construction and building maintenance , as well as asbestos abatement . the general purposes of this study are ( a ) to correlate asbestos rea with various forms of lba for a large number of cases from varied industries , exposure scenarios , and fiber - types ; ( b ) to evaluate the linearity , precision , reliability and applicability of both rea and lba for these different scenarios with limited asbestos exposure parameter information ; and ( c ) to demonstrate a validation procedure for rea methods . rea , also known as exposure reconstruction , is the application of well - established scientific methodologies , models and judgments to facilitate understanding of exposures and risk ( armstrong et al . , 2009 ) . although the technique has been formalized ( aiha , 2008 ; armstrong et al . , 2009 ) , associated reliability is related to the competency and experience of the industrial hygienist or exposure scientist . an exposure reconstruction , as with any model established as a scientific hypothesis , needs to be tested or validated empirically to assess the quality of the model ( armstrong et al . , 2009 ; gauch , 2003 ; hewett et al . , 2006 ; lin et al . , 2004 ; rodricks , 2011 ; vandentorren et al important criteria for industrial hygienists and exposure scientists who perform exposure reconstructions include relevant experience in the methodology , demonstrated competency and relevant field experience . the methods of exposure assessment , in general , apply to the methods and principles of rea . thus , the study of the precision , reliability and applicability of rea is also applicable to industrial hygiene exposure assessment , in general , since exposure assessment is typically not performed via contemporaneous air sampling , but involves consideration of models , historical data from similarly exposed groups and judgment ( aiha , 2008 ) . our paper , therefore , has application to assessment of industrial hygiene judgment to evaluate potential inhalation exposures with only limited information concerning details of exposure such as ventilation . other researchers have correlated estimated cumulative asbestos exposure with lung fiber burden , but not asbestos body burden . while such work has been important , the number of subjects tended to be relatively small , the cumulative exposure estimation methods are not always clearly stated , and these types of exposure estimates are sometimes based on estimated category ranges rather than the actual estimated values ( lin et al . , 2004 ; rowlands et al . , 1982 ; iwatsubo et al . , 1998 ; rdelsperger et al . , 1999 , 2001a , b ; lacourt et al . no study to date has utilized such a large dataset for analysis as is reported herein for asbestos , and the studies usually have not addressed correlation between individual rater estimates . finally , detailed analysis of the linearity of the methods and the effect of asbestos fiber - type on such correlations has not been reported . our assessment evaluated both the rea process as well as asbestos lba by the pathologist in a statistically robust manner . asbestos fiber - type issues that affect comparison between industrial hygiene exposure estimation and lba were also evaluated and explored . since all methods of characterizing past asbestos exposures have variability , it is important to estimate precision of the methods . of equal importance is to evaluate potential sources of bias between the methods . correspondingly , not only have we explored the correlation between the industrial hygiene raters and asbestos lung burden , but we also evaluated the linearity between cumulative exposure assessment and the pathology assessment methods . one of the authors ( vlr ) reviewed his consultation database to identify cases in which lung fiber analysis had been performed and for which information was available regarding occupational exposure to asbestos , including duration of exposure . cases in the database had been categorized occupationally into one of 23 categories , which included 12 industries , seven occupational categories and four non - occupational categories as previously described ( roggli et al . , 2002a ) , although categories were later simplified for statistical analysis in this publication . work history and exposure profile information was obtained upon review of medical records and other relevant documents . the latter included plaintiff s answers to interrogatories , work history sheets , work summaries or plaintiff s discovery depositions . typically , a qualitative description of exposure categorization with associated details was summarized in six words or less in the database . lung tissue was analyzed for mineral fiber content as previously described ( roggli , 2004 ) ( duke university pathology laboratory , durham , nc ) . in brief , samples were selected avoiding areas of tumor , consolidation and hemorrhage as much as possible . when whole lungs or lobes were available , samples were selected from the lung periphery to include a portion of visceral pleura . when only paraffin blocks were available , the block containing the most lung parenchyma without tumor , consolidation or hemorrhage was selected . sample weights ranged from 0.1 gram to 0.3 gram wet , with most samples being near to 0.3 gram . the residue was collected on 0.4 pore - size nuclepore filters ( ge healthcare life sciences , piscataway , nj ) , which were mounted on glass slides for asbestos body quantification by light microscopy . asbestos bodies were counted at a magnification of 200 and were distinguished from non - asbestos ferruginous bodies as previously described ( roggli , 2004 ) . results were reported as asbestos bodies per gram of wet lung tissue ( ab / g ) . filters were mounted on carbon discs for the analysis of fiber concentrations by scanning electron microscopy ( sem ) and determination of fiber - types by energy dispersive x - ray analysis as previously described ( roggli , 2004 ) . fibers 5 m or greater in length were counted at a screening magnification of 1000 , and counting was continued until 100 fields or 200 fibers were encountered , whichever came first . results were separately reported as asbestos bodies and uncoated fibers per gram ( f / g ) of wet lung tissue . for a 0.3 gram sample , the detection limit by sem was typically 440490 f / g of wet lung tissue . fibers were categorized by energy dispersive x - ray analysis as amosite , crocidolite , chrysotile , tremolite , actinolite , anthophyllite or specific non - asbestos mineral fibers . for every sample , reported concentrations of the specified mineral types were determined as distinct percentages of the total fibers present per wet gram of lung tissue . generally , for samples that yielded a non - detectable concentration , a value was not included in the analysis . a consultation database was provided by the pathologist with a brief description of asbestos exposure , the report date , age at time of report , gender and an estimate of the number of years of asbestos exposure . information was provided on 363 individuals . from that information , the approximate year of birth could be derived , which helped provide potential asbestos exposure information . names were withheld , and the pathologist s analytical data were removed prior to cumulative exposure estimation by the industrial hygiene raters . examples of asbestos exposure information provided included : painter / carpenter / maintenance man , 20 yearsbrake mechanic , 27 yearsasbestos plant worker , 27 yearsinsulator , 24 + yearsasbestos cloth weaver , 6 months and shipyard worker , 2 yearsshipyard insulator , 5 yearsjoiner , shipyard , 35 yearscarman railroad , 35 yearshousehold contact , husband , power plant worker , 29 yearspipefitter , 27 yearskent cigarettes , 2 yearshousehold contact ( husband and parents - shipyard workers ) , 2 to 3 years painter / carpenter / maintenance man , 20 years brake mechanic , 27 years asbestos plant worker , 27 years asbestos cloth weaver , 6 months and shipyard worker , 2 years shipyard insulator , 5 years joiner , shipyard , 35 years carman railroad , 35 years household contact , husband , power plant worker , 29 years kent cigarettes , 2 years household contact ( husband and parents - shipyard workers ) , 2 to 3 years the information related to the 363 cases was provided to four experienced certified industrial hygienists ( larry birkner , james rasmuson , fred boelter , william dyson ) , referred to as the raters . the raters had extensive experience estimating and measuring asbestos exposures , totaling more than 100 years , and were well experienced in industries and processes that had associated asbestos exposures . the raters were instructed to estimate the cumulative asbestos exposure in units of fiber per cubic centimeter years ( f / cc - years ) for each of the cases . they were not instructed on which method to use in developing their estimates , but utilized methods consistent with exposure reconstruction methods developed and recommended by the american industrial hygiene association ( aiha ) that are summarized in various aiha and other publications ( aiha , 2006 ; rodricks , 2011 ) . these methods include the use of ranges of measured eight - hour time - weighted average ( twa ) asbestos exposures associated with similar exposure groups ( segs ) , taking into account data quality issues , sampling times and factors that might change the applicability of measurements to a particular exposure scenario ( modification factors ) ( armstrong et al . , 2009 ) . the raters performed the rea blind with respect to other raters and blind with respect to analyzed lung burden data . either the rater best estimate or an average value between the lower and upper estimates was utilized in the analysis if a best estimate was not provided . for a number of cases , various raters indicated in their summaries that there was insufficient information to provide an exposure estimate . out of the 363 case reports , one of the exposure estimates of the 329 cases was found to be ambiguous and was therefore excluded from analysis . therefore , rater correlation was conducted for 328 or 90% of the cases provided by the pathologist . ambiguity of laboratory data for one subject caused the number to be reduced to a maximum 327 for subjects related to the statistical analysis involving correlations between asbestos lung burden and the exposure estimates . for 308 of the 327 cases , asbestos fiber - type information associated with lung fiber burden analysis was available . the consultation database contained total mineral fibers per wet gram by sem and total coated mineral fibers per wet gram by sem , besides asbestos bodies per wet gram of lung tissue . the ratios of asbestos fibers and asbestos bodies to mineral fibers and coated mineral fibers were determined from provided fiber - type analysis to transform the data into units of asbestos fibers per wet gram of lung tissue ( f / g ) and sem asbestos bodies per gram of wet lung tissue ( sem ab / g ) . histograms of the various laboratory lung burden and rater estimate values were examined and found to be distributed closer to log - normal than normal . therefore , the data were transformed logarithmically ( base 10 ) prior to statistical analysis . logarithmically transforming the data can mask relatively large arithmetic differences in large numbers and can accentuate small arithmetic differences in low values , but the transformation appears to have been necessary and helpful due to the distribution of the data and wide range of associated values . thus , for example , log10(ab / g ) is designated as ab / g . table 1.variable definitions.variable ( s)descriptiontypeunitsa , b , c , dthe a , b , c , and d variables refer to sets of individual cumulative exposure estimates for each of the four raters that were logarithmically - transformed for the 328 cases.continuouslog10 ( f / cc - years)expexp refers to the population of 328 arithmetically averaged , log - transformed exposure estimates from each rater . exp = ( a + b + c + d)/4 for each of the 328 cases for which all raters submitted an estimate.continuouslog10 ( f / cc - years)ab / glog10 transformed values of the set of linearly averaged light microscopy laboratory determinations of the number of asbestos bodies per wet gram of lung tissue for each case for each of 327 cases.continuouslog10 ( ab / g)sem ab / glog10 transformed values of the set of linearly averaged sem laboratory determinations of the number of asbestos bodies per wet gram of lung tissue . analytical data was available for 277 out of the 327 cases.continuouslog10 ( sem ab / g)f / glog10 transformed values of the set of linearly averaged sem laboratory determinations of the number of asbestos fibers longer than 5 per wet gram of lung tissue for each case for 308 overlapping cases with the 327 cases.continuouslog10 ( f / g)laba combination variable of ab / g , sem ab / g , and f / g that was transformed into the same units as ab / g . more on this variable is found in the discussion , below , and in table 2.continuouslog10 ( ab / g)etthe data set was further reduced from the original categorization into 11 exposure types ( or categories ) for analysis of covariance ( ancova ) ( rosner , 2006 ) . the main asbestos exposure types were based on occupation , but other categories such as household exposure and the type of asbestos product from which exposures occurred were also utilized . these exposure type ( et ) categories are defined in figure 4 , along with numbers of associated cases.classsingle integers ( dummy variable)ftfor 308 of the 327 cases , asbestos fiber - type information associated with lung fiber burden analysis was available . these four categories are defined in figure 4 , along with numbers of associated cases for each category , which allowed correlation by ancova.classsingle integers ( dummy variable ) variable definitions . when logarithmic values of two variables are correlated with a slope of 1 , when the slope is different from 1 , a non - linear relationship exists between the arithmetic values . therefore for purposes of our analysis , relationships between variables are defined as linear if no significant differences exist from a slope of 1 for the correlation . in order to determine the population estimates of the variable , lab ( a combined laboratory result variable defined in table 1 ) , relationships were determined between ab / g and both sem ab / g and f / g based on regression analysis and ab / g equivalence was calculated for sem ab / g and f / g . table 2.correlation of ab / g as a function of ( sem ab / g ) and f / g . coefficients were utilized to calculate lab , a combined function of ab / g , sem ab / g and f / g , all in the same units of ab / g.independent variablen ( all available lab analysis ) from original data setcorrelation coefficient ( r)slope ( standard error)intercept ( standard error)sem ab / g2770.911.17 ( 0.031)1.21 ( 0.13)f / g3410.871.29 ( 0.040)2.72 ( 0.18 ) correlation of ab / g as a function of ( sem ab / g ) and f / g . coefficients were utilized to calculate lab , a combined function of ab / g , sem ab / g and f / g , all in the same units of ab / g . identical units of log10 ( ab / g ) were thus obtained for all three lung burden analysis by converting sem ab / g values and f / g determinations to estimates of ab / g utilizing the slopes and intercepts found in table 2 . the conversions were performed with more significant figures than the rounded values shown in table 2 , as is also true of all statistical tests that were performed . lab was then determined for each of the available cases by averaging the converted values of ab / g ( no conversion needed for this variable ) , sem ab / g equivalence and f / g equivalence ( all log - transformed ) based on data that was available for each of the three methods . for some cases , lab was a function of one or two of the laboratory method results , instead of all three . although both sem ab / g and f / g had significantly different slopes from one , as shown in table 2 , indicating some degree of non - linearity between the two variables and ab / g , another test of linearity is the regression between the laboratory variables and exp ( defined in table 1 ) , with and without adjusting for confounding factors like asbestos fiber - type . correlation between raters and the laboratory analysis were determined by both regression and ancova analysis . ancova allows comparison of the values of various lung burden - dependent variables at constant exp values for the different exposure classes . the intraclass correlation coefficient ( icc ) was determined to further evaluate reproducibility of the exposure reconstructions performed by the raters and the overall degree of reliability ( rosner , 2011 ) . rea estimates associated with each subject , obtained from each of the participating experienced industrial hygienists were treated as replicate measures of the exposures associated with the same subject for our analysis . the icc was also calculated for each of the laboratory determinations of asbestos body ( ab / g and sem ab / g ) and fiber burden ( f / g ) lung tissue analysis . the icc is a statistical measure used to evaluate the reproducibility of measured or estimated continuous variable values such as replicate analytical determinations and has also been applied to evaluate continuous variable rater determinations . the icc was calculated as follows with log - transformed values : where = intraclass correlation coefficient , \usepackage { } \begin{document } $ $ & bsolhsub;def&bsolhsub;newpage{&bsolhsub;vfill & bsolhsub;break } & bsolhsub;nopagenumbers & dollar ; & bsolhsub;sigma _ b^2 & dollar ; & bsolhsub;newpage & bsolhsub;end $ $ \end{document } is the between sample variance and \usepackage { } \begin{document } $ $ & bsolhsub;def&bsolhsub;newpage{&bsolhsub;vfill & bsolhsub;break } & bsolhsub;nopagenumbers & dollar ; & bsolhsub;sigma _ w^2 & dollar ; & bsolhsub;newpage & bsolhsub;end $ $ \end{document } is the within replicate sample variance . as \usepackage { } \begin{document } $ $ & bsolhsub;def&bsolhsub;newpage{&bsolhsub;vfill & bsolhsub;break } & bsolhsub;nopagenumbers & dollar ; & bsolhsub;sigma _ w^2 & dollar ; & bsolhsub;newpage & bsolhsub;end $ $ \end{document } approaches zero , approximates 1 and as \usepackage { } \begin{document } $ $ & bsolhsub;def&bsolhsub;newpage{&bsolhsub;vfill & bsolhsub;break } & bsolhsub;nopagenumbers & dollar ; & bsolhsub;sigma _ w^2 & dollar ; & bsolhsub;newpage & bsolhsub;end $ $ \end{document } increases , approximates 0 . typically , < 0.40 indicates poor reproducibility , 0.4 < 0.75 indicates fair to good reproducibility , and > 0.75 indicates excellent reproducibility ( rosner , 2006 ) . in addition , the spearman brown correction factor ( wuensch , 2010 ) was applied to the calculated rater icc to estimate the reliability and reproducibility of the log - averaged exposure - estimates : where = intraclass correlation coefficient and j is the number of raters . similarly , for the analytical pathology results , where replicate measures were reported for distinct ab and fiber burden samples , an analysis of reproducibility was performed by calculating associated icc values . to determine whether increasing the number of variables was responsible for increasing correlation as class variables were added , ancova analysis was performed with the class variable integers randomized from a random number generator for the bounded ranges of the variables for the various analysis . the randomization caused ancova r - values to approach that of the correlation associated with simple regression . in addition , unadjusted r - values were compared with adjusted r - values ( that computationally compensated for the additional number of variables ) . differences in r no greater than about 0.01 were found except for the ancova analysis that utilized 15 class variables . in that instance , a change in r of about 0.015 was found . verification of all regression , ancova , and p value determinations was accomplished generally by a second statistician who utilized statistica 9.1 software ( statsoft , tulsa , ok ) . for all tests of significance , two - sided 95% confidence interval tests were performed unless otherwise noted . tests for normality of residual error distribution associated with the various correlations were determined with the wilk figure 1 illustrates a sample correlation plot involving the regression of the logarithm of an individual rater s exposure estimates with the individual exp values for which the r - value was 0.94 , the slope was 0.90 and the intercept was 0.13 . similar plots for the other three raters had r - values varying between 0.90 and 0.93 ( 0.90 , 0.93 and 0.93 ) . the 95% confidence interval ( variation of mean estimate ) is shown with the inner dotted lines , and the 95% prediction interval ( 95% variation of individual data points from the mean estimate ) is shown by the outer dotted lines in figure 1 . the relatively narrow confidence interval for the relationship is due in part to the large n associated with this analysis . ideally , the regression plots such as that shown in figure 1 would have a slope not significantly different from one and an intercept not significantly different from zero if there was only random variability between raters across all exposure levels and categories . were not far from one , three of the four slopes were significantly different from one . in addition , while the intercepts were not far from zero , they varied from 0.04 to 0.21 ( 0.21 , 0.13 , 0.04 and 0.21 ) and three were significantly different from zero . individual comparisons between all of the four raters on an individual basis ( six comparisons , based on the potential number of comparison combinations ) yielded a range of r - values between 0.76 and 0.90 ( 0.76 , 0.79 , 0.80 , 0.83 , 0.87 and 0.90 ) . figure 1.correlation of a rater s exposure estimates with exp . figure 2 demonstrates the histogram and a probit plot of the residual error for the figure 1 regression , along with numerical values for the quantiles associated with the probit plot . first , since the regression was performed on logarithmically - transformed data , it can be seen that the residual error approximates a log - normal distribution , but is not statistically verified ( p < 0.0001 ) . the quantile data indicate that for the upper and lower 5% and 95% bounds , the rater estimate is within an approximate ( arithmetic rather than logarithmic ) factor of three of the mean estimated data of all four raters , and 50% of the comparisons are within a factor of 1.5 . figure 2.residual error histogram with probit plot and quantile data for regression of a sample rater s exposure estimates with exp . residual error histogram with probit plot and quantile data for regression of a sample rater s exposure estimates with exp . further analysis between the log - transformed rater estimates yielded excellent agreement and reliability according to established guidelines ( rosner , 2006 ) with an icc - value of 0.76 , not far from the upper bound of the very good range ( icc = 0.75 ) . the four - rater average log exposure estimates were found to have an adjusted spearman brown icc of 0.92 , indicating excellent reliability . collectively , the rater estimates ( exp function ) had an icc value that were in the same range as that of the laboratory pathology determinations . this demonstrates and verifies the reliability of rater panels in favor of individual exposure assessments although useful exposure assessments may be completed by a single , experienced and qualified industrial hygienist . fiber burden analysis within the duke university pathology laboratory resulted in icc values above 0.75 , indicating excellent reliability and reproducibility as shown in table 3 . as can be seen from the data summary , additional cases besides those evaluated by the raters were utilized to calculate icc statistics for the pathology analysis . table 3.intraclass icc for asbestos bodies and fiber burden analysis.data setcorresponding variablenumber of casescases with replicatesreplicates per caseiccasbestos bodies in lung tissue : light microscopyab / g36316620.91asbestos bodies in lung tissue : semsem ab / g1249 * 5420.90free asbestos fibers in lung tissue > 5 by semf / g1249 * 6720.88*additional data were obtained from the pathologist for the purpose of calculating the laboratory iccs . additional data were obtained from the pathologist for the purpose of calculating the laboratory iccs . regressions of ab / g , sem ab / g , f / g and lab as a function of exp yielded the statistics shown in table 4 . table 4.correlation of the logarithm of various lung asbestos body and fiber burden determinations with average log of rater estimates ( exp).regression dependent variableoverall significance r slopeab / g p < 0.00010.651.22 ( significantly different from 1 , p = 0.004)sem ab / g p < 0.00010.650.87 ( barely significantly different from 1 , p = 0.040)f / g p < 0.00010.660.81 ( significantly different from 1 , p = 0.0004)lab ( normalized to ab / g units ) p < 0.00010.671.13 ( barely significantly different from 1 , p = 0.045 ) correlation of the logarithm of various lung asbestos body and fiber burden determinations with average log of rater estimates ( exp ) . table 4 indicates that each of the specified analytical methodologies is a good predictor of asbestos exposure within the indicated precision . slope values are slightly above and slightly below one , indicating reasonable linearity with estimated exposure estimates ( exp ) , but improved linearity can be obtained by compensating for fiber - type , as demonstrated below . as lab provided a slightly better correlation than the other three dependent variables that were tested , and it appeared to provide the most linearity , it was selected as the primary dependent variable to be studied further by ancova methods . nevertheless , the data appear to indicate that the light microscopy asbestos body determination generally yields results about as reliable as combining the three determinations into the single lab variable . as expected , lower correlation is found between lab and exp than for either laboratory repeatability studies or rater comparison correlations , e.g. the correlation demonstrated in figure 1 , because of variability in breathing rates associated with various occupations , varying fiber types , ages of exposure , individual fiber retention , individual efficiency of asbestos body production and perhaps other factors . some of these sources of variability can be evaluated and compensated by introduction of the class variables defined in table 1 , such as et ( related to breathing rate and asbestos fiber - type ) and ft determined by lung fiber burden analysis . this is demonstrated in the analysis described further . nevertheless , reasonable correlation is found ( r = 0.67 ) based on the large degree of potential variability associated with these potential confounders , and the correlation is significant ( p < 0.0001 ) . however , not taking into account all sources of variability , allows only explanation of 45% ( r ) of the variability , and 55% of the variation in the data remains unexplained . this variation is apparently largely related to the factors described above , given the excellent correlation between separate raters and the excellent correlation between replicate laboratory analysis . some of the variability in figure 3 is likely caused by fiber - type percentage differences between various asbestos formulations utilized in varying occupations , varying associated breathing rates and differing times between exposure and lba . the exposure class variable , et , defined in table 1 was added to study some of these effects , primarily asbestos fiber - type ( but also average breathing rate differences between various occupations and situations ) . for example , a shipyard insulator was typically exposed to a significant proportion of amosite while an automobile mechanic s exposures ( at least while performing automobile work ) typically involved chrysotile ( friction products and gaskets ) . therefore , a much higher percentage of inhaled asbestos fibers ( and resultant asbestos bodies ) would be expected to be retained in the lung for the shipyard insulator compared with the automobile mechanic , because of the much shorter half - life of chrysotile in the human lung ( weeks or months ) compared with amphibole asbestos ( decades , if not a lifetime ) ( roggli , 2004 ; churg , 1998 ; craighead & gibbs , 2008 ) . for any given estimated cumulative exposure ( exp ) , ancova provides a tool to examine the magnitude of how lab varies as a function of the various et classes , including the fiber - type . figure 4 illustrates the ancova correlation , a type of simultaneous regression , when the exposure type ( et ) categories are added to the correlation as shown in figure 3 . somewhat overall improved correlation was found ( r = 0.72 compared with the r = 0.67 associated with figure 3 ) . the correlation lines shown in figure 4 for the various occupational class variables are parallel because the interaction term in the ancova model between exp and et was not significant ( p = 0.37 ) and was therefore not included in the analysis . however , the correlation as shown in figure 4 had an associated p - value of < 0.0001 , and the class variable term had an associated p - value of 0.0005 . therefore , figure 4 shows lab as a function of the continuous variable , exp and the class variable , et , but the function does not include the product of exp and et ( exp*et ) . of importance , the slope of the parallel lines in figure 4 is not significantly different from one ( 0.96 , p = 0.66 ) . however , when the correlation lines are not forced to be parallel , the categories involving extensive amphibole asbestos exposure such as shipyard insulators are steeper than the slope for other categories , and the slope for the automobile and friction product work is relatively flat , because chrysotile fibers are not easily converted to asbestos bodies in the lung , and chrysotile fibers , for the most part , are not retained in the lung ( roggli & sharma , 2004 ) . figure 4.ancova correlation of lab as function of the continuous variable exp and the class variable et , no interaction term . shipyard insulator ( n = 10 ) , 2 . power plant worker ( n = 7 ) , 3 . secondary household exposures ( n = 18 ) , 7 . miscellaneous and mixed trade ( n = 72 ) , 8 . construction , piping , and boiler trades except shipyard ( n = 63 ) , 9 . building occupant ( n = 5 ) , 10 . refinery workers ( n = 6 ) , and 11 . automobile and friction product related ( n = 11 ) . colors of data points correspond to the colors of the parallel lines . ancova correlation of lab as function of the continuous variable exp and the class variable et , no interaction term . shipyard insulator ( n = 10 ) , 2 . power plant worker ( n = 7 ) , 3 . secondary household exposures ( n = 18 ) , 7 . miscellaneous and mixed trade ( n = 72 ) , 8 . construction , piping , and boiler trades except shipyard ( n = 63 ) , 9 . building occupant ( n = 5 ) , 10 . refinery workers ( n = 6 ) , and 11 . automobile and friction product related ( n = 11 ) . colors of data points correspond to the colors of the parallel lines . the order of the exposure categories ( parallel lines ) is shown in the caption of figure 4 , from highest equivalent asbestos body concentration per unit of cumulative asbestos exposure to the lowest . as might be expected , the shipyard insulator , shipyard worker , power plant worker and insulator categories indicate relatively high lab values as a function of exp compared with some of the other asbestos exposure categories . this is especially true when comparing the parallel line associated with likely automobile and friction product chrysotile exposures with the apparently amphibole asbestos - exposed classes except for refinery workers . the standard errors associated with statistical parameters of figure 4 are shown in table 5 , from which significant differences between the class plot lines can be derived . for example , there is a significant difference between each of the upper four amphibole asbestos classes and the automobile / friction product class based on a two - sided t - test with unequal variances assumed for each of the classes . additionally , a one - sided t - test shows significance between the automobile / friction product class and each of the secondary household , miscellaneous and mixed trades and the construction trade classes . expectedly , there is no difference between the building occupant class and the automobile / friction product class . unexpectedly , there is also no difference between the refinery workers class and the automobile / friction product class , but an n of six is relatively small for the refinery workers , and the exact nature of the work that they performed is unknown . table 5.standard errors associated with the data presented in figure 4.exposure type ( et ) or ( parameter)intercept or ( slope)standard error n ( overall slope)(0.957)0.098shipyard insulator3.070.4410power plant worker2.400.467asbestos insulator2.210.3641shipyard worker2.170.3089asbestos manufacturing , except friction products1.980.515secondary household exposures1.950.3618miscellaneous and mixed trade1.830.3072construction , piping , and boiler trades , except shipyard1.820.3163building occupant1.640.515refinery workers1.190.486automobile and friction product related1.070.2811 standard errors associated with the data presented in figure 4 . similar results as those found in figure 4 were found if the other variables , ab / g , sem ab / g or f / g were substituted for lab as the dependent variable in the ancova analysis . however , with the exception of ab / g , an overall slope significantly lower than one was found , with slightly reduced correlation ( r - values of 0.68 and 0.69 for sem ab / g and f / g , respectively ) . the slope for ab / g was 1.04 , and the correlation coefficient was 0.70 . to further test the hypothesis that fiber - type ( ft ) was a significant cause of variability in the correlation of exp estimates with lab , ancova was performed with ft as a class variable . the results are shown in figure 5 , along with various statistics and p values in table 6 . the overall correlation improved to r = 0.75 . in this instance , the interaction term between exp and ft ( exp*ft ) was significant ( p = 0.0001 ) , with p values for ft and exp equal to 0.0005 and < 0.0001 , respectively . as expected , where significant amphibole asbestos was involved in the exposures ( ft class variable 1 ) , the slope was not significantly different from one , but was overall significantly different from zero with p < 0.0001 . the majority of data points ( 216 out of 321 available fiber - type analysis ) were associated with ft class 1 . when significant amphiboles were present in ft class 2 , but apparently not as significant for ft class 1 , the slope was lower at 0.68 , but still not significantly different from one and also different from zero . for ft classes 3 and 4 , relatively flat correlation lines ( slopes of 0.33 and 0.27 ) were found , with slopes significantly different from one , but not different from zero . these slopes were probably not different from zero since the predominant asbestos fiber - type associated with exposure was often likely to have been chrysotile for ft , classes 3 and 4 . however , when the database was examined for classes 3 and 4 , little , if any , chrysotile was still present in the lungs for individual subjects at the time of analysis . moreover , even for relatively high chrysotile exposures such as those associated with asbestos insulators , only six of 49 insulator cases had detectable remaining lung chrysotile fibers . other typically non - commercial ( north america ) asbestos fiber types such as tremolite , actinolite and anthophyllite often remained in the lungs of the cases associated with ft classes 3 and 4 , but apparently these asbestos fibers were not proportionally related to the primary asbestos exposures associated with the cases , based on the ancova results represented in figure 5 . figure 5.lab as function of exp and fiber for four fiber type classes ( with interaction ) . 1 . commercial amphibole asbestos , majority fiber type , no chrysotile ( + ) , n = 216 2 . commercial amphibole asbestos , majority fiber type , chrysotile detected ( x ) , n = 30 . 3 . amosite and/or crocidolite detected , but not majority asbestos fiber - type ( o ) , n = 35 . 4 . no crocidolite or amosite detected ( ) , n = 40 . lab as function of exp and fiber for four fiber type classes ( with interaction ) . 1 . commercial amphibole asbestos , majority fiber type , no chrysotile ( + ) , n = 216 2 . commercial amphibole asbestos , majority fiber type , chrysotile detected ( x ) , n = 30 . 3 . amosite and/or crocidolite detected , but not majority asbestos fiber - type ( o ) , n = 35 . 4 . no crocidolite or amosite detected ( ) , n = 40 . in figure 5 , the difference in slopes for ft classes 1 and 2 were significant with a one - sided t - test , but were marginally significant with a two - sided test ( p = 0.0545 ) . the slope for class 1 was significantly different from the slopes associated with ft classes 3 and 4 . the only exposure type ( et ) of figure 4 that clearly and predominantly represented chrysotile exposures is represented by et class 11 , asbestos exposures related to automobile maintenance and friction product manufacture . if the hypothesis that the relatively flat relationships observed for ft classes 3 and 4 in figure 5 are because of significant chrysotile exposure as a part of the exp variable , a regression of lab as a function of exp for only values in et class 11 should yield a relatively flat response , with little correlation . indeed when this was done , an r - value of only 0.01 was found with a slope of 0.01 , essentially flat , but with a standard error of 0.46 for the slope since the exp estimates varied broadly ( relating to exposures varying between parts delivery , automobile mechanics , brake mechanics and the manufacture of friction products ) . since lung asbestos fiber burden is often utilized instead of asbestos body analysis or the two are used in conjunction to characterize past subject asbestos exposures , the ancova correlation demonstrated in figure 5 was also performed separately for ab / g , f / g and sem ab / g as dependent variables . this was done to evaluate whether the pathological markers of exposure behaved similarly with increasing asbestos exposure and to determine the degree of linearity of the response . comparison results for the three dependent variables with an interaction term ( exp*ft ) that has statistical significance ( which allows slope comparisons for the four ft classes ) are summarized in table 6 , and a comparison of both slope and intercepts for the ab / g and f / g analysis are shown in figure 6 . table 6.comparison of ancova correlation with exp and exp*ft for each of the dependent variables , lab , ab / g , and f / g.dependent variablerslope 1 ( std error)diff from 1 ? ( p)lab0.751.11 ( 0.085)0.68 ( 0.20)0.34 ( 0.25)0.27 ( 0.21)no ( 0.21)no ( 0.11)yes ( 0.009)yes ( 0.0006)yes ( < 0.0001)yes ( 0.0007)no ( 0.18)no ( 0.20)ab / g0.751.06 ( 0.094)0.66 ( 0.22)0.42 ( 0.28)0.54 ( 0.23)no ( 0.49)no ( 0.12)yes ( 0.036)yes ( 0.046)yes ( < 0.0001)yes ( 0.0027)no ( 0.13)yes ( 0.022)f / g0.700.88 ( 0.069)0.58 ( 0.16)0.20 ( 0.20)0.20 ( 0.20)no ( 0.095)yes ( 0.0086)yes ( < 0.0001)yes ( < 0.0001)yes ( < 0.0001)yes ( 0.0003)no ( 0.32)no ( 0.31 ) comparison of asbestos body and asbestos fiber burden ancova correlations . comparison of ancova correlation with exp and exp*ft for each of the dependent variables , lab , ab / g , and f / g . as can be seen from table 6 , somewhat similar results are found , regardless of whether the dependent variables ab / g , f / g , or the combination variable , lab is employed . for example , for the case of the dependent variable , ab / g , the slope for the first fiber - type category was 1.06 , not different from one , and r was 0.75 , the same value as that associated with the lab dependent variable shown in figure 5 . when sem ab / g was utilized as the dependent variable , the variability of the slopes increased , and the interaction term associated with defining slope values lost significance ( p = 0.07 ) . / g as the dependent variable , somewhat similar results as those associated with figure 5 were found , with r = 0.70 , and the slopes for categories 1 and 2 , not significantly different from one . significant differences between the slopes for the ab / g and f / g - ancova plots summarized in table 6 were similar to , but not precisely the same as that summarized above for the data in figure 5 . when ab / g was the dependent variable , differences between slope 1 and each of slopes 3 and 4 were observed , but a difference between slopes 1 and 2 was not as clear with a one - sided t - test p value of 0.051 . as before , differences between slopes 2 , 3 , and 4 were not observed . there are some differences in the residual error distribution of the analysis , which can be explained by differences in individual tendency to coat asbestos fibers in the lung to produce asbestos bodies ( figure 7 ) . as can be seen for a number of cases , the number of ab / g is under predicted , based on ft and exp . this is most likely related to the known phenomenon that some individuals tend to coat a much higher percentage of asbestos fibers than others ( roggli & sharma , 2004 ) . thus , the f / g residual deviation ( log ( f / g ) ) appears normally distributed . based on our data , although a slightly better correlation with exp and ft is found for ab / g , f / g may be a more reliable indicator of past amphibole asbestos exposure , because of coating differences . for example , the histogram for f / g in figure 7 implies that lower and upper 5% and 95% bounds are within about a factor of 13 and 11 of the mean values on an arithmetic basis . although the error distribution between raters appears to be smaller , if equal variation is attributed to both the lab analysis and the rater estimates , the data represented by each of the two methods is within about a factor of 8.5 of mean estimates in the correlation . additionally , the combination variable lab provides equal correlation as ab / g , but also includes and provides a compromise between the better normality found for sem ab / g and f / g . figure 7.residual error histograms for f / g and ab / g as a function of exp , ft and exp*ft . residual error histograms for f / g and ab / g as a function of exp , ft and exp*ft . when ancova was performed for lab as a function of exp with both of the et and ft class variables included , as well as the exp*ft interaction term , an r value of 0.77 was determined with significance found for exp ( p < 0.0001 ) , et ( p = 0.0001 ) , ft ( p < 0.0001 ) and ft*exp ( p = 0.0042 ) . an r - value of 0.77 implies that 60% of the variability in the dependent variable is accounted or explained although the correlation was not dramatically improved from simpler models , and a portion of the improvement could be attributed to the increase in the number of variables . our data suggest that industrial hygiene estimates of past asbestos exposures can be effectively performed , even with limited information based on self - reporting and is approximately linear with dose . fundamentally , there is no difference between an rea for past chrysotile exposures than for past amphibole asbestos exposures . thus , although correlation between only amphibole asbestos and rea was found , but no such correlation was found for chrysotile exposures , we believe that chrysotile exposure estimation by experienced industrial hygienists can be performed as reliably as for amphibole asbestos . in addition , our data suggest that lung asbestos body and asbestos fiber burden analysis is a valid and linear relative indicator of past cumulative amphibole fiber exposure . earlier comparative studies have indicated that industrial hygiene judgment and individual - reported asbestos exposure histories combine to provide the most effective exposure reconstruction process and that this process is superior to sole utilization of job exposure matrices ( gramond et al . , 2012 ) . thus , because the information provided for the exposure assessment had the charateristics of a job exposure matrix although occupation and exposure duration have elements of self - reporting , better inter - rater correlation and improved rater correlation with asbestos lung burden would have been expected if the raters had more detailed exposure information . nevertheless , this study suggests that when industrial hygienists with sufficient competency and experience perform the assessment , the measured precision generally appears better than inter and intra - worker exposure variance within an seg based on individual air samples ( kromhout et al . , 1993 ) . this is related to the phenomenon that the confidence interval of long - term seg data typically has a known or implied long - term variance that is considerably less than worker - to - worker or day - to - day variability . our data also supports the hypothesis that location - specific air sampling is not required to reasonably estimate exposures ( roggli & sharma , 2004 ) . assuming adequate sampling of lung tissue , our data also suggest that asbestos body determination by light microscopy yields useful information regarding past asbestiform amphibole exposures , comparable to the more elaborate asbestos fiber burden determination by electron microscopy , which also primarily is an indicator of past amphibole asbestos exposure . chrysotile , while having apparent lower potency with respect to mesothelioma than amphibole asbestos , can increase risk of asbestosis and remains classified as a human carcinogen . chrysotile exposures are of particular current public health interest because of continued world production of the mineral , especially in the former soviet union , brazil and china , and utilization of asbestos products , particularly in construction materials such as roofing products throughout much of the developing world . there is no effective substitute for industrial hygiene estimation of past cumulative chrysotile exposures , in most instances ( consensus report , 1997 ) ; thus , validation of cumulative asbestos exposure estimates is critical . tremolite fibers , which are a common amphibole ( but not necessarily an asbestiform fiber ) contaminant of chrysotile , have been suggested to be a reasonably good marker of past chrysotile exposure for miners ( roggli et al . , 2002b ) . however , estimated cumulative chrysotile exposure has been only poorly correlated with chrysotile fiber ( r = 0.40 ) and tremolite fiber lung burdens ( r = 0.49 ) for chrysotile miners and millers ( rowlands et al . , 1982 ) . while our analysis does not include miner and miller exposures , it does include manufacturing and end - user exposures that have been typical to north america . pathologists continue to characterize past chrysotile exposures via lung fiber burden analysis , but most of the chrysotile does not persist in the lung and only a small percentage results in asbestos body formation . even for amphibole asbestos exposures , the linearity and correlation of asbestos fiber and asbestos body concentrations in the lung with each other and with respect to past cumulative exposure has been questioned , and the degree of correlation may depend on ratios of amosite to crocidolite and whether the subject has asbestosis ( roggli , 2004 ; roggli et al . , 2002a ) . in the past , often such correlation attempts have involved total ferruginous bodies with total lung fiber burden data without differentiation of whether the ferruginous bodies are asbestos bodies or whether the lung fiber burden data is with respect to all fibers or to asbestos fibers . however , our study suggests that the various lung burden indicators appear to be linear with total amphibole asbestos exposure , which has not been previously demonstrated , although asbestos bodies have been shown to be a relatively good marker of past exposure to long amphibole fibers ( roggli , 2004 ) . although our data indicate a very good to excellent correlation between industrial hygienists estimate of lifetime cumulative exposure and the asbestos lung burden data , in general , based on the determined icc , factors such as individual variable fiber retention , individual variable fiber coating efficiency and varying time between exposure and laboratory analysis , as well as site - to - site variation of fiber content within the lung help explain the remaining significant unexplained variance . it must be remembered that an r - value of 0.75 , shown in table 6 , still leaves 44% of the variability unexplained , but some of the unexplained variability is related to exposure variability within segs and the limited exposure history information provided . in terms of measuring lung fiber content by phase contrast light microscopy , morgan & holmes ( 1983 , 1984 ) reported as much as a five to ten fold site - to - site variation . furthermore , there is interlaboratory variation depending upon methodological factors , which include the digestion procedure , recovery procedure , analytical procedure and reporting of results ( roggli , 2004 ) . such variability has been documented in an international inter - laboratory counting trial reported by gylseth et al . the purpose of lung fiber and asbestos body burden analysis is to determine the cumulative asbestos fiber content of an individual s lungs at the time that the tissue sample was obtained . this will differ from an individual s lifetime cumulative inhalational exposure in relation to the clearance of fibers from the lung . for amphibole fibers , the cumulative exposure correlates with the lba because the half - life for clearance of amphiboles is prolonged ( on the order of decades ) ( churg , 1998 ; roggli , 2004 ) . strictly speaking , a high level of agreement between the raters does not necessarily indicate high accuracy . however , the linearity of the exposure estimates with the lung burden data varying over almost four orders of magnitude and the approximate linearity and agreement between various raters suggests that good or at least reasonable accuracy was demonstrated in the exposure estimations . in addition , the relationships based on our exposure assessment are also consistent with estimated cumulative background asbestos levels in the united states ; extrapolating our data from the relationship between ab / g and exp to the range of 020 ab / g yields estimates that are reasonably consistent with previous estimates of potential ranges of cumulative lifetime asbestos background exposures ( atsdr , 2001 ) . examples of potential applications of our study include estimation of the relationship between workplace and related household asbestos exposures , relative workplace - to - workplace and/or job - to - job comparisons , estimation of historical general population background exposures in units of f / cc - years based on pathology analysis of non - occupationally exposed individuals and determination of asbestos risk assessment potency factors ( for example km in mesothelioma risk assessment procedures ) from cohort lung burden analysis when little or no air monitoring data is available , or when asbestos lung burden data is available to validate historical air monitoring data . application of the procedures described here , however , will have to include the inherent variability as measured in this publication , and estimation of exposure or dose - response parameters should be determined as ranges with confidence limits . another application of our study is underscored by the observed non - linear dose - response relationships observed in both meta - analysis of cohort studies for lung cancer and mesothelioma ( hodgson & darnton , 2000 ; berman & crump , 2008 ) as well as for case - control studies for mesothelioma ( iwatsubo et al . , 1998 ; lacourt et al . , 2010 ; rdelsperger et al . , 2001b however , a linear dose - response for mesothelioma was observed when asbestos fiber lba was employed to estimate relative exposures between cases and controls where amphibole asbestos was hypothesized to be the primary risk factor , and crocidolite appeared to be the primary asbestos amphibole ( rdelsperger et al . , 1999 ) . thus , the observed linearity of lba with estimated exposure for amphibole asbestos in our study adds credence to a linear dose - response relationship between mesothelioma and amphibole asbestos exposure at relatively low exposure levels . in addition , lba apparently can provide an objective means to eliminate recall bias in case - control studies . finally , exposure assessment ( including rea ) training represents an important curriculum for industrial hygienists . a portion of the consultation database has been used by the authors as a resource in a class exercise at the conclusion of american industrial hygiene association ( aiha ) professional development classes ( pdcs ) on exposure reconstruction . demonstration of competency in rea by industrial hygiene and pathology comparison will improve accuracy and reliability of estimates even when qualitatively characterizing past exposures . overall , our data suggest that past cumulative asbestos exposure estimations can be performed reliably by experienced industrial hygienists . naturally , there is some variability in both the lba procedure and the industrial hygiene estimation process , as demonstrated in our analysis . fifty - six percent of the variability between the correlation of industrial hygiene estimates and pathology determinations is explained , when a class variable accounting for variable fiber type is included in the correlation , but higher correlation was found between industrial hygiene raters , as demonstrated by determined icc values . the reasons for the unexplained variability between industrial hygiene estimations and laboratory lung burden analysis include varying times between exposure and fiber burden analysis for the various cases and individual differences in fiber retention and fiber coating tendency . industrial hygiene panels and utilization of empirical data rather than use of anecdotal information can potentially further increase reliability and precision . however , our current data also suggest that lung asbestos body and amphibole fiber burden analysis is a valid and linear relative indicator of past cumulative asbestiform amphibole fibers . the light microscopy determination of lung tissue asbestos bodies appears to be almost as good an indicator of past asbestiform amphibole fiber exposures as electron microscopy determination of asbestos fiber lung burden . lba appears to be a satisfactory relative indication of past asbestiform amphibole fiber exposures for case - control studies , and industrial hygiene reconstructed exposure estimates can also be satisfactorily utilized . our research does not settle the issue of whether asbestos disease dose - response relationships are linear or non - linear , but provides additional information for further research . all authors consult with law firms and have testified as experts in cases involving asbestos . no funding from any outside sources was provided for any aspect of this research , design of the study , analysis of the data or preparation of the manuscript .

contexta detailed evaluation of the correlation and linearity of industrial hygiene retrospective exposure assessment ( rea ) for cumulative asbestos exposure with asbestos lung burden analysis ( lba ) has not been previously performed , but both methods are utilized for case - control and cohort studies and other applications such as setting occupational exposure limits.objective(a ) to correlate rea with asbestos lba for a large number of cases from varied industries and exposure scenarios ; ( b ) to evaluate the linearity , precision , and applicability of both industrial hygiene exposure reconstruction and lba ; and ( c ) to demonstrate validation methods for rea.methodsa panel of four experienced industrial hygiene raters independently estimated the cumulative asbestos exposure for 363 cases with limited exposure details in which asbestos lba had been independently determined . lba for asbestos bodies was performed by a pathologist by both light microscopy and scanning electron microscopy ( sem ) and free asbestos fibers by sem . precision , reliability , correlation and linearity were evaluated via intraclass correlation , regression analysis and analysis of covariance . plaintiff s answers to interrogatories , work history sheets , work summaries or plaintiff s discovery depositions that were obtained in court cases involving asbestos were utilized by the pathologist to provide a summarized brief asbestos exposure and work history for each of the 363 cases.resultslinear relationships between rea and lba were found when adjustment was made for asbestos fiber - type exposure differences . significant correlation between rea and lba was found with amphibole asbestos lung burden and mixed fiber - types , but not with chrysotile . the intraclass correlation coefficients ( icc ) for the precision of the industrial hygiene rater cumulative asbestos exposure estimates and the precision of repeated laboratory analysis were found to be in the excellent range . the icc estimates were performed independent of specific asbestos fiber-type.conclusionsboth rea and pathology assessment are reliable and complementary predictive methods to characterize asbestos exposures . correlation analysis between the two methods effectively validates both rea methodology and lba procedures within the determined precision , particularly for cumulative amphibole asbestos exposures since chrysotile fibers , for the most part , are not retained in the lung for an extended period of time .

Introduction Methods Results Discussion Conclusions Declaration of interest

in particular , cumulative retrospective exposure assessment ( rea ) of asbestos has allowed dose response characterization of cohorts and individuals with respect to asbestosis , lung and other cancers , as well as mesothelioma . the determination of reliability and linearity of either industrial hygiene exposure estimation or asbestos lung burden analysis ( lba ) to characterize past asbestos exposures is necessary to effectively perform either cohort or case control studies and risk assessment of exposed individuals . the general purposes of this study are ( a ) to correlate asbestos rea with various forms of lba for a large number of cases from varied industries , exposure scenarios , and fiber - types ; ( b ) to evaluate the linearity , precision , reliability and applicability of both rea and lba for these different scenarios with limited asbestos exposure parameter information ; and ( c ) to demonstrate a validation procedure for rea methods . thus , the study of the precision , reliability and applicability of rea is also applicable to industrial hygiene exposure assessment , in general , since exposure assessment is typically not performed via contemporaneous air sampling , but involves consideration of models , historical data from similarly exposed groups and judgment ( aiha , 2008 ) . other researchers have correlated estimated cumulative asbestos exposure with lung fiber burden , but not asbestos body burden . while such work has been important , the number of subjects tended to be relatively small , the cumulative exposure estimation methods are not always clearly stated , and these types of exposure estimates are sometimes based on estimated category ranges rather than the actual estimated values ( lin et al . no study to date has utilized such a large dataset for analysis as is reported herein for asbestos , and the studies usually have not addressed correlation between individual rater estimates . finally , detailed analysis of the linearity of the methods and the effect of asbestos fiber - type on such correlations has not been reported . asbestos fiber - type issues that affect comparison between industrial hygiene exposure estimation and lba were also evaluated and explored . correspondingly , not only have we explored the correlation between the industrial hygiene raters and asbestos lung burden , but we also evaluated the linearity between cumulative exposure assessment and the pathology assessment methods . the latter included plaintiff s answers to interrogatories , work history sheets , work summaries or plaintiff s discovery depositions . filters were mounted on carbon discs for the analysis of fiber concentrations by scanning electron microscopy ( sem ) and determination of fiber - types by energy dispersive x - ray analysis as previously described ( roggli , 2004 ) . a consultation database was provided by the pathologist with a brief description of asbestos exposure , the report date , age at time of report , gender and an estimate of the number of years of asbestos exposure . names were withheld , and the pathologist s analytical data were removed prior to cumulative exposure estimation by the industrial hygiene raters . examples of asbestos exposure information provided included : painter / carpenter / maintenance man , 20 yearsbrake mechanic , 27 yearsasbestos plant worker , 27 yearsinsulator , 24 + yearsasbestos cloth weaver , 6 months and shipyard worker , 2 yearsshipyard insulator , 5 yearsjoiner , shipyard , 35 yearscarman railroad , 35 yearshousehold contact , husband , power plant worker , 29 yearspipefitter , 27 yearskent cigarettes , 2 yearshousehold contact ( husband and parents - shipyard workers ) , 2 to 3 years painter / carpenter / maintenance man , 20 years brake mechanic , 27 years asbestos plant worker , 27 years asbestos cloth weaver , 6 months and shipyard worker , 2 years shipyard insulator , 5 years joiner , shipyard , 35 years carman railroad , 35 years household contact , husband , power plant worker , 29 years kent cigarettes , 2 years household contact ( husband and parents - shipyard workers ) , 2 to 3 years the information related to the 363 cases was provided to four experienced certified industrial hygienists ( larry birkner , james rasmuson , fred boelter , william dyson ) , referred to as the raters . the raters were instructed to estimate the cumulative asbestos exposure in units of fiber per cubic centimeter years ( f / cc - years ) for each of the cases . they were not instructed on which method to use in developing their estimates , but utilized methods consistent with exposure reconstruction methods developed and recommended by the american industrial hygiene association ( aiha ) that are summarized in various aiha and other publications ( aiha , 2006 ; rodricks , 2011 ) . out of the 363 case reports , one of the exposure estimates of the 329 cases was found to be ambiguous and was therefore excluded from analysis . ambiguity of laboratory data for one subject caused the number to be reduced to a maximum 327 for subjects related to the statistical analysis involving correlations between asbestos lung burden and the exposure estimates . for 308 of the 327 cases , asbestos fiber - type information associated with lung fiber burden analysis was available . the ratios of asbestos fibers and asbestos bodies to mineral fibers and coated mineral fibers were determined from provided fiber - type analysis to transform the data into units of asbestos fibers per wet gram of lung tissue ( f / g ) and sem asbestos bodies per gram of wet lung tissue ( sem ab / g ) . histograms of the various laboratory lung burden and rater estimate values were examined and found to be distributed closer to log - normal than normal . table 1.variable definitions.variable ( s)descriptiontypeunitsa , b , c , dthe a , b , c , and d variables refer to sets of individual cumulative exposure estimates for each of the four raters that were logarithmically - transformed for the 328 cases.continuouslog10 ( f / cc - years)expexp refers to the population of 328 arithmetically averaged , log - transformed exposure estimates from each rater . exp = ( a + b + c + d)/4 for each of the 328 cases for which all raters submitted an estimate.continuouslog10 ( f / cc - years)ab / glog10 transformed values of the set of linearly averaged light microscopy laboratory determinations of the number of asbestos bodies per wet gram of lung tissue for each case for each of 327 cases.continuouslog10 ( ab / g)sem ab / glog10 transformed values of the set of linearly averaged sem laboratory determinations of the number of asbestos bodies per wet gram of lung tissue . analytical data was available for 277 out of the 327 cases.continuouslog10 ( sem ab / g)f / glog10 transformed values of the set of linearly averaged sem laboratory determinations of the number of asbestos fibers longer than 5 per wet gram of lung tissue for each case for 308 overlapping cases with the 327 cases.continuouslog10 ( f / g)laba combination variable of ab / g , sem ab / g , and f / g that was transformed into the same units as ab / g . more on this variable is found in the discussion , below , and in table 2.continuouslog10 ( ab / g)etthe data set was further reduced from the original categorization into 11 exposure types ( or categories ) for analysis of covariance ( ancova ) ( rosner , 2006 ) . the main asbestos exposure types were based on occupation , but other categories such as household exposure and the type of asbestos product from which exposures occurred were also utilized . these exposure type ( et ) categories are defined in figure 4 , along with numbers of associated cases.classsingle integers ( dummy variable)ftfor 308 of the 327 cases , asbestos fiber - type information associated with lung fiber burden analysis was available . although both sem ab / g and f / g had significantly different slopes from one , as shown in table 2 , indicating some degree of non - linearity between the two variables and ab / g , another test of linearity is the regression between the laboratory variables and exp ( defined in table 1 ) , with and without adjusting for confounding factors like asbestos fiber - type . correlation between raters and the laboratory analysis were determined by both regression and ancova analysis . the intraclass correlation coefficient ( icc ) was determined to further evaluate reproducibility of the exposure reconstructions performed by the raters and the overall degree of reliability ( rosner , 2011 ) . the icc was also calculated for each of the laboratory determinations of asbestos body ( ab / g and sem ab / g ) and fiber burden ( f / g ) lung tissue analysis . the icc is a statistical measure used to evaluate the reproducibility of measured or estimated continuous variable values such as replicate analytical determinations and has also been applied to evaluate continuous variable rater determinations . similarly , for the analytical pathology results , where replicate measures were reported for distinct ab and fiber burden samples , an analysis of reproducibility was performed by calculating associated icc values . further analysis between the log - transformed rater estimates yielded excellent agreement and reliability according to established guidelines ( rosner , 2006 ) with an icc - value of 0.76 , not far from the upper bound of the very good range ( icc = 0.75 ) . table 3.intraclass icc for asbestos bodies and fiber burden analysis.data setcorresponding variablenumber of casescases with replicatesreplicates per caseiccasbestos bodies in lung tissue : light microscopyab / g36316620.91asbestos bodies in lung tissue : semsem ab / g1249 * 5420.90free asbestos fibers in lung tissue > 5 by semf / g1249 * 6720.88*additional data were obtained from the pathologist for the purpose of calculating the laboratory iccs . table 4 indicates that each of the specified analytical methodologies is a good predictor of asbestos exposure within the indicated precision . slope values are slightly above and slightly below one , indicating reasonable linearity with estimated exposure estimates ( exp ) , but improved linearity can be obtained by compensating for fiber - type , as demonstrated below . as lab provided a slightly better correlation than the other three dependent variables that were tested , and it appeared to provide the most linearity , it was selected as the primary dependent variable to be studied further by ancova methods . some of these sources of variability can be evaluated and compensated by introduction of the class variables defined in table 1 , such as et ( related to breathing rate and asbestos fiber - type ) and ft determined by lung fiber burden analysis . some of the variability in figure 3 is likely caused by fiber - type percentage differences between various asbestos formulations utilized in varying occupations , varying associated breathing rates and differing times between exposure and lba . therefore , a much higher percentage of inhaled asbestos fibers ( and resultant asbestos bodies ) would be expected to be retained in the lung for the shipyard insulator compared with the automobile mechanic , because of the much shorter half - life of chrysotile in the human lung ( weeks or months ) compared with amphibole asbestos ( decades , if not a lifetime ) ( roggli , 2004 ; churg , 1998 ; craighead & gibbs , 2008 ) . however , when the correlation lines are not forced to be parallel , the categories involving extensive amphibole asbestos exposure such as shipyard insulators are steeper than the slope for other categories , and the slope for the automobile and friction product work is relatively flat , because chrysotile fibers are not easily converted to asbestos bodies in the lung , and chrysotile fibers , for the most part , are not retained in the lung ( roggli & sharma , 2004 ) . for example , there is a significant difference between each of the upper four amphibole asbestos classes and the automobile / friction product class based on a two - sided t - test with unequal variances assumed for each of the classes . additionally , a one - sided t - test shows significance between the automobile / friction product class and each of the secondary household , miscellaneous and mixed trades and the construction trade classes . unexpectedly , there is also no difference between the refinery workers class and the automobile / friction product class , but an n of six is relatively small for the refinery workers , and the exact nature of the work that they performed is unknown . to further test the hypothesis that fiber - type ( ft ) was a significant cause of variability in the correlation of exp estimates with lab , ancova was performed with ft as a class variable . other typically non - commercial ( north america ) asbestos fiber types such as tremolite , actinolite and anthophyllite often remained in the lungs of the cases associated with ft classes 3 and 4 , but apparently these asbestos fibers were not proportionally related to the primary asbestos exposures associated with the cases , based on the ancova results represented in figure 5 . amosite and/or crocidolite detected , but not majority asbestos fiber - type ( o ) , n = 35 . amosite and/or crocidolite detected , but not majority asbestos fiber - type ( o ) , n = 35 . indeed when this was done , an r - value of only 0.01 was found with a slope of 0.01 , essentially flat , but with a standard error of 0.46 for the slope since the exp estimates varied broadly ( relating to exposures varying between parts delivery , automobile mechanics , brake mechanics and the manufacture of friction products ) . this was done to evaluate whether the pathological markers of exposure behaved similarly with increasing asbestos exposure and to determine the degree of linearity of the response . for example , for the case of the dependent variable , ab / g , the slope for the first fiber - type category was 1.06 , not different from one , and r was 0.75 , the same value as that associated with the lab dependent variable shown in figure 5 . significant differences between the slopes for the ab / g and f / g - ancova plots summarized in table 6 were similar to , but not precisely the same as that summarized above for the data in figure 5 . there are some differences in the residual error distribution of the analysis , which can be explained by differences in individual tendency to coat asbestos fibers in the lung to produce asbestos bodies ( figure 7 ) . although the error distribution between raters appears to be smaller , if equal variation is attributed to both the lab analysis and the rater estimates , the data represented by each of the two methods is within about a factor of 8.5 of mean estimates in the correlation . an r - value of 0.77 implies that 60% of the variability in the dependent variable is accounted or explained although the correlation was not dramatically improved from simpler models , and a portion of the improvement could be attributed to the increase in the number of variables . thus , although correlation between only amphibole asbestos and rea was found , but no such correlation was found for chrysotile exposures , we believe that chrysotile exposure estimation by experienced industrial hygienists can be performed as reliably as for amphibole asbestos . earlier comparative studies have indicated that industrial hygiene judgment and individual - reported asbestos exposure histories combine to provide the most effective exposure reconstruction process and that this process is superior to sole utilization of job exposure matrices ( gramond et al . thus , because the information provided for the exposure assessment had the charateristics of a job exposure matrix although occupation and exposure duration have elements of self - reporting , better inter - rater correlation and improved rater correlation with asbestos lung burden would have been expected if the raters had more detailed exposure information . assuming adequate sampling of lung tissue , our data also suggest that asbestos body determination by light microscopy yields useful information regarding past asbestiform amphibole exposures , comparable to the more elaborate asbestos fiber burden determination by electron microscopy , which also primarily is an indicator of past amphibole asbestos exposure . chrysotile exposures are of particular current public health interest because of continued world production of the mineral , especially in the former soviet union , brazil and china , and utilization of asbestos products , particularly in construction materials such as roofing products throughout much of the developing world . there is no effective substitute for industrial hygiene estimation of past cumulative chrysotile exposures , in most instances ( consensus report , 1997 ) ; thus , validation of cumulative asbestos exposure estimates is critical . pathologists continue to characterize past chrysotile exposures via lung fiber burden analysis , but most of the chrysotile does not persist in the lung and only a small percentage results in asbestos body formation . even for amphibole asbestos exposures , the linearity and correlation of asbestos fiber and asbestos body concentrations in the lung with each other and with respect to past cumulative exposure has been questioned , and the degree of correlation may depend on ratios of amosite to crocidolite and whether the subject has asbestosis ( roggli , 2004 ; roggli et al . however , our study suggests that the various lung burden indicators appear to be linear with total amphibole asbestos exposure , which has not been previously demonstrated , although asbestos bodies have been shown to be a relatively good marker of past exposure to long amphibole fibers ( roggli , 2004 ) . although our data indicate a very good to excellent correlation between industrial hygienists estimate of lifetime cumulative exposure and the asbestos lung burden data , in general , based on the determined icc , factors such as individual variable fiber retention , individual variable fiber coating efficiency and varying time between exposure and laboratory analysis , as well as site - to - site variation of fiber content within the lung help explain the remaining significant unexplained variance . the purpose of lung fiber and asbestos body burden analysis is to determine the cumulative asbestos fiber content of an individual s lungs at the time that the tissue sample was obtained . however , the linearity of the exposure estimates with the lung burden data varying over almost four orders of magnitude and the approximate linearity and agreement between various raters suggests that good or at least reasonable accuracy was demonstrated in the exposure estimations . examples of potential applications of our study include estimation of the relationship between workplace and related household asbestos exposures , relative workplace - to - workplace and/or job - to - job comparisons , estimation of historical general population background exposures in units of f / cc - years based on pathology analysis of non - occupationally exposed individuals and determination of asbestos risk assessment potency factors ( for example km in mesothelioma risk assessment procedures ) from cohort lung burden analysis when little or no air monitoring data is available , or when asbestos lung burden data is available to validate historical air monitoring data . another application of our study is underscored by the observed non - linear dose - response relationships observed in both meta - analysis of cohort studies for lung cancer and mesothelioma ( hodgson & darnton , 2000 ; berman & crump , 2008 ) as well as for case - control studies for mesothelioma ( iwatsubo et al . , 2001b however , a linear dose - response for mesothelioma was observed when asbestos fiber lba was employed to estimate relative exposures between cases and controls where amphibole asbestos was hypothesized to be the primary risk factor , and crocidolite appeared to be the primary asbestos amphibole ( rdelsperger et al . a portion of the consultation database has been used by the authors as a resource in a class exercise at the conclusion of american industrial hygiene association ( aiha ) professional development classes ( pdcs ) on exposure reconstruction . fifty - six percent of the variability between the correlation of industrial hygiene estimates and pathology determinations is explained , when a class variable accounting for variable fiber type is included in the correlation , but higher correlation was found between industrial hygiene raters , as demonstrated by determined icc values . the reasons for the unexplained variability between industrial hygiene estimations and laboratory lung burden analysis include varying times between exposure and fiber burden analysis for the various cases and individual differences in fiber retention and fiber coating tendency . the light microscopy determination of lung tissue asbestos bodies appears to be almost as good an indicator of past asbestiform amphibole fiber exposures as electron microscopy determination of asbestos fiber lung burden . lba appears to be a satisfactory relative indication of past asbestiform amphibole fiber exposures for case - control studies , and industrial hygiene reconstructed exposure estimates can also be satisfactorily utilized .

water typically participates in molecular recognition and association . despite the importance of water in chemistry , biology , and nanosciences in general , our current understanding of its thermodynamic role in promoting or hampering the binding of a ligand to a receptor is still surprisingly limited . hydration and dewetting in model systems have been successfully investigated over varying length scales based on theory(1 ) and simulation , shedding transferable knowledge on the forces involved in water nonpolar confinment , including the influence of biomolecular geometry and topography . the study of model hydrophobic cavities was recently extended to hydrophobic ligand binding , encountered in biological recognition and drug design . experiments and molecular dynamics ( md ) simulations have demonstrated the general , dynamic nature of protein hydration . water can be absent(16 ) or , at least transiently , present(17 ) in nonpolar protein cavities or exchange between the bulk solvent and polar , hydrated cavities.(18 ) favorable free energy changes were calculated for tying up single , localized water molecules in the binding pockets of protein complexes . alternative hydration scenarios all imply favorable free energy changes upon transferring water from the bulk to a hydration site or confined volume . therefore , characterizing free energies is not sufficient to disentangle the determinants of different hydration behaviors . substantially more fundamental , transferable information could be gained through the underlying compensating enthalpyentropy terms ( i.e. , the thermodynamic signature ) . dunitz suggested that small , unfavorable entropic changes would accompany water binding to protein crystals by comparing standard entropies of anhydrous and dehydrated salts with that of bulk water.(21 ) vaitheeswaran et al . proposed that the configurational entropy of water clusters decreases when occupying spherical hydrophobic cavities,(2 ) a similar conclusion to that obtained by yin et al . for a nonpolar cavity in the tetrabraichion protein.(22 ) on the other hand , based on nuclear - magnetic - resonance data and a model for librational dynamics , denisov et al . suggested favorable entropy changes for disordered water permeating various protein cavities.(23 ) taken as a whole , these studies demonstrate that water thermodynamics is strictly dependent on the specific physicochemical properties of the confining environment . however , how such properties modulate cavityligand binding has not yet been addressed in this context . is water a passive player , only embedding cavityligand recognition , or a driving player ? answering this question essentially requires quantifying , in addition to water occupancy and dynamics , the underlying thermodynamic signatures . interpreting the molecular basis of measured free energy , enthalpy , and entropy changes upon proteinligand binding is a difficult task , due to the tight coupling between solvent and solute contributions . yet , new experiments indeed provide strong evidence of the role of water in driving cavityligand binding . from a computational standpoint , the major obstacle to estimate compensating enthalpy and entropy contributions is finite sampling , thus far confining entropy calculations either to partial single - molecule ligand or receptor(30 ) degrees of freedom , or to systems of reduced size . in the latter case , thermodynamic signatures can be obtained using potential of mean force ( pmf ) calculations , e.g. for methanemethane association in water , with the appealing advantage of characterizing not only two - state , thermodynamic differences but also the profile along the association coordinate , revealing free energy , enthalpy , and entropy barriers directly related with solvent reorganization at the atomic level . despite these important advances , water enthalpyentropy compensation regulating molecular recognition and association remains largely unexplored , mainly due to water dynamic correlation and the nonadditive character of its thermodynamic effects ( e.g. , see refs ( 13 , 35 , 36 ) ) . in particular , to our knowledge pmf approaches have not yet been applied to derive thermodynamic signatures of cavityligand binding . in this study , we derive complete thermodynamic signature profiles of cavityligand recognition using a straightforward approach based on pmf calculations and the temperature dependence of the free energy.(37 ) on one hand , we use the simplest possible model systems to generalize cavityligand association , i.e. explicitly solvated hemispherical cavities binding spherical ligands ( figure 1 ) . on the other hand , by employing cumulative microsecond sampling , free energy , enthalpy , and entropy can be estimated including a sizable water component , thus explicitly accounting for waterwater and watersolute coupling and correlating these effects with water density distribution . by cross - comparing seven systems with varying cavity and ligand physicochemical properties , our study reveals an unprecedented picture of hydration thermodynamics , opening appealing perspectives for estimating solvent entropy in complex systems and improving state - of - the - art models for molecular simulation . snapshot and schematic representation of the explicitly solvated hemispherical cavities and spherical ligands used in this study . the seven systems only differ for the charges on cavity , qc , and ligand , ql . a detailed description of the molecular models , system preparation , and simulation setup was reported for the ( n , n ) system.(37 ) the six new cavityligand systems employed in this work only differ for the qc and ql unit charges attributed to one cavity particle and/or the ligand particle , as schematically drawn in figure 1 . all systems considered contain 1030 water molecules and only differ by one charge at a time ( no other interaction parameter is changed ) . the tip4p water model(38 ) employed proved good agreement with the experimental phase diagram of water . md simulations were performed using the charmm software.(41 ) water density distribution maps were generated using the xfarbe software.(42 ) free energy changes along were estimated from the potential of mean force where kb is the boltzmann constant , t the absolute temperature , and p( ) the probability of finding the ligand particle at , which can be obtained from the ligand configurational space , (r ) . in this study , the umbrella sampling procedure(43 ) allowed effective sampling of (r ) along . pmf profiles were obtained by the weighted histogram analysis method.(44 ) in the ( npt ) ensemble , the gibbs free energy for moving the ligand from = to a given value reads using standard expressions , the corresponding entropy can be estimated numerically through the temperature dependence of the free energy , the corresponding enthalpy was determined as computational details were previously described,(37 ) together with the definition of decomposed interaction energies and a procedure to determine the statistical uncertainties on g , h , and ts profiles along ( error bars in figures 25 ) . thermodynamic signature profile and water density maps along the binding coordinate for a nonpolar ligand binding a nonpolar cavity ( n , n ) . left , top panel : gibbs free energy , g ( red ) , enthalpy , h ( blue ) , and entropic term , ts ( green ) are shown with their uncertainties ( vertical bars ) . left , bottom panel : water contribution to relative gibbs free energy , gw ( orange ) and decomposed energies for ligand - water , ulw ( green ) , cavity - water , ucw ( black ) , and waterwater , uww ( cyan ) interactions . water density ( * ) distribution maps are shown for key snapshots along using a color coding normalized with respect to bulk water , for which * = 1 . see movie s1 for the corresponding dynamic hydration video . a binding scenario , i.e. a favorable change in system free energy reached at a certain equilibrium binding distance , eq , was observed in five out of the seven investigated systems . figure 2 summarizes the results for the binding between a nonpolar cavity ( n ) and a nonpolar ligand ( n ) , referred to in the following as the ( n , n ) system . as the ligand moves toward the cavity , the system relative gibbs free energy , g , monotonically decreases from 0.4 nm to eq = 0.345 nm . the corresponding changes in system enthalpy , h , and entropic term , ts , are also displayed and show at first an increase of h ( or decrease of ts ) until 0.55 nm , followed by a rapid exchange of favorable / unfavorable compensating components at 0.4 nm . as suggested by the water density distribution maps , this rapid exchange results from the sudden cavity dehydration induced by the approaching ligand . the corresponding large favorable enthalpy change is the dominant contribution to the overall binding thermodynamics : the favorable g of 16.5 kj mol is dominated by enthalpy , h = 29.1 kj mol , with a counterbalancing ts = 12.6 kj mol ( table 1 ) . larger eq values for ( + , n ) , ( ,n ) , ( ,+ ) , and ( + , ) are due to solvent - separated binding . the molecular origin of such hydrophobic enthalpy - driven binding can be fully understood by characterizing the individual energy contributions arising from ligand or cavity hydration , as well as waterwater interactions ( figure 2 and table 1 ) . cavity dehydration that dominates binding is associated with a sizable , favorable energetic change resulting from new waterwater interactions formed upon expelling water molecules from the nonpolar cavity ( uww = 37 kj mol ) . this is the major factor determining the overall water contribution to the system free energy ( gw ) , making water the driving player in hydrophobic cavityligand association(37 ) with a gw = 10.5 kj mol , compared to significantly smaller direct cavityligand interactions ulc = 6 kj mol . our data imply that water entropy is more favorable for water inside the cavity than for water molecules forming more favorable ( and highly correlated ) waterwater interactions in the bulk , in agreement with favorable entropy changes proposed for disordered water permeating nonpolar protein cavities . however , it is the favorable enthalpy overcompensation of bulk waterwater interactions that drives nonpolar cavityligand binding in the ( n , n ) system . rather different thermodynamic profiles characterize the binding of the same nonpolar ligand to cavities at the end of which either a positive or negative net charge was introduced ( figure 3 , ( + , n ) and ( ,n ) systems ) . in both cases , g curves display characteristic barriers ( ( + , n ) and ( ,n ) : peaks of 4 and 6 kj mol at = 0.135 and 0.105 nm ) , yet binding is overall favorable ( g = 2.3 and 3.4 kj mol , table 1 ) . interestingly , similar g curves for the two systems result from substantially different enthalpyentropy compensation profiles . in the case of ( + , n ) , binding is entropy driven , resulting from h and ts of 11.7 and 14.0 kj mol . conversely , for ( ,n ) the enthalpy takes over the small entropic component , being h = 3.0 and ts = 0.4 kj mol . thermodynamic signature profiles and water density distribution maps along the ( + , n ) or ( ,n ) cavityligand binding coordinates . see figure 2 legend for color coding as well as movies s2 and s3 for dynamic hydration . in contrast to the ( n , n ) system , due to the presence of net cavity charges , cavity hydration is preserved in both systems . cavity water appears to be structured , and its displacement by the approaching ligand ( for < 0.4 nm ) is entropically favorable ( figure 3 ) . at the same time , water molecules persistently occupying the cavity bottom lose their interacting partners , and the underlying increase of uww is the main contribution to the enthalpy - dominated g barrier . when such a barrier is crossed , further reorganization of the confined water takes place as the nonpolar ligand moves closer to the cavity wall . in both ( + , n ) and ( ,n ) cases it ends with the formation of stable single hydration shells ; thus free energy minima correspond to solvent - separated rather than direct contact binding . the hydration shell formation is driven by enthalpy and entropically unfavorable , and the magnitude of these compensating terms depend on how different cavity charges promote water structure . charge asymmetry in this context is discussed in the charge asymmetry section ( discussion ) . the thermodynamic determinants for charged cavities binding oppositely charged ligands were also investigated ( figure 4 ) . despite a strong cavityligand electrostatic interaction , g changes are moderate , indicating a remarkable role of water electrostatic screening . in both ( ,+ ) and ( + , ) scenarios , the favorable g differences of 13.0 and 6.1 kj mol are exclusively enthalpy driven , resulting from h of 14.0 and 16.8 and ts of 0.9 and 10.7 kj mol , respectively ( table 1 ) . a gradual decrease of g along is accompanied by fluctuating enthalpy ( favorable ) and entropy ( unfavorable ) terms ( figure 4 ) . an oscillation period of 0.3 nm ( i.e water molecular size ) reflects the reorganization of discrete water hydration shells upon ligand binding . thermodynamic signature profiles and water density distribution maps along the ( ,+ ) or ( + , ) cavityligand binding coordinates . see figure 2 legend for color coding . in addition , the left , bottom panels report as well the change of cavityligand interaction energy , ucl ( dotted black ) . see movies s4 and s5 for dynamic hydration . cavity and ligand hydration is persistent throughout the binding process , as observed for ( + , n ) and ( ,n ) systems ( figures 3 and 4 ) . gw , ulw , and ucw follow monotonically increasing profiles along in both ( ,+ ) and ( + , ) systems , indicating that the unfavorable water free energy contribution largely arises from cavitywater and ligandwater interactions ( figure 4 ) . their changes upon binding are the largest among the seven systems considered ( gw = 153.0 and 123.9 kj mol , ulw = 117 and 80 kj mol , and ucw = 149 and 121 kj mol ) . unfavorable cavitywater electrostatic interactions are overcompensated by large , direct cavityligand interactions ( ucl = 166 and 130 kj mol ) . the second largest contribution to enthalpy - driven binding arises from creating more favorable waterwater interactions ( uww = 114 and 88 kj mol , the role of water in the screening of the strong cavityligand interactions can be explained by considering the strong dipole generated by their separated charges . in the bulk region of both ( ,+ ) and ( + , ) systems , the overall electrostatic field is dominated by the ligand charge and orients water dipoles favorably toward the ligand , yet unfavorably for the cavity . as a result , the charged cavity interacts favorably only with the ordered water dipoles in the region between cavity and ligand . upon cavityligand association this region becomes smaller , leading to a remarkable increase of ucw , which occurs as a relatively long - range effect ( see figure 4 , < 1.0 nm ) . furthermore , as the distance between cavity and ligand charges reduces ( < 0.3 nm ) , the resulting electrostatic field gradually vanishes in the bulk ; thus water molecules start to orient preferentially due to mutual hydrogen bonding rather than to the interaction with the ligand . this interpretation is supported as well by comparison with ( n,+ ) and ( n, ) systems : in the absence of electrostatic screening significantly smaller , unfavorable ulw changes are observed for identical ligands ( see the next section and table 1 ) . charge asymmetry from our data is discussed in the charge asymmetry section ( discussion ) . therefore , understanding the thermodynamic determinants is as relevant for binding as it is for rejection scenarios . such ligand rejection is observed for ( + ) and ( ) ligands approaching a nonpolar cavity ( figure 5 ) . a monotonically increasing ( unfavorable ) profile for g in both ( n,+ ) and ( n, ) systems is accompanied by monotonically increasing h ( and decreasing ts ) for < 0.7 nm . overall , binding is prevented largely because of enthalpy penalties ( h of 45.8 and 75.7 kj mol ) , overcoming a large , favorable entropy compensation ( ts of 32.9 and 55.8 kj mol ) . thermodynamic signature profiles and water density distribution maps for ligand rejection along the ( n,+ ) or ( n, ) cavityligand coordinates . see figure 2 legend for color coding as well as movies s6 and s7 for dynamic hydration . the enthalpy contribution to the unfavorable water free energy , gw , mostly arises from the loss of waterwater interactions ( uww values of 57 and 79 kj mol ) . water density distribution maps suggest that increasing uww and progressively more favored ucw values are due to the charged ligands dragging their hydration shells deep into the nonpolar cavity . partial ligand dehydration , evidenced by increasing ulw energies and the change of slope of g profiles , takes place at 0.0 nm , as the hydration shells are sterically unable to fit into the confining cavity volume . this process is substantially different compared with hydrophobic ligand dehydration in the ( n , n ) system ( figure 2 and figure 5 ) . as expected , the removal of charged ligands from the bulk solvent is entropically favorable . water is highly ordered around the ligands already in the bulk ; thus no entropy penalty needs to be paid upon dragging the ordered hydration shells into a nonpolar cavity . noticeable differences in enthalpy and entropy terms for a ( ) vs ( + ) ligand support the key role of water in cavityligand specificity , as discussed in the charge asymmetry section ( discussion ) . a variety of thermodynamic scenarios and driving effects for model cavityligand recognition was revealed based on our simulations , as summarized in terms of the overall changes in g , h , and ts ( figure 6 ) . a common feature is the fundamental role of water as an active player in determining binding or rejection . though quantitative results are system dependent , on a qualitative level more complex cases of macromolecular recognition are likely a multivariate combination of the scenarios presented herein . one can imagine that the variability of corresponding thermodynamic signatures would broaden for complex biomolecules , as a result of the multifaceted hydrophobicity of the binding partners , roughness , flexibility , and topography of the macromolecular surfaces . water is clearly an active player in six out of seven cases in which ligand binding / rejection is driven either by enthalpy ( blue label ) or by entropy ( green label ) . reported a thermodynamic analysis for six small nonpolar compounds binding the nonpolar cavity of mouse major urinary protein - i.(45 ) measured thermodynamic changes closely resemble the scenario we find for the ( n , n ) system.(37 ) musah et al . measured thermodynamic changes upon binding for an engineered heterocyclic cation - binding site.(46 ) this cavity , created by mutation of a hydrophobic tryptophan residue to a glycine , presents a buried , negatively charged aspartic acid residue crucial for binding and displays similar volume and hydration to those of our ( , + ) model system.(18 ) among the seven systems investigated , the latter is the most closely related to the experimental data of enthalpy - driven binding . however , a more extensive , direct comparison of our data with water thermodynamics in proteinligand binding is hampered by the complexity of experimental interpretation . water thermodynamics depends on solute charge asymmetry ( e.g. , see refs ( 31 , 5053 ) ) , occasionally with unexpected macroscopic consequences.(54 ) a quantitative interpretation of ion hydration thermodynamics is still widely debated in the current literature.(55 ) however , the interpretation of structure - making and structure - breaking trends is well consolidated . for this reason we use it here to explain evident charge asymmetry from our data . we can interpret charge asymmetry considering the different dipole orientation of the first two hydration shells . water molecules preferentially orient their oxygen atoms toward a positive charge or their hydrogen atoms ( and dipoles ) toward a negative charge . small ions of high charge density are structure forming ( kosmotropes ) , i.e. increasing the enthalpy ( decreasing the entropy ) of surrounding water with respect to bulk . in contrast , large monovalent ions of low charge density are structure breaking ( chaotropes ) with contributions of opposite sign . the ( + ) ligand employed in our simulations has similar parameters to those of the chaotropic cs cation , the ( ) ligand intermediate between cl and f anions , which are moderately kosmotropic.(55 ) the most evident thermodynamic effects of charge asymmetry were observed for the ( n,+ ) vs ( n, ) systems overall . as suggested by collins et al.,(51 ) we notice qualitative differences within the first two hydration shells as the ligand moves from = 1.1 nm toward the cavity ( figure 5 ) . for the ( + ) ligand the reorganization of its second hydration shell is accompanied by an unfavorable entropy change , as expected for a chaotropic ion for which hydration is stabilized by entropy . on the contrary , for the ( ) ligand such reorganization requires an unfavorable enthalpy change , as expected for a kosmotropic ion whose hydration is enthalpy stabilized . it is worth noting that , at this initial stage , enthalpyentropy compensation is perfect ; i.e. , no free energy change occurs . charge asymmetry in the ( n,+ ) vs ( n, ) systems is also evident as the final ligand dehydration takes place . water interaction is stronger with a ( ) vs ( + ) ligand because water hydrogen atoms can more closely approach the charge than oxygen atoms . consistently , the ( ) ligand appears to be generally better hydrated and a comparatively higher enthalpy penalty has to be paid upon its dehydration ( table 1 ) . the corresponding favorable change in entropy is also larger for the ( ) vs ( + ) ligand , but insufficient for enthalpy compensation , leading to an overall more positive free energy change upon dehydration . this is in line with previously reported data of ionic hydration.(52 ) comparison between ( + , n ) and ( ,n ) scenarios shows interesting asymmetry for water reorganization upon binding ( figure 3 , < 0.135 and 0.105 nm , respectively ) . in the ( + , n ) system the favorable decrease of uww inverts its slope ( increases from 0.1 to eq = 0.085 nm ) , while in the ( + , n ) system water reorganization is accompanied by the monotonic decrease of enthalpy until eq = 0.105 nm . possibly , the positively charged cavity confers to the approaching nonpolar ligand properties characteristic of a chaotropic ion ( i.e. , a structure - breaking effect on water ) . thus , cavity water reorganization requires a larger entropy penalty for ( ,n ) vs ( + , n ) association ( figure 3 ) . charge asymmetry in the ( ,+ ) and ( + , ) systems is evident in the proximity of the bound states ( see figure 4 , inset panels ) . in the ( ,+ ) system , the g minimum at eq = 0.135 nm results from an h minimum and a ts maximum corresponding to an optimal hydration shell between cavity and ligand . conversely , in the ( + , ) system this is not the case , consistently with the interpretation that the kosmotropic nature of the ( ) ligand weakens near the ( + ) cavity charge , thus reducing the enthalpy - dominated order of the surrounding water molecules . we used a straightforward computational approach and extensive sampling to derive complete thermodynamic signatures of model cavityligand recognition . free energy , enthalpy , and entropy estimates were obtained along the association coordinate for a set of seven systems with varying cavity and ligand physicochemical properties . this approach gives access to coupled solutesolvent thermodynamics along the association coordinate , thus allowing investigation of the fundamental link of molecular recognition thermodynamics with cavity and ligand hydration / dewetting . qualitatively different cavityligand binding scenarios and driving effects were revealed , despite the simplicity of the model systems employed , and will help the interpretation and design of new proteinligand binding experiments . a common feature is the generally underestimated key role of water as an active player in determining ligand binding or rejection . we discussed our data also in the context of the asymmetric nature of water thermodynamics for opposite charges . remarkably , our results suggest that overall cavityligand recognition and binding propensity do not need to be ( or are not limited to ) the result of direct cavityligand interactions . for example , waterwater interactions play a key role as well in the electrostatic binding of an oppositely charged cavity and ligand through water electrostatic screening . due to this effect , the resulting overall free energy change is of similar magnitude for hydrophobic and electrostatic - driven binding . in more complex biomolecular association , involving multivariate combinations of the scenarios derived herein , water could determine the subtle balance among driving forces of different physical natures , thus broadening the array of available binding mechanisms . our approach could be generalized to tackle the challenge of solvent entropy estimation in complex biological systems and drug design , starting from hostguest systems of treatable size . overall , this study suggests that the understanding of cavityligand recognition relies on an improved description of water thermodynamics and opens excellent possibilities for developing new implicit solvent and coarse - grained models toward gaining a more realistic representation of solvation properties . in particular , a novel level - set variational implicit solvent model(56 ) is being parametrized based on our data .

we use explicit solvent molecular dynamics simulations to estimate free energy , enthalpy , and entropy changes along the cavityligand association coordinate for a set of seven model systems with varying physicochemical properties . owing to the simplicity of the considered systems we can directly investigate the role of water thermodynamics in molecular recognition . a broad range of thermodynamic signatures is found in which water ( rather than cavity or ligand ) enthalpic or entropic contributions appear to drive cavityligand binding or rejection . the unprecedented , nanoscale picture of hydration thermodynamics can help the interpretation and design of proteinligand binding experiments . our study opens appealing perspectives to tackle the challenge of solvent entropy estimation in complex systems and for improving molecular simulation models .

Introduction Methods Results Discussion Conclusion

water typically participates in molecular recognition and association . despite the importance of water in chemistry , biology , and nanosciences in general , our current understanding of its thermodynamic role in promoting or hampering the binding of a ligand to a receptor is still surprisingly limited . hydration and dewetting in model systems have been successfully investigated over varying length scales based on theory(1 ) and simulation , shedding transferable knowledge on the forces involved in water nonpolar confinment , including the influence of biomolecular geometry and topography . experiments and molecular dynamics ( md ) simulations have demonstrated the general , dynamic nature of protein hydration . water can be absent(16 ) or , at least transiently , present(17 ) in nonpolar protein cavities or exchange between the bulk solvent and polar , hydrated cavities. (18 ) favorable free energy changes were calculated for tying up single , localized water molecules in the binding pockets of protein complexes . alternative hydration scenarios all imply favorable free energy changes upon transferring water from the bulk to a hydration site or confined volume . therefore , characterizing free energies is not sufficient to disentangle the determinants of different hydration behaviors . dunitz suggested that small , unfavorable entropic changes would accompany water binding to protein crystals by comparing standard entropies of anhydrous and dehydrated salts with that of bulk water. proposed that the configurational entropy of water clusters decreases when occupying spherical hydrophobic cavities,(2 ) a similar conclusion to that obtained by yin et al . for a nonpolar cavity in the tetrabraichion protein. (22 ) on the other hand , based on nuclear - magnetic - resonance data and a model for librational dynamics , denisov et al . suggested favorable entropy changes for disordered water permeating various protein cavities. (23 ) taken as a whole , these studies demonstrate that water thermodynamics is strictly dependent on the specific physicochemical properties of the confining environment . however , how such properties modulate cavityligand binding has not yet been addressed in this context . answering this question essentially requires quantifying , in addition to water occupancy and dynamics , the underlying thermodynamic signatures . interpreting the molecular basis of measured free energy , enthalpy , and entropy changes upon proteinligand binding is a difficult task , due to the tight coupling between solvent and solute contributions . yet , new experiments indeed provide strong evidence of the role of water in driving cavityligand binding . from a computational standpoint , the major obstacle to estimate compensating enthalpy and entropy contributions is finite sampling , thus far confining entropy calculations either to partial single - molecule ligand or receptor(30 ) degrees of freedom , or to systems of reduced size . in the latter case , thermodynamic signatures can be obtained using potential of mean force ( pmf ) calculations , e.g. for methanemethane association in water , with the appealing advantage of characterizing not only two - state , thermodynamic differences but also the profile along the association coordinate , revealing free energy , enthalpy , and entropy barriers directly related with solvent reorganization at the atomic level . despite these important advances , water enthalpyentropy compensation regulating molecular recognition and association remains largely unexplored , mainly due to water dynamic correlation and the nonadditive character of its thermodynamic effects ( e.g. in particular , to our knowledge pmf approaches have not yet been applied to derive thermodynamic signatures of cavityligand binding . in this study , we derive complete thermodynamic signature profiles of cavityligand recognition using a straightforward approach based on pmf calculations and the temperature dependence of the free energy. (37 ) on one hand , we use the simplest possible model systems to generalize cavityligand association , i.e. on the other hand , by employing cumulative microsecond sampling , free energy , enthalpy , and entropy can be estimated including a sizable water component , thus explicitly accounting for waterwater and watersolute coupling and correlating these effects with water density distribution . by cross - comparing seven systems with varying cavity and ligand physicochemical properties , our study reveals an unprecedented picture of hydration thermodynamics , opening appealing perspectives for estimating solvent entropy in complex systems and improving state - of - the - art models for molecular simulation . snapshot and schematic representation of the explicitly solvated hemispherical cavities and spherical ligands used in this study . the seven systems only differ for the charges on cavity , qc , and ligand , ql . a detailed description of the molecular models , system preparation , and simulation setup was reported for the ( n , n ) system. all systems considered contain 1030 water molecules and only differ by one charge at a time ( no other interaction parameter is changed ) . the tip4p water model(38 ) employed proved good agreement with the experimental phase diagram of water . (42 ) free energy changes along were estimated from the potential of mean force where kb is the boltzmann constant , t the absolute temperature , and p( ) the probability of finding the ligand particle at , which can be obtained from the ligand configurational space , (r ) . (44 ) in the ( npt ) ensemble , the gibbs free energy for moving the ligand from = to a given value reads using standard expressions , the corresponding entropy can be estimated numerically through the temperature dependence of the free energy , the corresponding enthalpy was determined as computational details were previously described,(37 ) together with the definition of decomposed interaction energies and a procedure to determine the statistical uncertainties on g , h , and ts profiles along ( error bars in figures 25 ) . thermodynamic signature profile and water density maps along the binding coordinate for a nonpolar ligand binding a nonpolar cavity ( n , n ) . left , top panel : gibbs free energy , g ( red ) , enthalpy , h ( blue ) , and entropic term , ts ( green ) are shown with their uncertainties ( vertical bars ) . left , bottom panel : water contribution to relative gibbs free energy , gw ( orange ) and decomposed energies for ligand - water , ulw ( green ) , cavity - water , ucw ( black ) , and waterwater , uww ( cyan ) interactions . water density ( * ) distribution maps are shown for key snapshots along using a color coding normalized with respect to bulk water , for which * = 1 . a binding scenario , i.e. a favorable change in system free energy reached at a certain equilibrium binding distance , eq , was observed in five out of the seven investigated systems . as the ligand moves toward the cavity , the system relative gibbs free energy , g , monotonically decreases from 0.4 nm to eq = 0.345 nm . the corresponding changes in system enthalpy , h , and entropic term , ts , are also displayed and show at first an increase of h ( or decrease of ts ) until 0.55 nm , followed by a rapid exchange of favorable / unfavorable compensating components at 0.4 nm . as suggested by the water density distribution maps , this rapid exchange results from the sudden cavity dehydration induced by the approaching ligand . the corresponding large favorable enthalpy change is the dominant contribution to the overall binding thermodynamics : the favorable g of 16.5 kj mol is dominated by enthalpy , h = 29.1 kj mol , with a counterbalancing ts = 12.6 kj mol ( table 1 ) . larger eq values for ( + , n ) , ( ,n ) , ( ,+ ) , and ( + , ) are due to solvent - separated binding . the molecular origin of such hydrophobic enthalpy - driven binding can be fully understood by characterizing the individual energy contributions arising from ligand or cavity hydration , as well as waterwater interactions ( figure 2 and table 1 ) . this is the major factor determining the overall water contribution to the system free energy ( gw ) , making water the driving player in hydrophobic cavityligand association(37 ) with a gw = 10.5 kj mol , compared to significantly smaller direct cavityligand interactions ulc = 6 kj mol . our data imply that water entropy is more favorable for water inside the cavity than for water molecules forming more favorable ( and highly correlated ) waterwater interactions in the bulk , in agreement with favorable entropy changes proposed for disordered water permeating nonpolar protein cavities . however , it is the favorable enthalpy overcompensation of bulk waterwater interactions that drives nonpolar cavityligand binding in the ( n , n ) system . rather different thermodynamic profiles characterize the binding of the same nonpolar ligand to cavities at the end of which either a positive or negative net charge was introduced ( figure 3 , ( + , n ) and ( ,n ) systems ) . interestingly , similar g curves for the two systems result from substantially different enthalpyentropy compensation profiles . conversely , for ( ,n ) the enthalpy takes over the small entropic component , being h = 3.0 and ts = 0.4 kj mol . thermodynamic signature profiles and water density distribution maps along the ( + , n ) or ( ,n ) cavityligand binding coordinates . see figure 2 legend for color coding as well as movies s2 and s3 for dynamic hydration . in contrast to the ( n , n ) system , due to the presence of net cavity charges , cavity hydration is preserved in both systems . cavity water appears to be structured , and its displacement by the approaching ligand ( for < 0.4 nm ) is entropically favorable ( figure 3 ) . at the same time , water molecules persistently occupying the cavity bottom lose their interacting partners , and the underlying increase of uww is the main contribution to the enthalpy - dominated g barrier . when such a barrier is crossed , further reorganization of the confined water takes place as the nonpolar ligand moves closer to the cavity wall . in both ( + , n ) and ( ,n ) cases it ends with the formation of stable single hydration shells ; thus free energy minima correspond to solvent - separated rather than direct contact binding . the hydration shell formation is driven by enthalpy and entropically unfavorable , and the magnitude of these compensating terms depend on how different cavity charges promote water structure . despite a strong cavityligand electrostatic interaction , g changes are moderate , indicating a remarkable role of water electrostatic screening . a gradual decrease of g along is accompanied by fluctuating enthalpy ( favorable ) and entropy ( unfavorable ) terms ( figure 4 ) . thermodynamic signature profiles and water density distribution maps along the ( ,+ ) or ( + , ) cavityligand binding coordinates . in addition , the left , bottom panels report as well the change of cavityligand interaction energy , ucl ( dotted black ) . see movies s4 and s5 for dynamic hydration . gw , ulw , and ucw follow monotonically increasing profiles along in both ( ,+ ) and ( + , ) systems , indicating that the unfavorable water free energy contribution largely arises from cavitywater and ligandwater interactions ( figure 4 ) . their changes upon binding are the largest among the seven systems considered ( gw = 153.0 and 123.9 kj mol , ulw = 117 and 80 kj mol , and ucw = 149 and 121 kj mol ) . unfavorable cavitywater electrostatic interactions are overcompensated by large , direct cavityligand interactions ( ucl = 166 and 130 kj mol ) . the second largest contribution to enthalpy - driven binding arises from creating more favorable waterwater interactions ( uww = 114 and 88 kj mol , the role of water in the screening of the strong cavityligand interactions can be explained by considering the strong dipole generated by their separated charges . upon cavityligand association this region becomes smaller , leading to a remarkable increase of ucw , which occurs as a relatively long - range effect ( see figure 4 , < 1.0 nm ) . furthermore , as the distance between cavity and ligand charges reduces ( < 0.3 nm ) , the resulting electrostatic field gradually vanishes in the bulk ; thus water molecules start to orient preferentially due to mutual hydrogen bonding rather than to the interaction with the ligand . therefore , understanding the thermodynamic determinants is as relevant for binding as it is for rejection scenarios . a monotonically increasing ( unfavorable ) profile for g in both ( n,+ ) and ( n, ) systems is accompanied by monotonically increasing h ( and decreasing ts ) for < 0.7 nm . thermodynamic signature profiles and water density distribution maps for ligand rejection along the ( n,+ ) or ( n, ) cavityligand coordinates . see figure 2 legend for color coding as well as movies s6 and s7 for dynamic hydration . the enthalpy contribution to the unfavorable water free energy , gw , mostly arises from the loss of waterwater interactions ( uww values of 57 and 79 kj mol ) . water density distribution maps suggest that increasing uww and progressively more favored ucw values are due to the charged ligands dragging their hydration shells deep into the nonpolar cavity . noticeable differences in enthalpy and entropy terms for a ( ) vs ( + ) ligand support the key role of water in cavityligand specificity , as discussed in the charge asymmetry section ( discussion ) . a variety of thermodynamic scenarios and driving effects for model cavityligand recognition was revealed based on our simulations , as summarized in terms of the overall changes in g , h , and ts ( figure 6 ) . a common feature is the fundamental role of water as an active player in determining binding or rejection . though quantitative results are system dependent , on a qualitative level more complex cases of macromolecular recognition are likely a multivariate combination of the scenarios presented herein . one can imagine that the variability of corresponding thermodynamic signatures would broaden for complex biomolecules , as a result of the multifaceted hydrophobicity of the binding partners , roughness , flexibility , and topography of the macromolecular surfaces . water is clearly an active player in six out of seven cases in which ligand binding / rejection is driven either by enthalpy ( blue label ) or by entropy ( green label ) . reported a thermodynamic analysis for six small nonpolar compounds binding the nonpolar cavity of mouse major urinary protein - i. (46 ) this cavity , created by mutation of a hydrophobic tryptophan residue to a glycine , presents a buried , negatively charged aspartic acid residue crucial for binding and displays similar volume and hydration to those of our ( , + ) model system. (18 ) among the seven systems investigated , the latter is the most closely related to the experimental data of enthalpy - driven binding . however , a more extensive , direct comparison of our data with water thermodynamics in proteinligand binding is hampered by the complexity of experimental interpretation . water thermodynamics depends on solute charge asymmetry ( e.g. (54 ) a quantitative interpretation of ion hydration thermodynamics is still widely debated in the current literature. (55 ) however , the interpretation of structure - making and structure - breaking trends is well consolidated . for this reason we use it here to explain evident charge asymmetry from our data . we can interpret charge asymmetry considering the different dipole orientation of the first two hydration shells . small ions of high charge density are structure forming ( kosmotropes ) , i.e. the ( + ) ligand employed in our simulations has similar parameters to those of the chaotropic cs cation , the ( ) ligand intermediate between cl and f anions , which are moderately kosmotropic. as suggested by collins et al.,(51 ) we notice qualitative differences within the first two hydration shells as the ligand moves from = 1.1 nm toward the cavity ( figure 5 ) . for the ( + ) ligand the reorganization of its second hydration shell is accompanied by an unfavorable entropy change , as expected for a chaotropic ion for which hydration is stabilized by entropy . on the contrary , for the ( ) ligand such reorganization requires an unfavorable enthalpy change , as expected for a kosmotropic ion whose hydration is enthalpy stabilized . , no free energy change occurs . water interaction is stronger with a ( ) vs ( + ) ligand because water hydrogen atoms can more closely approach the charge than oxygen atoms . the corresponding favorable change in entropy is also larger for the ( ) vs ( + ) ligand , but insufficient for enthalpy compensation , leading to an overall more positive free energy change upon dehydration . (52 ) comparison between ( + , n ) and ( ,n ) scenarios shows interesting asymmetry for water reorganization upon binding ( figure 3 , < 0.135 and 0.105 nm , respectively ) . possibly , the positively charged cavity confers to the approaching nonpolar ligand properties characteristic of a chaotropic ion ( i.e. , a structure - breaking effect on water ) . charge asymmetry in the ( ,+ ) and ( + , ) systems is evident in the proximity of the bound states ( see figure 4 , inset panels ) . in the ( ,+ ) system , the g minimum at eq = 0.135 nm results from an h minimum and a ts maximum corresponding to an optimal hydration shell between cavity and ligand . conversely , in the ( + , ) system this is not the case , consistently with the interpretation that the kosmotropic nature of the ( ) ligand weakens near the ( + ) cavity charge , thus reducing the enthalpy - dominated order of the surrounding water molecules . we used a straightforward computational approach and extensive sampling to derive complete thermodynamic signatures of model cavityligand recognition . free energy , enthalpy , and entropy estimates were obtained along the association coordinate for a set of seven systems with varying cavity and ligand physicochemical properties . this approach gives access to coupled solutesolvent thermodynamics along the association coordinate , thus allowing investigation of the fundamental link of molecular recognition thermodynamics with cavity and ligand hydration / dewetting . qualitatively different cavityligand binding scenarios and driving effects were revealed , despite the simplicity of the model systems employed , and will help the interpretation and design of new proteinligand binding experiments . a common feature is the generally underestimated key role of water as an active player in determining ligand binding or rejection . we discussed our data also in the context of the asymmetric nature of water thermodynamics for opposite charges . remarkably , our results suggest that overall cavityligand recognition and binding propensity do not need to be ( or are not limited to ) the result of direct cavityligand interactions . due to this effect , the resulting overall free energy change is of similar magnitude for hydrophobic and electrostatic - driven binding . in more complex biomolecular association , involving multivariate combinations of the scenarios derived herein , water could determine the subtle balance among driving forces of different physical natures , thus broadening the array of available binding mechanisms . our approach could be generalized to tackle the challenge of solvent entropy estimation in complex biological systems and drug design , starting from hostguest systems of treatable size . overall , this study suggests that the understanding of cavityligand recognition relies on an improved description of water thermodynamics and opens excellent possibilities for developing new implicit solvent and coarse - grained models toward gaining a more realistic representation of solvation properties .

in conjunction with the annual meeting of the international society for extracellular vesicles ( isev ) , a satellite symposium on hiv , neuroaids , drug abuse & evs was held on 23 april 2015 in bethesda , md , usa . the workshop was organised by dr shilpa buch from the university of nebraska medical center in cooperation with dr kenneth witwer from the johns hopkins university , dr jeymohan joseph from the national institute of mental health and dr john satterlee from the national institute on drug abuse . dr witwer as the local chair for isev2015 provided the introductory remarks and opened up the session . while various agents have been identified as initiators of the disease process , it is now becoming clear that spread of inflammation and toxicity is a key hallmark feature of disease progression . in this light , spread of disease process by exosomes is gaining momentum . exosomes are globular , membrane bound extracellular nanovesicles ( 30100 nm range ) that are shed from almost all types of cells both during normal cellular functioning , and specifically in response to cellular stressors.[14 ] these small vesicles originally thought to be junk cellular debris , were first described by trams et al . when they observed smaller membrane bound vesicles within the larger endosomes ( later termed multi - vesicular bodies ; mvbs ) . exosomes to sheep reticulocytes when they were shown to be released into the extracellular environment following the fusion of the multi - vesicular bodies with the plasma membrane . release of these small vesicles into the extracellular environment was the proposed mechanism by which reticulocytes secreted the transferrin receptor . this proposed mechanism was further supported by in vitro analyses of sheep reticulocytes , which demonstrated selective loss of certain proteins from the maturing cells . an understanding of their role in various cell types has evolved immensely since the turn of the century . they are no longer viewed as waste bags ; instead , exosomes are thought to play an important role as cargo - carrying vesicles mediating communication among diverse cells and tissues , including the cns . exosomes are known to carry nucleic acids ( rna , mirna and dna ) , functional proteins ( including viral material ) and other cellular products.[810 ] the human immunodeficiency virus ( hiv ) is a lentivirus ( a subgroup of retrovirus ) that slowly works to destroy the host s immune system by invading t - cells , eventually hijacking them and using cellular machinery to perpetuate this process . this course of infection eventually leads to acquired immunodeficiency syndrome ( aids ) , which subjects the host to increased risk of opportunistic infections . the development of combined antiretroviral therapy ( cart ) has been a major advancement in the control of aids . the use of cart has significantly increased the life span and health of individuals living with hiv-1 . however , despite the successful control of virus replication by antiretroviral therapies , there continues to be an increase in the number of individuals afflicted with hiv - associated neurological disorders ( hand ) , likely due to the increased longevity of individuals infected with hiv-1 as well as the existence of viral sanctuaries like the cns , despite the presence of cart . these neurological disorders manifest as a spectrum from mild , in which the condition may go undetected and does not impair a person s daily functionality , to moderate , wherein the patient experiences progressive cognitive impairment affecting the ability to function normally . a growing body of evidence suggests that exosomes play a vital role in neuroinflammatory diseases . these small vesicles are likely important in cns communication as most cns cells secrete them . cell communication via exosomes could play an important role in pathogenesis due to its ability to quickly transmit disease - causing agents from one cell to another . indeed , exosomes have been associated with numerous neuroinflammatory diseases including parkinson s , alzheimer s , and creutzfield - jacob diseases . continued research into the role these vesicles play in disease progression is important for finding effective preventative and therapeutic options . dr kenneth witwer noted that retroviruses and extracellular vesicles were intimately related and that retroviruses can be seen as a hijacked extracellular vesicle . from another perspective , the extracellular vesicle is a virus - like particle , transferring proteins , nucleic acids , and other cargoes to recipient cells . with the advent of ultracentrifuge technology in the first part of the twentieth century , methods were rapidly developed for virus and other subcellular particle purification . perhaps the earliest indications of evs were particles obtained from uninfected , negative control preparations . hints as to the roles of these particles were revealed in the 1940s , when chargaff and west reported that ultracentrifuged fractions of blood from healthy individuals could confer clotting properties to the blood of haemophiliacs . two decades later , peter wolf described sudanophilic ( lipid ) particles derived from platelets , famously referring to them as platelet dust . he further described the necessity of diluting plasma or serum before centrifugation to allow optimal pelleting of particles , a principle now well known to the ev researcher . much more was learned about evs in bone , in body fluids , in cancers , and in the immune system over the next several decades . around the turn of the century and in following years , viruses and evs met again on three fronts . first , with the finding ( a re - discovery of sorts ) that viruses and evs copurify by stepped ultracentrifugation , leading to adaptation of new density gradient methods ( and others ) for virus / ev separation . second , with the idea that the hiv particle evades the immune system by mimicking a native exosome . and third , with the rise of the exrna hypothesis : the notion that evs , like viruses , could shuttle rna from a cell of origin to a recipient cell . in this environment , the stage was set for new investigations into the relationships of evs and retroviruses . dr jeymohan joseph gave a talk on nimh priorities in neuroaids and exosome research . he provided a brief overview of the neuroaids research programmes and priorities supported by the hiv neuropathogenesis , genetics and therapeutics branch , division of aids research . this branch supports an integrated programme of studies to investigate the pathophysiology and genetic factors that contribute to hand . support is also provided for developing novel therapeutic strategies to mitigate the cns complications of hiv infection , using information gathered from research into hiv neuropathogenesis . he then outlined the following areas of focus relating to extracellular vesicles and hand that have been identified by nimh as priority areas : assessing changes in the physiology of exosome release and cargo content from cns - derived cells in the setting of hand;studying the impact of exosomes as novel conduits for cell cell communication in the setting of hand ( for example , assessing the effect of exosomes derived from macrophages / astrocytes on neuronal apoptosis or neuroprotection);studying the impact of highly active antiretroviral therapy ( haart ) on the content , release and effect of exosomes derived from hiv-1 infected cells of the cns;studying the impact of exosome - mediated intercellular signalling on the development and maintenance of neural circuits in adult and paediatric populations in the setting of hiv-1 infection of the cns;assessing the potential role of exosomal cargo as biomarkers to serve as clinical diagnostics for hand;assessing the role of exosomes as delivery vehicles for cns - targeted therapeutics ; andstudying the role of exosomes in cross talk between the periphery and cns with particular focus on inflammatory mediators . assessing changes in the physiology of exosome release and cargo content from cns - derived cells in the setting of hand ; studying the impact of exosomes as novel conduits for cell cell communication in the setting of hand ( for example , assessing the effect of exosomes derived from macrophages / astrocytes on neuronal apoptosis or neuroprotection ) ; studying the impact of highly active antiretroviral therapy ( haart ) on the content , release and effect of exosomes derived from hiv-1 infected cells of the cns ; studying the impact of exosome - mediated intercellular signalling on the development and maintenance of neural circuits in adult and paediatric populations in the setting of hiv-1 infection of the cns ; assessing the potential role of exosomal cargo as biomarkers to serve as clinical diagnostics for hand ; assessing the role of exosomes as delivery vehicles for cns - targeted therapeutics ; and studying the role of exosomes in cross talk between the periphery and cns with particular focus on inflammatory mediators . dr vincent c. bond talked on cytokines associated with exosomes in hiv - infected individuals . he presented data showing that thp-1 cells when transfected with hiv-1 negative regulatory factor ( nef ) or exposed to exosomal nef protein demonstrated increased surface expression of il-15/il-15r in a dose - dependent manner . peripheral blood mononuclear cells ( pbmcs ) exposed to exosomal nef showed increased cell surface expression of interleukin 15 receptor alpha subunit ( il-15r ) on cd14 + monocytes , and cd38 on central and effector memory cd4 and cd8 t - lymphocytes . exposure of pbmcs to exosomes purified from plasma of viremic hiv-1 infected individuals significantly increased cell surface expression of il-15r on cd14 + monocytes compared to untreated cells , suggesting exnef contributes to immune activation via induction of il-15/il-15r in monocytes and macrophages . his work examined purified exosomes for levels of 21 cytokines and chemokines . these were compared to similar cytokine / chemokines in the exosome depleted plasma . most cytokines were markedly enriched in exosomes from hiv+ individuals relative to exosomes from negative controls and to exosome depleted plasma . the authors demonstrated the biological relevance of these plasma exosomes , exposing naive pbmcs to exosomes purified from hiv+ patients . their group observed induction of cd38 expression on naive and central memory cd4 and cd8 + t cells relative to plasma exosomes from negative controls . in their study , they observed that most cytokines were not in free form , but rather were associated with exosomes . additionally , cytokines were actively and selectively enriched in plasma exosomes ( distinct from virions ) isolated from hiv-1 infected individuals . interestingly , this mechanism has remarkable similarity to a host mechanism leading to immune privilege during pregnancy . this process has been found to be hijacked during carcinogenesis leading to immune modulation / inflammation and ultimately allowing tumour growth . it was thus suggested that this mechanism likely contributed to inflammation and viral propagation via bystander cell activation . dr shilpa buch gave a talk on trans - activator of transcription ( tat ) and opiates modulate hand : blaming the messengers . impairment of microglial functions , such as activation and migration , comprise the underlying features of hand . the author s previous studies demonstrated that hiv tat via induction of mir-9 , a highly conserved and brain enriched mirna ( mir ) , plays a pivotal role in regulating the microglial inflammatory response in the context of hand . the goal of the current study was to examine whether hiv tat mediated release of mir-9 released from astrocytes , which could also lead to impairment of other microglial functions such as cell migration . the authors demonstrated upregulation of mir-9 in the brains of siv / hiv - infected subjects . these findings were further validated by in situ hybridisation , demonstrating increased expression of mir-9 in the astrocytes in the brains of siv - infected macaques compared with uninfected controls . the authors further demonstrated that evs released from tat treated astrocytes could shuttle mir-9 , which in turn , could be taken up by neighbouring microglia leading to microglial migration , as evidenced by increased migration in boyden chambers . the authors identified mir-9 as a positive regulator of microglial migration via downregulation of the key target protein , monocyte chemotactic protein - induced protein 1 and its downstream signalling , the -catenin pathway . in agreement with the in vitro results , the in vivo data demonstrated increased microglial migration towards the lipopolysaccharides ( lps ) injection site in the brains of mice administered exogenous mir-9 compared with the control group . these data not only shed light on the mechanism(s ) underlying the roles of ev mirs on microglial function(s ) but also serve as a foundation for future development of nanovesicle and mir - based therapeutics for the treatment of cognitive decline observed in hand . dr norman haughey gave a talk on brain - derived exosomes regulate the peripheral immune response to brain injury . he presented his findings that adoptive transfer of evs into the striatum induced a liver cytokine response , and activated leukocytes , which , in turn , transmigrated to the site of brain injection in a manner that was indistinguishable from the response following a simple injection of il-1 into the striatum . the protein and/or mir cargo of evs was thus responsible for the observed effects on leukocyte migration . the next step involved stimulation of isolated neurons , astrocytes , microglia and oligodendrocytes with il-1 ( and a plethora of other stimuli ) , to monitor the release of evs . the goal of the study was to investigate whether there was a soluble mediator released from the brain that could enter into the periphery . herein efforts were focused on astrocytes since these cells have an intimate association with endothelial cells of the blood the premise of the study was that if evs released from astrocytes regulated the peripheral immune response to an inflammatory brain lesion , then these nm - sized particles would have to efficiently cross the bbb . it was found that astrocytes isolated from mice expressing gfp under the control of a gfap promoter released evs that were gfp positive . this cellular labelling of evs provided a means to easily track the fate of astrocyte shed evs . using the transwell bbb system to track the transit of gfp+ evs shed from astrocytes , it was found that the astrocyte evs shed in response to il-1 rapidly crossed to the luminal side of the barrier without disrupting the trans - barrier resistance . these results thus suggested that evs shed from astrocytes could rapidly enter into the circulation without a gross disruption of the integrity of the bbb . ultrastructural examination of these transwell systems by electron microscopy showed that il-1 stimulation induced the formation of multivesicular bodies in astrocytes that was followed by the release of evs that entered into endothelial cells . the cellular entrance of evs appeared to occur largely by a macropinocytosis mechanism with a lesser contribution by endocytosis . most of the evs simply appeared at either the extracellular or intracellular surface of endothelial cells as single particles with a few encapsulated in what appeared to be endocytic bodies . the authors confirmed these results in vivo showing that striatal injection of il-1 into gfap - gfp mice resulted in the release of gfp labelled evs that entered into circulation . ultrastructural examination of these brain tissues by electron microscopy and immunogold labelling of gfp showed gold - labelled particles the size of evs located in the cytosol of endothelial cells adjacent to the site of il-1 injection . these gold - labelled particles were observed in small numbers in saline injected mice , with a low - basal level of gfp labelled nm - sized particles in liver , lung and spleen , suggesting thereby a possible constitutive release of evs from astrocytes into the peripheral circulation . this basal release could simply be the removal of cellular debris for transportation to peripheral macrophages for degradation , or it could represent a continuous communication of brain with the peripheral systems . in either case , the numbers of labelled evs in these tissues was dramatically increased following il-1 injection into the striatum . it was thus concluded that a constitutive low - level release of evs from astrocytes is increased in response to il-1 , and that these evs rapidly cross the bbb , and are localised to multiple tissues including the liver , lung and spleen . to determine the mechanism(s ) by which evs regulate the peripheral immune response to inflammatory brain lesions a proteomic , lipidomic , and nanostring ( mir ) analysis of evs released from astrocytes in response to il-1 was performed . it was determined that the cargo of evs was complex and contained hundreds of distinct proteins , lipids and mirs . it was reasoned that a focus on any one of these components would be unlikely to provide adequate information on how these complex particles regulated immune function . bioinformatics approaches interrogating the entire molecular composition of evs was thus used to assess the cargo . as ppar is highly expressed in liver and tissues that oxidise fatty acids , and regulates cytokine expression through nf-b , efforts were focused on assessing this pathway in keeping with previous reports by daniel anthony et al.,[3540 ] demonstrating an acute phase response in liver and that nf-b was required to prime neutrophils for transmigration into the brain parenchyma following il-1 induced brain lesion.[3540 ] immunoprecipitation of chromatin ( chip ) isolated from liver samples of mice following striatal injection of il-1 demonstrated increased binding of the nf-b subunit c - rel to the promoter regions of ccl2 , il-1 , and tnf , but not to il-17 , consistent with the increased expression of inflammatory cytokines . administration of the ppar antagonist fenofibrate just prior to the induction of an inflammatory brain lesion with il-1 prevented not only the binding of nf - kb binding to ccl2 , il-1 and tnf promoters but also the induction of inflammatory cytokines transmigration of leukocytes into the brain parenchyma . involvement of ppar was thus confirmed in mice administered il-1 in conjunction with the nsmase2 inhibitor altenusin . in this study leukocyte influx is normally blocked , but peripheral administration of the ppar- antagonist gw6471 re - established leukocyte recruitment to the site of il-1 induced lesion . these findings thus suggested that peripheral activation of leukocytes in response to an inflammatory brain lesion can be regulated by astrocyte shed evs that enter into peripheral circulation to regulate the liver acute cytokine response through suppression of ppar. dr fatah kashanchi gave a talk on exosomes from latent hiv infections : spreading the message beyond infection . he stated that nearly one third of hiv - infected individuals develop neurocognitive deficits despite adequate cart and excellent virological control in the blood . this range of neurocognitive deficits is collectively referred to as hand , and in general , the number of productively infected cells in the brain is small compared to the amount of neuronal damage , and the neurons are not infected with the virus . he stated that hiv may traffic into the brain via blood monocytes ( trojan horse phenomenon ) early in the course of infection long before symptoms of aids appear , and in fact the virus can be detected in the cerebrospinal fluid ( csf ) soon after a primary infection . microglia , perivascular and meningeal macrophages , astrocytes , and neural stem cells are sites of viral infection in the human brain and they potentially secrete exosomes . exosomes play a key role in intercellular communication , as well as in the pathogenesis of neurodegenerative diseases , including neuronal degeneration . however , the role of exosomes released from hiv-1 infected cells in the neurological impairments associated with hiv infection is still unclear . hiv-1 and other viruses encode multiple small , regulatory non - coding rnas ( ncrnas ) and mirs that regulate host gene expression via exosomes . they have previously shown that exosomes containing non - coding rna , called trans - activating response ( tar ) element rna , enhance susceptibility of undifferentiated naive cells to hiv-1 infection . the current studies indicated that exosomes from hiv-1 infected primary cells are highly abundant with tar rna as detected by rt - real - time pcr . interestingly , up to 0.11.0 million copies of tar rna per microlitre were also detected in human infected serum and also from hiv-1 infected humanised mice , suggesting that tar rna may be very stable in vivo . tar rna was also present in four out of 10 csf samples ( generous gift of dr a. nath ; nih ) tested from hiv-1 infected patients under cart . incubation of exosomes from hiv-1 infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines , il-6 and tnf- , indicating that exosomes containing tar rna could play a direct role in control of cytokine gene expression and hand . the intact tar molecule was able to bind to pkr , tlr3 effectively , whereas 5 and 3 stems bound best to tlr3 , 7 and 8 , and none to pkr . binding of tar to pkr did not result in its phosphorylation and , therefore , tar may be a dominant negative decoy molecule . the tar rna molecule has a 23 base pair stem , which is not sufficient to activate pkr . interestingly , the entire 9.5 kb rna genome does not have a straight 30 base pair stem and loop structure , potentially acting as dominant negative for pkr ( or related innate immune complexes ) . furthermore , the single - stranded 5 or 3 processed stem rna binding to tlrs activates the nf-b pathway and regulates cytokine expression . finally , the binding of rna to the tlrs can activate a new form of ikk- , which may contribute to the release of free p50 and p65 nf-b and ultimately contribute to cytokine activation . collectively , these results imply that exosomes containing tar rna could directly affect the proinflammatory cytokine gene expression ( potentially in naive uninfected cells ) and may explain a possible mechanism of inflammation observed in hiv-1 infected patients . the effects may be further enhanced in tissues vs. blood stream , where the uninfected cells are constantly exposed to neighbouring infected exosomes . this may ultimately result in an initial phase of activation , but over time with continued exposure may cause exhaustion and cell death . dr lynn pulliam gave a talk on monocyte immune activation in hiv can alter end organ function via exosomes . she commented that the monocyte is a critical first line response to the peripheral immune reaction to hiv infection . the previous findings from dr pulliam s group showed that circulating monocytes from hiv - infected individuals undergoing antiretroviral therapy had a type 1 interferon profile with increased levels of lps in the plasma . while the interferon profile in some individuals correlates with cognitive impairment , the levels of lps do not , suggesting that while lps is present , it is not entirely responsible for neural cell dysfunction . the authors were interested in exploring how the activated monocytes might influence neural cell function , in particular how exosomes from activated monocytes might cause subtle changes in neural cells . exosomes normally interact with recipient cells and maintain homeostasis ; however , activation not only alters the parent cell but also the exosomes they secrete . preliminary characterisation of monocyte - derived exosomes showed that when monocytes were stimulated with ifn , lps or a combination of the two , there was no difference in exosome size or amount secreted . because exosomes contain abundant mir , we were interested in comparing the monocyte profile with macrophages . using mir arrays , we determined that the most abundant mir in monocytes is mir-223 , with this mir being decreased when monocytes differentiate into macrophages . their profiles were similar with the most significant change being an increased abundance of mir146a / b and mir155 in monocytes and exosomes from lps and i / l treatments . finally , the impact of activated monocyte - derived exosomes on primary human astrocytes was examined by staining for p65 as an indicator of nf - kb activation . exosomes from monocytes with no activation showed minimal p65 staining and looked the same as astrocytes not treated with exosomes . exosomes from ifn - treated monocytes had a strong cytoplasmic p65 response with little to no p65 nuclear translocation . exosomes from lps - treated monocytes showed increased cytoplasmic and nuclear translocation of p65 ; the i / l - activated monocyte exosomes had almost exclusively nuclear p65 staining with little to no cytoplasmic staining . the preliminary interpretation is that ifn - activated exosomes may elicit a protective response whereas ifn in the presence of lps promote nuclear translocation . the results showed that activated monocytes can secrete exosomes with altered mir profiles and that these exosomes can activate nf - kb . these preliminary findings highlight the impact that activated peripheral monocyte exosomes can have on distant neural cells without the parent cell being present . experts in the field focused on different aspects of exosome biology in keeping with the nih priorities outlined by drs joseph and satterlee . dr bond s work revealed that plasma cytokines from infected individuals did not exist in the free form , rather were associated with exosomes . additionally , cytokines are actively and selectively enriched in plasma exosomes ( distinct from virions ) from infected individuals . dr buch s group found that exosomes released from astrocytes contained abundant levels of various mirs , and that exosomal mirs could be taken up by microglia , resulting in their activation and thereby contributing to neuroinflammation . dr haughey s work implicated that evs regulate leukocyte activation in the peripheral immune system , thereby underscoring the intricacies of bidirectional communication system . dr pulliam s findings showed that activated monocytes could secrete exosomes with altered mir profiles and that these exosomes could activate the transcription factor nf - kb in astrocytes . interestingly , dr kashanchi presented that exosomes containing tar rna could directly affect the proinflammatory cytokine gene expression profile , and that this could explain a possible mechanism of inflammation observed in hiv-1 infected patients . all experts agreed that the expanding knowledge concerning exosome biogenesis , composition , function , and use continues to provide new insights into the normal physiology as well as disease pathogenesis . these small vesicles are secreted by many cell types , including nearly all the cns cells , and are key in regulating cell cell communication . such a mechanism can have important implications in the context of disease progression and pathogenesis . this workshop focused mainly on the possible link between exosomes , hiv-1 pathogenesis , hiv - associated disease and drug abuse . the ability to target mirs involved in hiv-1 pathogenesis that are released by the exosomes could provide a novel means of controlling the infection / inflammation , which in turn could mitigate many of the complications associated with neurocognitive disorders . further study is needed concerning the application of exosome therapeutics , both through these vesicles as a drug delivery mechanism and , as therapeutic targets themselves , in the context of battling hiv-1 infection and its associated cns complications .

abstractextracellular vesicles ( evs ) are globular , membrane bound nanovesicles ( 30100 nm range ) that are shed both during normal cellular functioning and under pathological conditions by most cell types . in recent years , there has been significant interest in the study of these vesicles as conduits for the delivery of information between cells from both analogous and disparate tissues . their ability to carry specialised cargo including signalling mediators , proteins , messenger rna and mirnas characterises these vesicles as primary facilitators of cell - to - cell communication and regulation . evs have also been demonstrated to play important roles in the field of cancer biology and metastasis . however , significant knowledge gaps exist in the role these vesicles play in the context of hiv infection and drug abuse . to foster discussion in this area a satellite symposium on hiv , neuroaids , drug abuse & evs , was held in conjunction with the annual meeting of the international society for extracellular vesicles ( isev ) in bethesda , in april 2015 . experts in hiv and drug abuse fields were invited to share their findings on the role of evs in hiv-1 infection and drug addiction . additional discussion included current areas of research in ev biology in hiv infection and drug abuse .

Introduction Summary of presentations Conclusions Disclosure statement

in conjunction with the annual meeting of the international society for extracellular vesicles ( isev ) , a satellite symposium on hiv , neuroaids , drug abuse & evs was held on 23 april 2015 in bethesda , md , usa . the workshop was organised by dr shilpa buch from the university of nebraska medical center in cooperation with dr kenneth witwer from the johns hopkins university , dr jeymohan joseph from the national institute of mental health and dr john satterlee from the national institute on drug abuse . while various agents have been identified as initiators of the disease process , it is now becoming clear that spread of inflammation and toxicity is a key hallmark feature of disease progression . in this light , spread of disease process by exosomes is gaining momentum . exosomes are globular , membrane bound extracellular nanovesicles ( 30100 nm range ) that are shed from almost all types of cells both during normal cellular functioning , and specifically in response to cellular stressors. when they observed smaller membrane bound vesicles within the larger endosomes ( later termed multi - vesicular bodies ; mvbs ) . exosomes to sheep reticulocytes when they were shown to be released into the extracellular environment following the fusion of the multi - vesicular bodies with the plasma membrane . release of these small vesicles into the extracellular environment was the proposed mechanism by which reticulocytes secreted the transferrin receptor . this proposed mechanism was further supported by in vitro analyses of sheep reticulocytes , which demonstrated selective loss of certain proteins from the maturing cells . an understanding of their role in various cell types has evolved immensely since the turn of the century . they are no longer viewed as waste bags ; instead , exosomes are thought to play an important role as cargo - carrying vesicles mediating communication among diverse cells and tissues , including the cns . exosomes are known to carry nucleic acids ( rna , mirna and dna ) , functional proteins ( including viral material ) and other cellular products. [810 ] the human immunodeficiency virus ( hiv ) is a lentivirus ( a subgroup of retrovirus ) that slowly works to destroy the host s immune system by invading t - cells , eventually hijacking them and using cellular machinery to perpetuate this process . the development of combined antiretroviral therapy ( cart ) has been a major advancement in the control of aids . however , despite the successful control of virus replication by antiretroviral therapies , there continues to be an increase in the number of individuals afflicted with hiv - associated neurological disorders ( hand ) , likely due to the increased longevity of individuals infected with hiv-1 as well as the existence of viral sanctuaries like the cns , despite the presence of cart . these neurological disorders manifest as a spectrum from mild , in which the condition may go undetected and does not impair a person s daily functionality , to moderate , wherein the patient experiences progressive cognitive impairment affecting the ability to function normally . cell communication via exosomes could play an important role in pathogenesis due to its ability to quickly transmit disease - causing agents from one cell to another . continued research into the role these vesicles play in disease progression is important for finding effective preventative and therapeutic options . dr kenneth witwer noted that retroviruses and extracellular vesicles were intimately related and that retroviruses can be seen as a hijacked extracellular vesicle . from another perspective , the extracellular vesicle is a virus - like particle , transferring proteins , nucleic acids , and other cargoes to recipient cells . with the advent of ultracentrifuge technology in the first part of the twentieth century , methods were rapidly developed for virus and other subcellular particle purification . perhaps the earliest indications of evs were particles obtained from uninfected , negative control preparations . hints as to the roles of these particles were revealed in the 1940s , when chargaff and west reported that ultracentrifuged fractions of blood from healthy individuals could confer clotting properties to the blood of haemophiliacs . much more was learned about evs in bone , in body fluids , in cancers , and in the immune system over the next several decades . around the turn of the century and in following years , viruses and evs met again on three fronts . first , with the finding ( a re - discovery of sorts ) that viruses and evs copurify by stepped ultracentrifugation , leading to adaptation of new density gradient methods ( and others ) for virus / ev separation . second , with the idea that the hiv particle evades the immune system by mimicking a native exosome . and third , with the rise of the exrna hypothesis : the notion that evs , like viruses , could shuttle rna from a cell of origin to a recipient cell . in this environment , the stage was set for new investigations into the relationships of evs and retroviruses . dr jeymohan joseph gave a talk on nimh priorities in neuroaids and exosome research . he provided a brief overview of the neuroaids research programmes and priorities supported by the hiv neuropathogenesis , genetics and therapeutics branch , division of aids research . support is also provided for developing novel therapeutic strategies to mitigate the cns complications of hiv infection , using information gathered from research into hiv neuropathogenesis . he then outlined the following areas of focus relating to extracellular vesicles and hand that have been identified by nimh as priority areas : assessing changes in the physiology of exosome release and cargo content from cns - derived cells in the setting of hand;studying the impact of exosomes as novel conduits for cell cell communication in the setting of hand ( for example , assessing the effect of exosomes derived from macrophages / astrocytes on neuronal apoptosis or neuroprotection);studying the impact of highly active antiretroviral therapy ( haart ) on the content , release and effect of exosomes derived from hiv-1 infected cells of the cns;studying the impact of exosome - mediated intercellular signalling on the development and maintenance of neural circuits in adult and paediatric populations in the setting of hiv-1 infection of the cns;assessing the potential role of exosomal cargo as biomarkers to serve as clinical diagnostics for hand;assessing the role of exosomes as delivery vehicles for cns - targeted therapeutics ; andstudying the role of exosomes in cross talk between the periphery and cns with particular focus on inflammatory mediators . assessing changes in the physiology of exosome release and cargo content from cns - derived cells in the setting of hand ; studying the impact of exosomes as novel conduits for cell cell communication in the setting of hand ( for example , assessing the effect of exosomes derived from macrophages / astrocytes on neuronal apoptosis or neuroprotection ) ; studying the impact of highly active antiretroviral therapy ( haart ) on the content , release and effect of exosomes derived from hiv-1 infected cells of the cns ; studying the impact of exosome - mediated intercellular signalling on the development and maintenance of neural circuits in adult and paediatric populations in the setting of hiv-1 infection of the cns ; assessing the potential role of exosomal cargo as biomarkers to serve as clinical diagnostics for hand ; assessing the role of exosomes as delivery vehicles for cns - targeted therapeutics ; and studying the role of exosomes in cross talk between the periphery and cns with particular focus on inflammatory mediators . dr vincent c. bond talked on cytokines associated with exosomes in hiv - infected individuals . these were compared to similar cytokine / chemokines in the exosome depleted plasma . the authors demonstrated the biological relevance of these plasma exosomes , exposing naive pbmcs to exosomes purified from hiv+ patients . this process has been found to be hijacked during carcinogenesis leading to immune modulation / inflammation and ultimately allowing tumour growth . the author s previous studies demonstrated that hiv tat via induction of mir-9 , a highly conserved and brain enriched mirna ( mir ) , plays a pivotal role in regulating the microglial inflammatory response in the context of hand . the goal of the current study was to examine whether hiv tat mediated release of mir-9 released from astrocytes , which could also lead to impairment of other microglial functions such as cell migration . the authors demonstrated upregulation of mir-9 in the brains of siv / hiv - infected subjects . these findings were further validated by in situ hybridisation , demonstrating increased expression of mir-9 in the astrocytes in the brains of siv - infected macaques compared with uninfected controls . the authors identified mir-9 as a positive regulator of microglial migration via downregulation of the key target protein , monocyte chemotactic protein - induced protein 1 and its downstream signalling , the -catenin pathway . in agreement with the in vitro results , the in vivo data demonstrated increased microglial migration towards the lipopolysaccharides ( lps ) injection site in the brains of mice administered exogenous mir-9 compared with the control group . these data not only shed light on the mechanism(s ) underlying the roles of ev mirs on microglial function(s ) but also serve as a foundation for future development of nanovesicle and mir - based therapeutics for the treatment of cognitive decline observed in hand . he presented his findings that adoptive transfer of evs into the striatum induced a liver cytokine response , and activated leukocytes , which , in turn , transmigrated to the site of brain injection in a manner that was indistinguishable from the response following a simple injection of il-1 into the striatum . the protein and/or mir cargo of evs was thus responsible for the observed effects on leukocyte migration . the next step involved stimulation of isolated neurons , astrocytes , microglia and oligodendrocytes with il-1 ( and a plethora of other stimuli ) , to monitor the release of evs . the goal of the study was to investigate whether there was a soluble mediator released from the brain that could enter into the periphery . herein efforts were focused on astrocytes since these cells have an intimate association with endothelial cells of the blood the premise of the study was that if evs released from astrocytes regulated the peripheral immune response to an inflammatory brain lesion , then these nm - sized particles would have to efficiently cross the bbb . this cellular labelling of evs provided a means to easily track the fate of astrocyte shed evs . using the transwell bbb system to track the transit of gfp+ evs shed from astrocytes , it was found that the astrocyte evs shed in response to il-1 rapidly crossed to the luminal side of the barrier without disrupting the trans - barrier resistance . these results thus suggested that evs shed from astrocytes could rapidly enter into the circulation without a gross disruption of the integrity of the bbb . ultrastructural examination of these transwell systems by electron microscopy showed that il-1 stimulation induced the formation of multivesicular bodies in astrocytes that was followed by the release of evs that entered into endothelial cells . the cellular entrance of evs appeared to occur largely by a macropinocytosis mechanism with a lesser contribution by endocytosis . most of the evs simply appeared at either the extracellular or intracellular surface of endothelial cells as single particles with a few encapsulated in what appeared to be endocytic bodies . the authors confirmed these results in vivo showing that striatal injection of il-1 into gfap - gfp mice resulted in the release of gfp labelled evs that entered into circulation . ultrastructural examination of these brain tissues by electron microscopy and immunogold labelling of gfp showed gold - labelled particles the size of evs located in the cytosol of endothelial cells adjacent to the site of il-1 injection . these gold - labelled particles were observed in small numbers in saline injected mice , with a low - basal level of gfp labelled nm - sized particles in liver , lung and spleen , suggesting thereby a possible constitutive release of evs from astrocytes into the peripheral circulation . this basal release could simply be the removal of cellular debris for transportation to peripheral macrophages for degradation , or it could represent a continuous communication of brain with the peripheral systems . in either case , the numbers of labelled evs in these tissues was dramatically increased following il-1 injection into the striatum . it was thus concluded that a constitutive low - level release of evs from astrocytes is increased in response to il-1 , and that these evs rapidly cross the bbb , and are localised to multiple tissues including the liver , lung and spleen . to determine the mechanism(s ) by which evs regulate the peripheral immune response to inflammatory brain lesions a proteomic , lipidomic , and nanostring ( mir ) analysis of evs released from astrocytes in response to il-1 was performed . it was determined that the cargo of evs was complex and contained hundreds of distinct proteins , lipids and mirs . it was reasoned that a focus on any one of these components would be unlikely to provide adequate information on how these complex particles regulated immune function . bioinformatics approaches interrogating the entire molecular composition of evs was thus used to assess the cargo . [3540 ] immunoprecipitation of chromatin ( chip ) isolated from liver samples of mice following striatal injection of il-1 demonstrated increased binding of the nf-b subunit c - rel to the promoter regions of ccl2 , il-1 , and tnf , but not to il-17 , consistent with the increased expression of inflammatory cytokines . administration of the ppar antagonist fenofibrate just prior to the induction of an inflammatory brain lesion with il-1 prevented not only the binding of nf - kb binding to ccl2 , il-1 and tnf promoters but also the induction of inflammatory cytokines transmigration of leukocytes into the brain parenchyma . involvement of ppar was thus confirmed in mice administered il-1 in conjunction with the nsmase2 inhibitor altenusin . in this study leukocyte influx is normally blocked , but peripheral administration of the ppar- antagonist gw6471 re - established leukocyte recruitment to the site of il-1 induced lesion . he stated that nearly one third of hiv - infected individuals develop neurocognitive deficits despite adequate cart and excellent virological control in the blood . this range of neurocognitive deficits is collectively referred to as hand , and in general , the number of productively infected cells in the brain is small compared to the amount of neuronal damage , and the neurons are not infected with the virus . he stated that hiv may traffic into the brain via blood monocytes ( trojan horse phenomenon ) early in the course of infection long before symptoms of aids appear , and in fact the virus can be detected in the cerebrospinal fluid ( csf ) soon after a primary infection . microglia , perivascular and meningeal macrophages , astrocytes , and neural stem cells are sites of viral infection in the human brain and they potentially secrete exosomes . exosomes play a key role in intercellular communication , as well as in the pathogenesis of neurodegenerative diseases , including neuronal degeneration . however , the role of exosomes released from hiv-1 infected cells in the neurological impairments associated with hiv infection is still unclear . they have previously shown that exosomes containing non - coding rna , called trans - activating response ( tar ) element rna , enhance susceptibility of undifferentiated naive cells to hiv-1 infection . collectively , these results imply that exosomes containing tar rna could directly affect the proinflammatory cytokine gene expression ( potentially in naive uninfected cells ) and may explain a possible mechanism of inflammation observed in hiv-1 infected patients . dr lynn pulliam gave a talk on monocyte immune activation in hiv can alter end organ function via exosomes . she commented that the monocyte is a critical first line response to the peripheral immune reaction to hiv infection . the previous findings from dr pulliam s group showed that circulating monocytes from hiv - infected individuals undergoing antiretroviral therapy had a type 1 interferon profile with increased levels of lps in the plasma . the authors were interested in exploring how the activated monocytes might influence neural cell function , in particular how exosomes from activated monocytes might cause subtle changes in neural cells . exosomes normally interact with recipient cells and maintain homeostasis ; however , activation not only alters the parent cell but also the exosomes they secrete . preliminary characterisation of monocyte - derived exosomes showed that when monocytes were stimulated with ifn , lps or a combination of the two , there was no difference in exosome size or amount secreted . their profiles were similar with the most significant change being an increased abundance of mir146a / b and mir155 in monocytes and exosomes from lps and i / l treatments . the preliminary interpretation is that ifn - activated exosomes may elicit a protective response whereas ifn in the presence of lps promote nuclear translocation . experts in the field focused on different aspects of exosome biology in keeping with the nih priorities outlined by drs joseph and satterlee . dr bond s work revealed that plasma cytokines from infected individuals did not exist in the free form , rather were associated with exosomes . dr haughey s work implicated that evs regulate leukocyte activation in the peripheral immune system , thereby underscoring the intricacies of bidirectional communication system . dr pulliam s findings showed that activated monocytes could secrete exosomes with altered mir profiles and that these exosomes could activate the transcription factor nf - kb in astrocytes . interestingly , dr kashanchi presented that exosomes containing tar rna could directly affect the proinflammatory cytokine gene expression profile , and that this could explain a possible mechanism of inflammation observed in hiv-1 infected patients . these small vesicles are secreted by many cell types , including nearly all the cns cells , and are key in regulating cell cell communication . such a mechanism can have important implications in the context of disease progression and pathogenesis . this workshop focused mainly on the possible link between exosomes , hiv-1 pathogenesis , hiv - associated disease and drug abuse . the ability to target mirs involved in hiv-1 pathogenesis that are released by the exosomes could provide a novel means of controlling the infection / inflammation , which in turn could mitigate many of the complications associated with neurocognitive disorders . further study is needed concerning the application of exosome therapeutics , both through these vesicles as a drug delivery mechanism and , as therapeutic targets themselves , in the context of battling hiv-1 infection and its associated cns complications .

in addition to traditional experimental , theoretical and computational scientific paradigms , increasing use of cyberspace ( 1 ) in scientific settings has prompted the development of a fourth scientific paradigm , widely known as } cyber paradigm ( 2 ) . in the computing context , } cyber is an internet - related prefix , which is added to a wide range of existing concepts , to describe any person , thing , idea or space relating to the digital world and global networks ( 3 ) . in recent studies , authors focus on rethinking the notion of modern science , by suggesting a range of cyber - related concepts , such as cyber trade , cyber security , cyber laws , cyber infrastructure , cyber learning and cyber medicine ( 4 - 9 ) . as koschmann ( 10 ) argues paradigm shifts in technology prompt the development of new theoretical and practical frameworks in science . several studies provide evidence that medical education systems , in view of the cyber - paradigm shift , dynamically incorporate cyberspace capabilities ( 11 - 14 ) . in fact , a number of well - known medical universities , such as oxford ( 15 ) , harvard ( 16 ) , johns hopkins ( 17 ) , sydney ( 18 ) and tokyo ( 19 ) used a wide range of cyberspace capabilities including database application , web - based learning , simulation models and intelligent learning games to increase their competitive advantage . according to health across reports ( 20 ) , over the past few decades , two revolutionary approaches have emerged as a new form of medical education : electronic medical education ( 2005 - 2010 ) and web - based medical education ( 2010 - 2020 ) . however , researchers have expressed that cyberspace will change health system s main objective of training physicians and medical education once again ( 21 ) . at present , in most countries , the establishment of intelligent government ( 22 ) has increased the options for optimal use of cyberspace features in health system and medical education . various authors have argued that the integration and sharing of policies , strategies , assets , infrastructure and human capital on cyberspace , can strengthen and expand the status of higher education ( in general ) and medical education ( in particular ) at international levels . however , it can imply interdisciplinary cooperation between information and communication technology system and health system which leads to intelligent and innovative approaches to the development of medical education on cyberspace . this article aimed to critically review the learning systems in cyberspace , to identify particular factors that have led to successful medical education in cyberspace . the main goal of this study was to identify and to understand the health system critical issues in terms of core components and mechanisms involved in the development of medical education in cyberspace . our assumption was that successful medical education in cyberspace required an integrated and balanced structure from higher levels to lower levels . in this study , we closely observed the principles of a critical literature review , based on the four - phase method adopted by carnwell and daly ( 23 ) . the critical methodology are regularly employed in the exploratory stage of research ( 24 ) to identify challenges , to get a sense of what cross - disciplinary studies have suggested and to provide directions for future research , and many studies used this method ( 25 - 27 ) . since studies of medical education development on cyberspace are still on exploratory phases , critical literature review was considered as the appropriate method to achieve the goal of our study . we did this review of the literature with a specific focus on health and cyber system functions ; it was associated with a systemic approach . critical literature review usually requires including many articles and related documents in detail , to understand the main issues from different perspectives , approaches and frameworks . boaro noted that critical review does not mean to provide just criticism ; summarizing and reducing the reviewed literature to main points and expressing the most important opinions is what a critical review does ( 28 ) . the four - phase method in critical literature review consists of a ) reviewing the literature of a wide area ; b ) exploring the key scope of the review ; c ) organizing the results into themes ; and d ) concluding and informing further studies . according to carnwell , the researcher begins with a clear definition of the research scope and a critical appraisal of the main literature ; then he or she attempts to expand the issue through seeing beyond what others have worked out , and so the researcher should identify particular components in the existing knowledge , as well as , potential knowledge gaps , next organizes the evidence in the results , discussion and conclusion and ultimately , provides directions for future research ( 29 ) . a search for reports and peer - reviewed publications was conducted in relevant databases , including pubmed , google scholar , eric ( active descriptor medical education ) and web of science . search terms included medical education , educational technology , health information technology and health systems plans . we also searched related terms such as delivery concepts in cyberspace ( internet , web , online , virtual and distance ) , learning concepts ( learning process , curriculum , learning environment and learning administer ) , health system concepts ( stewardship , governance and management ) . we restricted our search to reports published in or after 1990 , because the cyberspace developed in the 1990s , when the uses of the internet and digital communication were growing intensely and the term " cyberspace " was able to represent the many phenomena that were emerging ; the last date of search was 2015 . we also scanned organizational reports and conference proceedings , which were relevant to the present research objectives . this approach produced further articles , books and book chapters to be included in the study . due to the heterogeneity of the research field , it was very difficult to define the criteria applied to ensuring adequacy of studies in this research . in here , we point out that the inclusion criteria did not constitute a fixed set of standard criteria , but needed to be understood as broad criteria in this particular research . anyway , we keep in mind the basic principles that the research is based on openness , interaction between the researchers and providing a clear documentation of the search strategy , information collection and analysis ( 32 ) . we also observed the rules for a sound research and all decisions were made collectively ( 33 ) . inclusion criteria were used in selecting the studies : first , those studies were selected which addressed the topics of higher education or medical education in cyberspace and were relevant to the core components or mechanisms of health system and its functions at all levels ; second , the studies had to be published in english language and had open access . studies were excluded if they reported distance learning without internet use ( such as using compact disc or audiovisual materials ) and if their full access needed to be purchased . in the initial review after excluding irrelevant studies a total of 208 documents were selected that were clustered into six levels for the present study . the documents were too heterogeneous ; literature addressed a wide range of medical education topics in cyberspace , in addition to numerous interventions at disparate levels . we identified core dimensions , components and main indices observed in the studies ( table 1 ) . we also attempted to explain essential mechanisms that could be applied to the development of medical education in cyberspace ( table 2 ) . taxonomy in informatics is defined as the practice of information classification and big data categorization ; the approach in which ideas and issues are recognized , differentiated , and understood via clarification of the relationship between abstract and concrete constructs ( 34 ) . conceptual categories is an advanced method of the taxonomy - based approaches ( 35 ) . we addressed a six - level taxonomy as a key solution that can be applied to the success of medical education on cyberspace ; these include cyber level , governance level , ministry level , organization level , program level and performance level . the results were summarized and appraised in more details as follows : medical education on cyberspace : level , dimension , component and index critical mechanisms for development of medical education on cyberspace this level has formed based on integrated capabilities of interactive web and semantic web in digital world . according to stewart and colleagues ( 36 ) cyber technology includes a series of computer system , data storage system , data management system and those who are able to communicate with each other via advanced network systems . dimensions of this level have been strongly emphasized in the interdisciplinary literature ; from the perspective of the computing research association , cyber technology and related services has provided endless possibilities in academic landscape all around the world ( 37 ) . cyber infrastructure includes network infrastructure , computing infrastructure , communication infrastructure , information infrastructure , values infrastructure , political , social and economic infrastructures ( 38 - 41 ) . these " hard and soft cyber infrastructures are necessary for a successful medical education in cyberspace . hard infrastructure ( 42 ) refers to large cyber - physical systems which are the integrations of computation , networking , and physical processes and are necessary for the operational functions . soft infrastructure ( 43 ) refers to the management framework of a cyber - system , such as cyber policy making , cyber strategic planning , cyber financial system and system of governments . we emphasize that integrated cyber infrastructures provide a quantum leap for developing medical education in cyberspace . sawyer stated that the poor or incomplete design of cyber- infrastructure , even in small size , led to large problems in any system that depended on cyberspace ( 44 ) . designing , implantation and evaluation of medical education in cyberspace with a look to main indices such as availability , accessibility , flexibility , security and mobility is also considered a developmental mechanism at this level . medical education in cyberspace is still novel at specialized level , so it should receive more attention . although the cyber infrastructure as a key asset has a strategic importance in the health system , leaders have a less comprehensive attention to it . we suggest that they should take seriously their role and participate in policy making in cyber level through an interdisciplinary communication and network collaboration . obviously the governments have the highest political level in all countries that influence the future orientation on cyber - based learning systems . in recent years , some of the core government plans of these issues have been around to create intelligent infrastructures , to enhance collaboration and to work on readiness indices ( 45,46 ) . despite overwhelming evidence of policy initiatives in cyber issues , since 1980 , there have still been some gaps between policy - makers objectives and what actually happens at the point of policy implementation ( 47 ) , due to potential conflict of interest among leaders and stakeholders , which had a significant impact on implementation of formulated policies . conflict management can improve enforcement of rules , quality of decisions , financial approach and competitive advantage on a national and international scale . since the medical education on cyberspace consists of three structures including cyber system , learning system and medical system , different metaphors in these areas lead to important challenges such as overlapping powers and authority , interference of policies , practices and tasks , lack of cooperation among stakeholders and lack of accountability at this level . rasche suggested network governance model as the effective solution for stewardship of complex issues in complex systems ( 48 ) . however , the best practice for developing networked stewardship in health system ( especially on cyberspace ) has yet to be fully understood . researchers should attempt to bridge this gap by investigating the upstream process in health systems . we believe that integrated stewardship ( 49 ) is one of the key factors in governance level , which can have influences in the establishment and development of medical education in cyberspace . the optimized resource allocation and supported budgetary system are also critical mechanism for development of medical education on cyberspace . in addition , recognition and use of main index to supervise and assess this level is necessary ; the indices mentioned above can be good governance indicators , finance metrics and collaboration indicators . finally , we suggest integrated governance and stewardship approach as the most important mechanism for the development of medical education on cyberspace . by developing cyber technology , the roles and responsibility of ministers will take on new meaning . for example , in the development of medical education in cyberspace , the minister of information and communication technology ( ict ) is accountable for cyber issues ( 50 - 52 ) , therefore , they need to collaborate with health minister and to be aware of main health system s policies and medical education goals for accountability . currently , as nagy k. hanna ( 53 ) has emphasized , enhanced capacity of ministerial advisors for strategic analysis of learning technology , new learning environments and emerging education market is an essential key mechanism . some researchers maintain that a country leaders authority and the link with the chain of the organizational factors have an impact on promoting the capacity building and innovations ( 54,55 ) . thus professional cooperation at higher level according to richard heeks ( 56 ) cooperation and consultation at senior official s level can improve rule and standards effectiveness and transparency and accountability provide added value . in this regard , we emphasize the importance of the link between ministries . it seems , there is a deficiency on creating a common strategic perspective as a key mechanism at this level , particularly on determining available capacities and potential assets that may impact on main performance in cyberspace . sustainability , accountability , capacity building , competitive advantage were main indices that were emphasized for assessment of this level . also internal and external evaluation was significant mechanisms for the evaluation of developmental action at this level . according to carayannis & campbell ( 50 ) and smith & leydesdorff ( 51 ) , although the tricycle of government , industry and university cooperating together is a unique innovative ecosystem , their stability is affected by the political , economic , technological events . this is very important to consider and to prevent it through reinforcement of links between ministerial levels in the development of medical education on cyberspace . the idea of the university as a cyberspace leads to a great leap in higher education architecture . based on this change , virtualization in computational , organizational and geographical aspects , leads to integrated learning services . in cyber university model , introduced by lee and colleagues as the third generation of virtual universities , there is no physical space as university . these factors are stability , responsibility and quality ( strategic ) ; availability , interoperability and safety ( technical ) ; management , record keeping , finance , location ( instructional ) and other factors such as culture , curricula , procedural requirements , activities , performance , resources , infrastructure , human resources , suppliers , capacity , and budgets ( 57 ) . the concepts mentioned above are not new in medical education system , while applying them in the medical education in cyberspace is in the early stages . how should the medical education be designed , implemented , evaluated and developed on cyberspace ? which organizations have a significant role of upgrading the standards and protocols for cyber learning in the medical field ? what virtual , financial or human resources are needed for the formation of this level ? unfortunately , it seems that many medical organizations and medical education institutes have issued a variety of strategic documents to address cyber learning and research without a clear plan to respond to the above questions ( 58,59 ) . we are confident that education administrators can focus on best practices to achieve efficiency in medical education on cyberspace and develop it based on a professional perspective . it is suggested that for the development of medical education on cyberspace , managers pay more attention to the integration of organization architecture ( medical school , medical research center , medical center and community medicine ) via the service - oriented architecture approaches . service - oriented architecture is a logical framework based on the analysis of infrastructures , stakeholders and context for distribution of services in systems ( 60 ) . indicators identified to evaluate this level include interoperability , affordability , productivity , outcomes and impacts . this level formed based on the idea of global learning design ( gld ) that critical thinking as well as having different perspective on learning system is its prerequisite ( 61 ) . there are many choices for curriculum design on cyberspace which has attracted less attention , from a fully - controlled design to facilitating learner ownership in its design such as user generated learning environments system ( ugles ) . although learning content management and learning management systems are dominant in medical education , user generated learning will move ahead . since the development of such platform involves designing , programming and controlling of learning environment , scientists will face major challenges in future , especially by those users who had little computational thinking skills which are essential for deeper understanding of dynamic curriculum planning ( 62 - 63 ) . in addition , curriculum planning for medical education on cyberspace should observe moral dimensions ; professional development in this dimension is the main mechanism . as albaqami argued ( 64 ) , honesty , coordination , respect , reflexivity , competition , responsibility and loyalty are among the most important and well - known values at both traditional and cyber learning environments . therefore , to know principles and codes of ethics in the field of technology and medical education is necessary to develop medical education on cyberspace ; otherwise , unintended consequences may occur in relation to learning outcomes . this type of thinking about system quality is essential and it can be applied to the development of an actual medical education on cyberspace . . currently , innovative medical software applications such as medscape , visualdx , medcalc , labgear , uptodate , dropbox , resuscitation , etc . ( 65 ) are used more than before , while previous medical specialists and medical students were not even familiar with any of these applications ( 66 ) . interactive web and web modules applications ( 67 - 68 ) have been the main tools of learning toolkits in cyberspace , based on bloom s digital taxonomy ( 69 ) . it has covered most of the learning objectives and performance on cyberspace ( 69 - 70 ) . the first step of " call to action " at this level requires cyber - literacy . he defined cyber literacy as conceptual understandings of the nature of cyber world such as awareness of what you re doing online or consequences of the cyberspace activities . also she pointed out that the cyberspace should be navigated with awareness and emphasized on critical competencies for the learner to judge the legitimacy and credibility of cyber learning environments ( 71 ) . at this level , technology acceptance and user preferences the cyber generations are diverse in their learning performance which is related to personality traits , values , attitudes and interest of self - learning ; therefore , special attention on these individual differences for design and development of medical education on cyberspace is a useful step . this level has formed based on integrated capabilities of interactive web and semantic web in digital world . according to stewart and colleagues ( 36 ) cyber technology includes a series of computer system , data storage system , data management system and those who are able to communicate with each other via advanced network systems . dimensions of this level have been strongly emphasized in the interdisciplinary literature ; from the perspective of the computing research association , cyber technology and related services has provided endless possibilities in academic landscape all around the world ( 37 ) . cyber infrastructure includes network infrastructure , computing infrastructure , communication infrastructure , information infrastructure , values infrastructure , political , social and economic infrastructures ( 38 - 41 ) . these " hard and soft cyber infrastructures are necessary for a successful medical education in cyberspace . hard infrastructure ( 42 ) refers to large cyber - physical systems which are the integrations of computation , networking , and physical processes and are necessary for the operational functions . soft infrastructure ( 43 ) refers to the management framework of a cyber - system , such as cyber policy making , cyber strategic planning , cyber financial system and system of governments . we emphasize that integrated cyber infrastructures provide a quantum leap for developing medical education in cyberspace . sawyer stated that the poor or incomplete design of cyber- infrastructure , even in small size , led to large problems in any system that depended on cyberspace ( 44 ) . designing , implantation and evaluation of medical education in cyberspace with a look to main indices such as availability , accessibility , flexibility , security and mobility is also considered a developmental mechanism at this level . medical education in cyberspace is still novel at specialized level , so it should receive more attention . although the cyber infrastructure as a key asset has a strategic importance in the health system , leaders have a less comprehensive attention to it . we suggest that they should take seriously their role and participate in policy making in cyber level through an interdisciplinary communication and network collaboration . obviously the governments have the highest political level in all countries that influence the future orientation on cyber - based learning systems . in recent years , some of the core government plans of these issues have been around to create intelligent infrastructures , to enhance collaboration and to work on readiness indices ( 45,46 ) . despite overwhelming evidence of policy initiatives in cyber issues , since 1980 , there have still been some gaps between policy - makers objectives and what actually happens at the point of policy implementation ( 47 ) , due to potential conflict of interest among leaders and stakeholders , which had a significant impact on implementation of formulated policies . conflict management can improve enforcement of rules , quality of decisions , financial approach and competitive advantage on a national and international scale . since the medical education on cyberspace consists of three structures including cyber system , learning system and medical system , different metaphors in these areas lead to important challenges such as overlapping powers and authority , interference of policies , practices and tasks , lack of cooperation among stakeholders and lack of accountability at this level . rasche suggested network governance model as the effective solution for stewardship of complex issues in complex systems ( 48 ) . however , the best practice for developing networked stewardship in health system ( especially on cyberspace ) has yet to be fully understood . researchers should attempt to bridge this gap by investigating the upstream process in health systems . we believe that integrated stewardship ( 49 ) is one of the key factors in governance level , which can have influences in the establishment and development of medical education in cyberspace . the optimized resource allocation and supported budgetary system are also critical mechanism for development of medical education on cyberspace . in addition , recognition and use of main index to supervise and assess this level is necessary ; the indices mentioned above can be good governance indicators , finance metrics and collaboration indicators . finally , we suggest integrated governance and stewardship approach as the most important mechanism for the development of medical education on cyberspace . by developing cyber technology , the roles and responsibility of ministers will take on new meaning . for example , in the development of medical education in cyberspace , the minister of information and communication technology ( ict ) is accountable for cyber issues ( 50 - 52 ) , therefore , they need to collaborate with health minister and to be aware of main health system s policies and medical education goals for accountability . currently , as nagy k. hanna ( 53 ) has emphasized , enhanced capacity of ministerial advisors for strategic analysis of learning technology , new learning environments and emerging education market is an essential key mechanism . some researchers maintain that a country leaders authority and the link with the chain of the organizational factors have an impact on promoting the capacity building and innovations ( 54,55 ) . thus professional cooperation at higher level according to richard heeks ( 56 ) cooperation and consultation at senior official s level can improve rule and standards effectiveness and transparency and accountability provide added value . in this regard , we emphasize the importance of the link between ministries . it seems , there is a deficiency on creating a common strategic perspective as a key mechanism at this level , particularly on determining available capacities and potential assets that may impact on main performance in cyberspace . sustainability , accountability , capacity building , competitive advantage were main indices that were emphasized for assessment of this level . also internal and external evaluation was significant mechanisms for the evaluation of developmental action at this level . according to carayannis & campbell ( 50 ) and smith & leydesdorff ( 51 ) , although the tricycle of government , industry and university cooperating together is a unique innovative ecosystem , their stability is affected by the political , economic , technological events . this is very important to consider and to prevent it through reinforcement of links between ministerial levels in the development of medical education on cyberspace . the idea of the university as a cyberspace leads to a great leap in higher education architecture . based on this change , virtualization in computational , organizational and geographical aspects , leads to integrated learning services . in cyber university model , introduced by lee and colleagues as the third generation of virtual universities , there is no physical space as university . the complexity of modern universities increases under the influence of some factors . these factors are stability , responsibility and quality ( strategic ) ; availability , interoperability and safety ( technical ) ; management , record keeping , finance , location ( instructional ) and other factors such as culture , curricula , procedural requirements , activities , performance , resources , infrastructure , human resources , suppliers , capacity , and budgets ( 57 ) . the concepts mentioned above are not new in medical education system , while applying them in the medical education in cyberspace is in the early stages . how should the medical education be designed , implemented , evaluated and developed on cyberspace ? which organizations have a significant role of upgrading the standards and protocols for cyber learning in the medical field ? what virtual , financial or human resources are needed for the formation of this level ? unfortunately , it seems that many medical organizations and medical education institutes have issued a variety of strategic documents to address cyber learning and research without a clear plan to respond to the above questions ( 58,59 ) . we are confident that education administrators can focus on best practices to achieve efficiency in medical education on cyberspace and develop it based on a professional perspective . it is suggested that for the development of medical education on cyberspace , managers pay more attention to the integration of organization architecture ( medical school , medical research center , medical center and community medicine ) via the service - oriented architecture approaches . service - oriented architecture is a logical framework based on the analysis of infrastructures , stakeholders and context for distribution of services in systems ( 60 ) . indicators identified to evaluate this level include interoperability , affordability , productivity , outcomes and impacts . this level formed based on the idea of global learning design ( gld ) that critical thinking as well as having different perspective on learning system is its prerequisite ( 61 ) . there are many choices for curriculum design on cyberspace which has attracted less attention , from a fully - controlled design to facilitating learner ownership in its design such as user generated learning environments system ( ugles ) . although learning content management and learning management systems are dominant in medical education , user generated learning will move ahead . since the development of such platform involves designing , programming and controlling of learning environment , scientists will face major challenges in future , especially by those users who had little computational thinking skills which are essential for deeper understanding of dynamic curriculum planning ( 62 - 63 ) . in addition , curriculum planning for medical education on cyberspace should observe moral dimensions ; professional development in this dimension is the main mechanism . as albaqami argued ( 64 ) , honesty , coordination , respect , reflexivity , competition , responsibility and loyalty are among the most important and well - known values at both traditional and cyber learning environments . therefore , to know principles and codes of ethics in the field of technology and medical education is necessary to develop medical education on cyberspace ; otherwise , unintended consequences may occur in relation to learning outcomes . this type of thinking about system quality is essential and it can be applied to the development of an actual medical education on cyberspace . currently , innovative medical software applications such as medscape , visualdx , medcalc , labgear , uptodate , dropbox , resuscitation , etc . ( 65 ) are used more than before , while previous medical specialists and medical students were not even familiar with any of these applications ( 66 ) . interactive web and web modules applications ( 67 - 68 ) have been the main tools of learning toolkits in cyberspace , based on bloom s digital taxonomy ( 69 ) . it has covered most of the learning objectives and performance on cyberspace ( 69 - 70 ) . the first step of " call to action " at this level requires cyber - literacy . cyber literacy as conceptual understandings of the nature of cyber world such as awareness of what you re doing online or consequences of the cyberspace activities . also she pointed out that the cyberspace should be navigated with awareness and emphasized on critical competencies for the learner to judge the legitimacy and credibility of cyber learning environments ( 71 ) . at this level , technology acceptance and user preferences the cyber generations are diverse in their learning performance which is related to personality traits , values , attitudes and interest of self - learning ; therefore , special attention on these individual differences for design and development of medical education on cyberspace is a useful step . our results have covered two areas including technological and institutional structure which are critical for medical education on cyberspace . based on the evidence , conceptualization of medical education in cyber space as a systemic approach is essential for effective design , implementation , evaluation and development of many related topics in health system ( 72 - 77 ) . in this study , we introduced cyber medical education , as a set of policies , decisions , strategies , processes , programs , performances and interactions in the health system architecture . based on the above evidence , a complex map of the critical points of the medical education system on cyberspace , which was highlighted in all six taxonomy levels , emerged in our study . it is necessary to emphasize on the building of a network bridging between all dimensions , components and indices of medical education system on cyberspace ( as mentioned in table 1 ) although it will be difficult without creative viewpoints and integrative platforms . several mechanisms were also identified for improving and developing medical education on cyberspace ( see table 2 ) . these include mechanisms related to policy and strategy formation , cyber infrastructure management , network collaboration , cyber medical education services provision , functions and performance assessment as mentioned above . in this sense , our investigation was in line with some findings on the capabilities of the cyberspace to create new opportunities in the field of medical education ( 7,11 - 14,58 - 66 ) and also toward the challenges and threats that other researchers mentioned in their studies ( 5,6,25,40,67 - 73 ) . a health system must quickly adapt to cyber science and provide medical education within cyberspace at different levels ( 78 - 80 ) . we developed a new taxonomy for medical education on cyberspace which took into account health system structure . we believe it is useful for the understanding of medical education on cyberspace and it helps with provision of an integrated framework for development . although cyberspace creates new opportunities in medical education system , adaptation solutions are fragmented in various sectors . it is important that all aspects of the health systems be involved in the development of cyber - based medical education . without this convergence , we will be blind to the decisions made by others within the system . the health system should also involve those sectors that are critical to achieving better learning on cyberspace . integrated planning , governance and administration are pivotal to the promotion of the medical education on cyberspace . it is clear that any kind of direct or indirect decisions related to governance level can impact on the whole medical education system . the promotion of integrated governance with a fundamental focus on policy / strategy frameworks will be generating novel innovations in future . our study has a number of limitations such as : a ) the sensitivity of literature searches ( despite using experienced researchers and study protocol , some articles might not have been identified and therefore not included in the body of the study ) ; in this case , our group s judgments in design and analysis increased scientific rigor . b ) it was difficult to make combining results due to the wide variety of studies in the area ; this work relies on researchers expertise and experience in judgment about quality and utility to spotlight key features of learning on cyberspace that we believe have power to advance the field of medical education and c ) the important dialogue in our context ( iran ) was n't mentioned , we published opinion of iranian experts in another article . future research can explore how stewards at upstream levels impact on the development of medical education in cyberspace , what leaders say about medical education in cyberspace and the role of cyberspace in clinical learning in a health system . our study has a number of limitations such as : a ) the sensitivity of literature searches ( despite using experienced researchers and study protocol , some articles might not have been identified and therefore not included in the body of the study ) ; in this case , our group s judgments in design and analysis increased scientific rigor . b ) it was difficult to make combining results due to the wide variety of studies in the area ; this work relies on researchers expertise and experience in judgment about quality and utility to spotlight key features of learning on cyberspace that we believe have power to advance the field of medical education and c ) the important dialogue in our context ( iran ) was n't mentioned , we published opinion of iranian experts in another article . future research can explore how stewards at upstream levels impact on the development of medical education in cyberspace , what leaders say about medical education in cyberspace and the role of cyberspace in clinical learning in a health system .

introduction : over the past few decades , two revolutionary approaches have emerged as a new form of medical education : electronic medical education and web - based medical education . a number of well - known medical institutions , such as harvard and johns hopkins used a wide range of cyberspace capabilities to increase their competitiveness . researchers have expressed that cyberspace will change health system s main objective of training physicians and medical education . we conducted this study to identify the health system critical considerations on core issues , involving the development of medical education on cyberspace . methods : in order to conduct this study , we observed the steps of a critical literature review , combined with the four - phase method adopted by carnwell and daly . we focused on particular literature on health and cyber system functions ; it was associated with systemic approach . results : we developed a six - level taxonomy , cyber level , governance level , ministerial level , organizational level , program level and performance level , as a key solution that can be applied for the success of medical education on cyberspace . the results were summarized and appraised in more details . conclusion : medical education on cyberspace is a complex interdisciplinary system . it is important that all aspects of the health systems be involved as integral to the development of cyber based medical education ; without this convergence , we will be confused by the decisions made by others within the system . health system should also communicate with those external sectors that are critical to achieving better learning on cyberspace . integrated planning , governance and management of medical education in cyberspace are pivotal elements for the promotion .

Introduction Methods Results Discussion Conclusion

in addition to traditional experimental , theoretical and computational scientific paradigms , increasing use of cyberspace ( 1 ) in scientific settings has prompted the development of a fourth scientific paradigm , widely known as } cyber paradigm ( 2 ) . in the computing context , } cyber is an internet - related prefix , which is added to a wide range of existing concepts , to describe any person , thing , idea or space relating to the digital world and global networks ( 3 ) . in recent studies , authors focus on rethinking the notion of modern science , by suggesting a range of cyber - related concepts , such as cyber trade , cyber security , cyber laws , cyber infrastructure , cyber learning and cyber medicine ( 4 - 9 ) . several studies provide evidence that medical education systems , in view of the cyber - paradigm shift , dynamically incorporate cyberspace capabilities ( 11 - 14 ) . in fact , a number of well - known medical universities , such as oxford ( 15 ) , harvard ( 16 ) , johns hopkins ( 17 ) , sydney ( 18 ) and tokyo ( 19 ) used a wide range of cyberspace capabilities including database application , web - based learning , simulation models and intelligent learning games to increase their competitive advantage . according to health across reports ( 20 ) , over the past few decades , two revolutionary approaches have emerged as a new form of medical education : electronic medical education ( 2005 - 2010 ) and web - based medical education ( 2010 - 2020 ) . however , researchers have expressed that cyberspace will change health system s main objective of training physicians and medical education once again ( 21 ) . at present , in most countries , the establishment of intelligent government ( 22 ) has increased the options for optimal use of cyberspace features in health system and medical education . various authors have argued that the integration and sharing of policies , strategies , assets , infrastructure and human capital on cyberspace , can strengthen and expand the status of higher education ( in general ) and medical education ( in particular ) at international levels . however , it can imply interdisciplinary cooperation between information and communication technology system and health system which leads to intelligent and innovative approaches to the development of medical education on cyberspace . this article aimed to critically review the learning systems in cyberspace , to identify particular factors that have led to successful medical education in cyberspace . the main goal of this study was to identify and to understand the health system critical issues in terms of core components and mechanisms involved in the development of medical education in cyberspace . our assumption was that successful medical education in cyberspace required an integrated and balanced structure from higher levels to lower levels . in this study , we closely observed the principles of a critical literature review , based on the four - phase method adopted by carnwell and daly ( 23 ) . since studies of medical education development on cyberspace are still on exploratory phases , critical literature review was considered as the appropriate method to achieve the goal of our study . we did this review of the literature with a specific focus on health and cyber system functions ; it was associated with a systemic approach . critical literature review usually requires including many articles and related documents in detail , to understand the main issues from different perspectives , approaches and frameworks . the four - phase method in critical literature review consists of a ) reviewing the literature of a wide area ; b ) exploring the key scope of the review ; c ) organizing the results into themes ; and d ) concluding and informing further studies . according to carnwell , the researcher begins with a clear definition of the research scope and a critical appraisal of the main literature ; then he or she attempts to expand the issue through seeing beyond what others have worked out , and so the researcher should identify particular components in the existing knowledge , as well as , potential knowledge gaps , next organizes the evidence in the results , discussion and conclusion and ultimately , provides directions for future research ( 29 ) . we also searched related terms such as delivery concepts in cyberspace ( internet , web , online , virtual and distance ) , learning concepts ( learning process , curriculum , learning environment and learning administer ) , health system concepts ( stewardship , governance and management ) . due to the heterogeneity of the research field , it was very difficult to define the criteria applied to ensuring adequacy of studies in this research . inclusion criteria were used in selecting the studies : first , those studies were selected which addressed the topics of higher education or medical education in cyberspace and were relevant to the core components or mechanisms of health system and its functions at all levels ; second , the studies had to be published in english language and had open access . the documents were too heterogeneous ; literature addressed a wide range of medical education topics in cyberspace , in addition to numerous interventions at disparate levels . we also attempted to explain essential mechanisms that could be applied to the development of medical education in cyberspace ( table 2 ) . we addressed a six - level taxonomy as a key solution that can be applied to the success of medical education on cyberspace ; these include cyber level , governance level , ministry level , organization level , program level and performance level . the results were summarized and appraised in more details as follows : medical education on cyberspace : level , dimension , component and index critical mechanisms for development of medical education on cyberspace this level has formed based on integrated capabilities of interactive web and semantic web in digital world . these " hard and soft cyber infrastructures are necessary for a successful medical education in cyberspace . soft infrastructure ( 43 ) refers to the management framework of a cyber - system , such as cyber policy making , cyber strategic planning , cyber financial system and system of governments . we emphasize that integrated cyber infrastructures provide a quantum leap for developing medical education in cyberspace . designing , implantation and evaluation of medical education in cyberspace with a look to main indices such as availability , accessibility , flexibility , security and mobility is also considered a developmental mechanism at this level . medical education in cyberspace is still novel at specialized level , so it should receive more attention . although the cyber infrastructure as a key asset has a strategic importance in the health system , leaders have a less comprehensive attention to it . since the medical education on cyberspace consists of three structures including cyber system , learning system and medical system , different metaphors in these areas lead to important challenges such as overlapping powers and authority , interference of policies , practices and tasks , lack of cooperation among stakeholders and lack of accountability at this level . we believe that integrated stewardship ( 49 ) is one of the key factors in governance level , which can have influences in the establishment and development of medical education in cyberspace . the optimized resource allocation and supported budgetary system are also critical mechanism for development of medical education on cyberspace . finally , we suggest integrated governance and stewardship approach as the most important mechanism for the development of medical education on cyberspace . for example , in the development of medical education in cyberspace , the minister of information and communication technology ( ict ) is accountable for cyber issues ( 50 - 52 ) , therefore , they need to collaborate with health minister and to be aware of main health system s policies and medical education goals for accountability . some researchers maintain that a country leaders authority and the link with the chain of the organizational factors have an impact on promoting the capacity building and innovations ( 54,55 ) . it seems , there is a deficiency on creating a common strategic perspective as a key mechanism at this level , particularly on determining available capacities and potential assets that may impact on main performance in cyberspace . according to carayannis & campbell ( 50 ) and smith & leydesdorff ( 51 ) , although the tricycle of government , industry and university cooperating together is a unique innovative ecosystem , their stability is affected by the political , economic , technological events . this is very important to consider and to prevent it through reinforcement of links between ministerial levels in the development of medical education on cyberspace . the concepts mentioned above are not new in medical education system , while applying them in the medical education in cyberspace is in the early stages . unfortunately , it seems that many medical organizations and medical education institutes have issued a variety of strategic documents to address cyber learning and research without a clear plan to respond to the above questions ( 58,59 ) . we are confident that education administrators can focus on best practices to achieve efficiency in medical education on cyberspace and develop it based on a professional perspective . it is suggested that for the development of medical education on cyberspace , managers pay more attention to the integration of organization architecture ( medical school , medical research center , medical center and community medicine ) via the service - oriented architecture approaches . there are many choices for curriculum design on cyberspace which has attracted less attention , from a fully - controlled design to facilitating learner ownership in its design such as user generated learning environments system ( ugles ) . since the development of such platform involves designing , programming and controlling of learning environment , scientists will face major challenges in future , especially by those users who had little computational thinking skills which are essential for deeper understanding of dynamic curriculum planning ( 62 - 63 ) . in addition , curriculum planning for medical education on cyberspace should observe moral dimensions ; professional development in this dimension is the main mechanism . as albaqami argued ( 64 ) , honesty , coordination , respect , reflexivity , competition , responsibility and loyalty are among the most important and well - known values at both traditional and cyber learning environments . therefore , to know principles and codes of ethics in the field of technology and medical education is necessary to develop medical education on cyberspace ; otherwise , unintended consequences may occur in relation to learning outcomes . this type of thinking about system quality is essential and it can be applied to the development of an actual medical education on cyberspace . interactive web and web modules applications ( 67 - 68 ) have been the main tools of learning toolkits in cyberspace , based on bloom s digital taxonomy ( 69 ) . it has covered most of the learning objectives and performance on cyberspace ( 69 - 70 ) . he defined cyber literacy as conceptual understandings of the nature of cyber world such as awareness of what you re doing online or consequences of the cyberspace activities . at this level , technology acceptance and user preferences the cyber generations are diverse in their learning performance which is related to personality traits , values , attitudes and interest of self - learning ; therefore , special attention on these individual differences for design and development of medical education on cyberspace is a useful step . these " hard and soft cyber infrastructures are necessary for a successful medical education in cyberspace . soft infrastructure ( 43 ) refers to the management framework of a cyber - system , such as cyber policy making , cyber strategic planning , cyber financial system and system of governments . we emphasize that integrated cyber infrastructures provide a quantum leap for developing medical education in cyberspace . designing , implantation and evaluation of medical education in cyberspace with a look to main indices such as availability , accessibility , flexibility , security and mobility is also considered a developmental mechanism at this level . medical education in cyberspace is still novel at specialized level , so it should receive more attention . although the cyber infrastructure as a key asset has a strategic importance in the health system , leaders have a less comprehensive attention to it . since the medical education on cyberspace consists of three structures including cyber system , learning system and medical system , different metaphors in these areas lead to important challenges such as overlapping powers and authority , interference of policies , practices and tasks , lack of cooperation among stakeholders and lack of accountability at this level . we believe that integrated stewardship ( 49 ) is one of the key factors in governance level , which can have influences in the establishment and development of medical education in cyberspace . the optimized resource allocation and supported budgetary system are also critical mechanism for development of medical education on cyberspace . finally , we suggest integrated governance and stewardship approach as the most important mechanism for the development of medical education on cyberspace . for example , in the development of medical education in cyberspace , the minister of information and communication technology ( ict ) is accountable for cyber issues ( 50 - 52 ) , therefore , they need to collaborate with health minister and to be aware of main health system s policies and medical education goals for accountability . in this regard , we emphasize the importance of the link between ministries . it seems , there is a deficiency on creating a common strategic perspective as a key mechanism at this level , particularly on determining available capacities and potential assets that may impact on main performance in cyberspace . this is very important to consider and to prevent it through reinforcement of links between ministerial levels in the development of medical education on cyberspace . the concepts mentioned above are not new in medical education system , while applying them in the medical education in cyberspace is in the early stages . how should the medical education be designed , implemented , evaluated and developed on cyberspace ? unfortunately , it seems that many medical organizations and medical education institutes have issued a variety of strategic documents to address cyber learning and research without a clear plan to respond to the above questions ( 58,59 ) . we are confident that education administrators can focus on best practices to achieve efficiency in medical education on cyberspace and develop it based on a professional perspective . it is suggested that for the development of medical education on cyberspace , managers pay more attention to the integration of organization architecture ( medical school , medical research center , medical center and community medicine ) via the service - oriented architecture approaches . in addition , curriculum planning for medical education on cyberspace should observe moral dimensions ; professional development in this dimension is the main mechanism . as albaqami argued ( 64 ) , honesty , coordination , respect , reflexivity , competition , responsibility and loyalty are among the most important and well - known values at both traditional and cyber learning environments . therefore , to know principles and codes of ethics in the field of technology and medical education is necessary to develop medical education on cyberspace ; otherwise , unintended consequences may occur in relation to learning outcomes . this type of thinking about system quality is essential and it can be applied to the development of an actual medical education on cyberspace . it has covered most of the learning objectives and performance on cyberspace ( 69 - 70 ) . cyber literacy as conceptual understandings of the nature of cyber world such as awareness of what you re doing online or consequences of the cyberspace activities . also she pointed out that the cyberspace should be navigated with awareness and emphasized on critical competencies for the learner to judge the legitimacy and credibility of cyber learning environments ( 71 ) . at this level , technology acceptance and user preferences the cyber generations are diverse in their learning performance which is related to personality traits , values , attitudes and interest of self - learning ; therefore , special attention on these individual differences for design and development of medical education on cyberspace is a useful step . our results have covered two areas including technological and institutional structure which are critical for medical education on cyberspace . based on the evidence , conceptualization of medical education in cyber space as a systemic approach is essential for effective design , implementation , evaluation and development of many related topics in health system ( 72 - 77 ) . in this study , we introduced cyber medical education , as a set of policies , decisions , strategies , processes , programs , performances and interactions in the health system architecture . based on the above evidence , a complex map of the critical points of the medical education system on cyberspace , which was highlighted in all six taxonomy levels , emerged in our study . it is necessary to emphasize on the building of a network bridging between all dimensions , components and indices of medical education system on cyberspace ( as mentioned in table 1 ) although it will be difficult without creative viewpoints and integrative platforms . several mechanisms were also identified for improving and developing medical education on cyberspace ( see table 2 ) . these include mechanisms related to policy and strategy formation , cyber infrastructure management , network collaboration , cyber medical education services provision , functions and performance assessment as mentioned above . in this sense , our investigation was in line with some findings on the capabilities of the cyberspace to create new opportunities in the field of medical education ( 7,11 - 14,58 - 66 ) and also toward the challenges and threats that other researchers mentioned in their studies ( 5,6,25,40,67 - 73 ) . we developed a new taxonomy for medical education on cyberspace which took into account health system structure . we believe it is useful for the understanding of medical education on cyberspace and it helps with provision of an integrated framework for development . it is important that all aspects of the health systems be involved in the development of cyber - based medical education . without this convergence , we will be blind to the decisions made by others within the system . the health system should also involve those sectors that are critical to achieving better learning on cyberspace . integrated planning , governance and administration are pivotal to the promotion of the medical education on cyberspace . it is clear that any kind of direct or indirect decisions related to governance level can impact on the whole medical education system . our study has a number of limitations such as : a ) the sensitivity of literature searches ( despite using experienced researchers and study protocol , some articles might not have been identified and therefore not included in the body of the study ) ; in this case , our group s judgments in design and analysis increased scientific rigor . b ) it was difficult to make combining results due to the wide variety of studies in the area ; this work relies on researchers expertise and experience in judgment about quality and utility to spotlight key features of learning on cyberspace that we believe have power to advance the field of medical education and c ) the important dialogue in our context ( iran ) was n't mentioned , we published opinion of iranian experts in another article . future research can explore how stewards at upstream levels impact on the development of medical education in cyberspace , what leaders say about medical education in cyberspace and the role of cyberspace in clinical learning in a health system . our study has a number of limitations such as : a ) the sensitivity of literature searches ( despite using experienced researchers and study protocol , some articles might not have been identified and therefore not included in the body of the study ) ; in this case , our group s judgments in design and analysis increased scientific rigor . b ) it was difficult to make combining results due to the wide variety of studies in the area ; this work relies on researchers expertise and experience in judgment about quality and utility to spotlight key features of learning on cyberspace that we believe have power to advance the field of medical education and c ) the important dialogue in our context ( iran ) was n't mentioned , we published opinion of iranian experts in another article . future research can explore how stewards at upstream levels impact on the development of medical education in cyberspace , what leaders say about medical education in cyberspace and the role of cyberspace in clinical learning in a health system .

molecular recognition is one of the most important chemical events in biological systems and has been mimicked in supramolecular chemistry as , for example , artificial enzymes [ 13 ] . most of the supramolecular systems for molecular recognition are composed of molecularly dispersed states in homogeneous solutions . molecular recognition in living systems occurs mostly at interfacial environments such as membrane surfaces , enzyme reaction sites , or at the interior of the dna double helix . why have biological systems adopted interfacial environments for molecular recognition ? a part of the answer was provided by pioneering work by kunitake and co - workers who compared binding efficiency between guanidinium and phosphate in three different environments ( figure 1 ) . binding constants of adenosine monophosphate ( amp ) to guanidinium functionality in aqueous aggregates such as micelle or bilayer vesicles were evaluated at 1010 m. these values are significantly larger than those between molecularly - dispersed guanidinium and phosphate in water ( 1.4 m ) . surprisingly , a large enhancement in binding constant was reported for binding of amp to guanidinium groups when embedded at a water surface . these results clearly indicate that molecular recognition can be achieved much more efficiently at an appropriate interface . in order to further understand these findings , sakurai and co - workers considered theoretical aspects of molecular recognition at the air water interface using a quantum chemical approach including reaction field calculations combined with am1 molecular orbital methods [ 79 ] . guanidinium host and phosphate guest were placed in various positions at a model interface consisting of a lipid layer ( low dielectric , = 2 ) and water phase ( high dielectric , = 80 ) . the calculated binding energies depended significantly on the position of the binding site relative to the two - phase boundary . a large binding constant was obtained when the binding site was located in the lipid phase , while positioning the guanidinium deep in the water phase resulted in a very small binding energy . even when the hydrogen bonding site was positioned in the water phase , the site is affected electronically by the low dielectric lipid layer , which strengthens intermolecular hydrogen bonding and electrostatic interactions . as a result , the binding constant increases significantly at the border between lipid and aqueous phases . these quantum chemical calculations suggested advantageous aspects of interfacial environments for efficient molecular recognition of substances in aqueous media . molecular recognition at interfaces not only has relevance to biological systems but also is important for modern applications such as high sensitivity sensors . selective binding of guest molecules in solution to host molecules located at a solid surface is crucial for electronic and photonic detection of target substances . in response to these demands , molecular recognition at interfaces has been researched extensively during the past two decades using langmuir monolayers , self - assembled monolayers , and lipid assemblies as recognition media . in this review , a molecular monolayer on a water surface provides an attractive medium for an aqueous guest to interact with host sites at the surface of the functionalized monolayer , while langmuir monolayers can be also regarded as media appropriate for research on molecular recognition between membrane components . binding of aqueous ionic species to host monolayer surfaces is one of the most popular targets in this research area , as summarized in a review by kruppa and kning . okahata and co - workers investigated binding of calcium ions to phospholipid monolayers through the combined study of surface pressure ( ) - molecular area ( a ) isotherm measurement , electrochemical response , and quartz crystal microbalance ( qcm ) . reported selective detection of iodide in admixture with other halogen anions by using the change in aggregation mode within a monolayer of n - confused tetraphenylporphyrin , which induces drastic variation in the absorption spectrum of the porphyrin . leblanc and co - workers demonstrated metal ion binding on mixed monolayers of peptide amphiphiles having glyhisgly - related peptide sequence based on combinatorial surface - chemistry . monolayers can be transferred onto solid supports by using the langmuir - blodgett ( lb ) technique sometimes leading to useful sensing materials . matsunaga and co - workers used an lb film composed of 4-n - dodecyl-6-(2-thiazolylazo)resorcinol for naked - eye detection of submicromolar levels of cadmium ions . this probe membrane exhibited visual color transition , forming a series of reddish - orange to pinkish - purple complexes with cadmium , over a wide concentration range ( 0.0444.5 m ) . binding of neutral substances to langmuir monolayers has been also extensively investigated , as seen in the rather ambiguous interaction with poly(vinylpyrrolidone ) reported by ariga et al . and in the highly specific recognition of 1-phenylethylamine reported by miyashita and co - workers . some of the most attractive guest molecules among neutral guests are the nucleic acid bases and related molecules . pioneering work on molecular recognition of aqueous nucleic acid base was performed by kitano and ringsdorf who demonstrated changes in the -a isotherm of an adenine - functionalized monolayer upon addition of thymidine to the subphase . subsequently , direct evidence for hydrogen bond formation in a wide variety of systems including recognition of nucleotides , nucleic acid bases , and their analogues , was obtained by kunitake and co - workers using analytical techniques such as fourier - transform ir ( ft - ir ) and x - ray photoelectron spectroscopy ( xps ) . these target guests often have multiple binding sites so that translational freedom of molecules embedded at the air - water interface is advantageous for recognition of such guest molecules . in an example of a binary system , recognition of flavin mononucleotide ( fmn ) by mixed monolayers of dialkylmelamine and a ternary recognition system has been similarly designed where one flavin adenine dinucleotide ( fad ) molecule has the potential to bind two guanidinium molecules at phosphate groups , one orotate molecule at adenine sites , and a diaminotriazine at an isoalloxiazine ring . formation of multi - point recognition sites in the mixed monolayer does not require great synthetic effort for the preparation of complicated covalently - linked host compounds , which is usually obstacle for research of molecular recognition . a combination of surface pressure studies with brewster angle microscopy ( bam ) imaging and grazing incidence x - ray diffraction ( gixd ) measurements proved to be optimal for the characterization of the change in structure and phase behavior during the interfacial recognition process . complementary hydrogen bonding of two thymine molecules by one melamine host molecule is deduced from the chemical structure of both components . liang and co - workers also made considerable effort to analyse molecular recognition of nucleic acid base related molecules at the air - water interface . some proteins , especially antibodies , are capable of performing highly selective molecular recognition tasks . their recognition sites are composed of well - harmonized assemblies of peptide segments . similarly assembled binding sites can be generated within monolayers at the air - water interface , as demonstrated by kunitake and co - workers in their systematic research on recognition of aqueous peptides by peptide - amphiphile monolayers ( figure 2 ) . an appropriate interval between host sites is the crucial point for successful recognition of the guest peptide molecules . monoalkyl oligoglycine amphiphiles spread on water form strong hydrogen bonds within the monolayers and are not capable of accommodating external guest peptides contained in the subphase ( a ) . conversely , peptide binding together with guest selection was achieved in monolayers of dialkyl peptide amphiphiles in which the dialkylamine moiety was connected to the glycylglycinamide head group through a terephthaloyl unit , which endows an appropriate interval between the host peptide sites ( b ) . introduction of additional host functional groups that can interact with guest c - terminal or n - terminal should enhance the binding efficiency ( c ) [ 2931 ] . experimental results suggest that binding sites could be constructed spontaneously by an induced - fit mechanism and this similar concept has been used by other researchers . leblanc and co - workers mimicked the binding site of acetylcholinesterase using a peptide - amphiphile with the phetrpserhisglu segment and an octadecyloxy chain for selective recognition of paraoxon , sodium dihydrogen phosphate or 4-nitrophenyl phosphate disodium . systematic investigation by -a isotherm measurements revealed that the presence of phe and trp may be crucial for interaction with paraoxon . the same research group further investigated this recognition system using infrared reflection - absorption spectroscopy ( ir - ras ) of the peptide - amphiphile langmuir monolayer on subphases containing paraoxon . it was revealed that the molecular recognition in this system involved - interactions between the nitrobenzene group of paraoxon and aromatic groups in the peptide - amphiphile . izhaky and addadi also demonstrated the importance of spontaneous assembly of binding sites through stereoselective recognition by monoclonal antibodies of two - dimensional monolayers of cholesterol spread at the air - water interface . zadmard and schrader reported nanomolar protein detection using an octadecanoic acid monolayer with a small amount ( 0.13 equiv ) of one or two different calixarene receptors , adorned with charged functional groups at their upper rims . the recognition process requires a self - assembly of multiple calixarene units over the protein surface , which bind the protein in a cooperative fashion . similarly , schuster et al . investigated the enzymatic interplay of porcine pancreatic phospholipase a2 on a monolayer composed of dimyristoylphosphatidylethanolamine ( dmpe ) . oliviera and co - workers researched chitosan and phospholipid interactions using langmuir and lb films as cell membrane models . saccharide species are also important bioactive components . for example , recognition of saccharide is crucial in processes such as virus infection , and research on recognition of saccharide species has been recently investigated at the air - water interface . recognition of saccharides at the air - water interface was initiated in the late 1980s and 1990s and is exemplified in the cyclic resorcinol tetramer host reported by kurihara et al . . this was followed by several examples with synthetic hosts such as phenylboronic acid or a macrocyclic sugar cluster . . recently , miyahara and kurihara reported rational design of lb films that are capable of electrochemical detection of redox - active saccharides ( figure 3 ) . the boronic acid - functionalized , electroconductive lb film was fabricated by polymerization of a 1:10 mixture of a carotenoid type amphiphile and a polymerizable amphiphile . the selectivity of the electrode for sugar derivatives was achieved by attaching the molecular recognition site to the conjugated molecule . the conducting molecule with binding site , that is , the molecular wire with the connecting terminal , as designed in this study should open the possibility of designing well - defined complex molecular devices based on the available library of molecular assembly chemistry . binding of an aqueous guest to multiple amphiphiles in a monolayer should lead to formation of specific patterns in two - dimension . the novel concept of two - dimensional molecular patterning was initiated by kunitake and co - workers . in the first example , aqueous dicarboxylate can bind to two dialkylguanidinium molecules , and a spacer between the two carboxylates of the aqueous template affects the crystallinity of the guanidinium amphiphiles . monolayers transferred from aqueous oxalate ( no methylene spacer ) displayed a crystalline arc , whereas only an amorphous halo was observed for the monolayer from the pure water subphase . a rigid oxalate template bound the two guanidinium molecules and effectively weakened electrostatic repulsion among cationic guanidinium molecules through stoichiometric binding . when malonate [ ( ch2 ) spacer ] or succinate [ ( ch2)2 spacer ] was used as a template , the electron diffraction pattern could be assigned to a crystalline state , but the arc in the patterns broadened . a further increase in the spacer length [ glutarate with ( ch2)3 spacer and adipate with ( ch2)4 spacer ] again induced the amorphous phase of the monolayer . melamine and barbituric acid ( or related combinations ) alternately bind through complementary hydrogen bonding , resulting in a molecular ribbon [ 4749 ] , which was kinetically analyzed by vollhardt and co - workers using a sophisticated elemental set - up . an afm molecular image of the didodecylmelamine monolayer bound to the barbituric template could be easily observed , although the corresponding monolayer transferred from pure water was too fragile for afm observation . an oblique array of methyl terminals was observed . for molecular patterning with an aqueous template molecule having multiple binding sites , flavin adenine dinucleotide ( fad ) was selected as an aqueous template and a recognition system with the guanidinium amphiphile and the orotate amphiphile was investigated ( figure 4 ) [ 52 , 53 ] . afm images of the formed complex transferred onto a mica surface showed the periodic brighter and darker portions that correspond to higher and lower regions of the monolayer surface , respectively . the binding of fad with the guanidinium / orotate mixed monolayer should dispose two functional units at the same level through binding to the template fad molecule where the flavin unit in fad may be buried under the phosphate group owing to the conformational flexibility of the fad molecule , and resulting in a height difference between the two terminal methyl groups . the air - water interface provides a flexible medium where conformational changes of amphiphiles are unobstructed . therefore , regulation of molecular recognition based on conformational changes of host amphiphiles is one of the distinguishing characteristics of molecular recognition . for example , higuchi et al . reported ph control of molecular recognition at the air - water interface . the poly(l - alanine ) graft chains attached to amphiphilic polymer poly(allylamine ) on water yielded sites which bind specifically with aqueous l - alanine rather than its d isomer . the specific binding of l - alanine to the monolayer was completely dependent on the structure of the monolayer , which can be regulated by variation of ph . realized mechanically controlled molecular recognition through dynamic formation of a cavity structure using host molecules , known as steroid cyclophanes [ 5660 ] . the steroid cyclophane molecule used in this research contains a 1,6,20,25-tetraaza[6.1.6.1]-paracyclophane cyclic core connected to four steroid moieties through a flexible l - lysine spacer with cholic acid ( see figure 5 ) . in a monolayer at low pressures , it forms an open conformation in order to contact the hydrophilic face efficiently to the water phase . compression of the monolayer forces the steroid cyclophane in the monolayer into a more compact conformation . as a result reversible binding ( capture and release ) of an aqueous fluorescent guest ( 6-(p - toluidino)naphthalene-2-sulfonate ( tns ) was demonstrated by a repeated compression and expansion of the steroid cyclophane monolayer . according to repeated pressure application during the compression - expansion cycle , a periodic change in the fluorescence intensity was detected , because tns emits fluorescence strongly in a hydrophobic host interior . the same research group applied a related system to aqueous molecular assemblies and realized modulation of emission wavelength . the concept of dynamic function at the air - water interface has been recently similarly reported by other research groups . resorcinarene cavitands with four quinoxaline bridges are a family of macrocycles that adopt a contracted , vase - type conformation , capable of guest inclusion , whereas they switch to an expanded , kite - type conformation with a large flat surface under other conditions . stoddart , ho , and co - workers demonstrated that redoxcontrollable molecular shuttles , in the shape of amphiphilic , bistable rotaxanes , switch mechanically under chemical stimulus even when contained in closely packed langmuir films . more recently , michinobu et al . reported control of enantioselectivity of amino acid recognition by dynamic motion of a polycholesteryl - substituted cyclen complex host molecule at the air - water interface . the octacoordinate sodium complex has two possible quadruple helicate structures ( see figure 6 ) . helicity is influenced by the chirality of the side arms especially when ordered or aggregated at the supramolecular level . application of lateral pressure should affect the helix structure and , consequently , the diastereomeric stability of complexes with guest molecules . the k values of d - leucine are always greater than those of l - leucine , indicating that the cyclen monolayers have a stronger interaction with d - leucine . conversely , the values of l - valine are smaller than those of d - valine at low surface pressure but exceed them at 2223 mn m. in other words , chiral recognition in the cyclen monolayers with valine changes from the d- to l - form upon compression . it is remarkable that such small difference in the chemical structure between leucine and valine can be distinguished by the dynamic process of monolayer formation . apart from the air - water interface , various interfacial environments provide media useful for molecular recognition and its related functions . in particular , self - assembled monolayers ( sam ) are often used for sensor applications because the sam structures facilitate contact with artificial devices such as electrodes and field effect transistors , as has been reviewed by reinhoudt and co - workers . okahata and co - workers developed a siloxane - linked monolayer attached to a porous glass surface for permeation control [ 6668 ] . they applied the same structure onto a tin dioxide electrode , where insertion of alkylalcohol into the alkylsiloxane monolayer could be electrochemically detected . geiger and co - workers reported use of an enhanced surface second harmonic generation ( shg ) signal for detection of the toxic metal pollutant chromium(vi ) at custom - made amino acid functionalized fused quartz / water interfaces . chromate adsorption isotherms recorded at ph 7 were suggestive of an intramolecular chelation mechanism that would be important when four or more hydrogen - bonding moieties were displayed toward the incoming chromate . credo et al . reported a method to manipulate conductance using molecular recognition at a sam surface . as illustrated in figure 7 , a binder molecule , diacyl 2,6-diaminopyridine decanethiolate ( dap , figure 7a ) was inserted into a background monolayer of decanethiolate on au(111 ) using replacement lithography . electroactive functionalization of the monolayer was then achieved through binding of the complementary ferrocene - terminated uracil to the binder molecule . current - voltage properties of the patterned region were monitored by using an stm tip . noncovalent self - assembly provides a potential method to install and subsequently remove electroactive functionality in molecular electronics systems . kitano and taira used sam structures of cyclodextrin derivatives for detection of bisphenol - type substances . they examined complexation of various kinds of bisphenols by a sam of thiolated -cyclodextrin on a gold electrode by cyclic voltammetry using hydroquinone as a probe . on the basis of the inhibitory effect of bisphenols on the inclusion of hydroquinone by the surface - confined cyclodextrin , use of -cyclodextrin and hexasodium calixarene hexasulfonic acid as host structures were also reported by the same research group . willner and co - workers demonstrated photochemical imprinting of molecular recognition sites for phenoxynaphthacene quinone in sam assembled on au surfaces . the primary step of their approach includes the assembly of the trans - phenoxynaphthacenequinone monolayer , followed by the rigidification of the monolayer with long chain alkanethiols generating a densely packed quinone monolayer . the second process involves the photoisomerization of the monolayer to the ana - quinone state , followed by the nucleophilic displacement of the quinone with butylamine . the binding of phenoxynaphthacenequinone to the imprinted recognition sites reveals selectivity , and structurally related substrates did not associate with the imprinted sites . combining recognition events with appropriate analytical methods can yield sensor devices suitable for biological applications . zeng , wang , and co - workers demonstrated that an unlabeled carbohydrate mass sensor in combination with lectin - bacterial o - antigen recognition can be used for detection of high molecular weight bacterial targets with remarkably high sensitivity and enhanced specificity . a functional mannose self - assembled monolayer in combination with lectin concanavalin a ( con a ) was used as the molecular recognition element for detection of escherichia coli w1485 using qcm as a transducer . whitesides and co - workers reported the synthesis of bifunctional polyacrylamides containing pendant vancomycin and fluorescein groups , and the use of these polymers to direct antibodies against fluorescein to sam presenting d - alanine - d - alanine groups . liu and amiridis studied the interaction of avidin with biotin on functionalized quartz surfaces terminated with 3-aminopropyltrimethoxysilane , 2,2-(ethylenedioxy)bis(ethylenediamine ) , and fourth generation amine - terminated polyamidoamine dendrimers , using the ftir - ras technique . advincula and co - workers reported nanomolar detection and specific recognition of pinacolyl methylphosphonate , a hydrolysis product and an analog of a relatively persistent class of toxic nerve agents . in this system a modified polyamidoamine carbazole / cu dendrimer , which is electrochemically cross - linked on a self - assembled monolayer ( sam ) modified au substrate , acted as an active sensing element for trapping the nerve agent analogs . bunker , stoddart , and co - workers developed supramolecular machines using sam structures in which molecular configurations can be reversibly programmed using electrochemical stimuli . interactions between the tethered tetracationic cyclophane host cyclobis(paraquat - p - phenylene ) and dissolved -electron - rich guest molecules , such as tetrathiafulvalene , were reversibly switched by oxidative electrochemistry . as reviewed by rotello and co - workers , sam structures can be prepared on size - controlled nanoparticles , providing scaffolds for sensing target molecules . sanchez - cortes and co - workers reported the use of 25,27-diethyl - dithiocarbamic-26,28-dihydroxy - p - tert - butylcalixarene in the functionalization of ag nanoparticles for pyrene detection by surface - enhanced raman scattering ( sers ) . immiscible liquids produce a dynamic interface such as the water - oil interface that have been also used as media for specific molecular recognition . kitamura and co - workers investigated molecular recognition mediated by hydrogen - bonding interactions at a water - ccl4 interface by means of time - resolved total internal reflection ( tir ) fluorescence spectroscopy . in the presence of n , n - dioctadecyl- [ 1,3,5]triazine-2,4,6-triamine ( dtt ) in the ccl4 phase , the fluorescence decay profiles of riboflavin in aqueous phase were significantly modified , as compared with those observed in the absence of dtt , with the relevant amplitude varying with the concentration of dtt . watarai and co - workers reported a molecular recognition system of the interfacial aggregation of monocationic palladium(ii)-2-(5-bromo-2-pyridylazo)-5-diethylaminophenol complex with neutral diazine or purine bases at the toluene - water system . the formation of interfacial aggregates of these complexes was investigated by centrifugal liquid membrane ( clm)/uv - vis spectroscopy , clm / resonance raman spectroscopy and optical microscopy . the palladium(ii)-2-(5-bromo-2-pyridylazo)-5-diethylaminophenol complex formed interfacial aggregates preferentially with purine bases ( adenine and guanine ) . alkali metal ion recognition with [ 2-hydroxy-5-(4-nitrophenylazo)phenyl]-methyl-15-crown-5 at the heptane - water interface was investigated by teramae and co - workers , using in situ second harmonic generation ( shg ) spectroscopy . it was revealed experimentally that the na and k complexes were flatter while the li complex exhibited a lift - up orientation at the heptane - water interface . because of its relevance to biological systems , molecular recognition at the surface of aqueous lipid bilayers has also been investigated [ 8691 ] . sasaki et al . reported lead ion recognition by a crown ether functionalized lipid membrane . the receptor - lipid with the crown ether at the head group and a pyrene fluorescent tag on the hydrophobic tail was synthesized and incorporated into bilayers of distearylphosphatidylcholine . the functionalized bilayer exhibited selective affinity for lead ions in aqueous buffered solution ( ph 7.4 ) and a fluorescence response that was linear over the concentration range 10 to 10 m metal ions . recognition and binding of lead ions at the membrane surface resulted in a rapid and prominent reorganization of the receptor - lipids in the membrane that was measurable at both the macro- and nanoscales . removal of the lead ions , through the addition of edta , resulted in recovery of the original fluorescence and the reaggregation of structures in the membrane . darcy , ravoo , and co - workers synthesized amphiphilic cyclodextrins through 6-s - alkylation of the primary side and introduction of a poly(ethylene glycol ) chain to the secondary side of - , - , and -cyclodextrins , which formed nonionic bilayer vesicles in aqueous solution . molecular recognition of a hydrophobic anion ( adamantane carboxylate ) by the formed cyclodextrin vesicles was investigated by using capillary electrophoresis . the increase in electrophoretic mobility occurred when the hydrophobic anions bind to the nonionic cyclodextrin vesicles . jelinek and co - workers proposed a molecular system in which interactions between antibodies and peptide epitopes displayed at a biomimetic membrane interface can be detected through induction of visible , rapid color transitions . the colorimetric assembly consists of a phospholipid / polydiacetylene matrix anchoring a hydrophobic peptide displaying the epitope at its n - terminus . the colorimetric transitions observed in the assembly , corresponding to perturbation of the polydiacetylene framework , are induced only upon recognition of the displayed epitope by its specific antibody present in the aqueous solution . this system could be utilized for studying antigen - antibody interactions and peptide - protein recognition , epitope mapping , and rapid screening of biological and chemical libraries . fabricated chemiresistive gas sensors by deposition of 50 nm thick films of cobalt phthalocyanine and metal - free phthalocyanine on interdigitated gold electrodes via organic molecular beam epitaxy . belfort and co - workers prepared two - dimensional surface molecular imprinting method using water - in - oil emulsion photo - polymerization on a microporous polypropylene substrate that was used to separate the bronchodilator , theophylline , from the mild stimulant , caffeine , both of similar chemical structure . kim et al . proposed a method for fabrication of patterned hydrogel microwells functionalized at their bases with antibodies to promote specific immobilization of lymphocytes . ma and co - workers reported an efficient silica coating process to prepare silica - coated gold nanorods . the subsequent covalent bioconjugation of amino - functionalized gold nanorod films with goat anti - human - immunoglobulin g ( anti - h - igg ) was successfully employed for the colorimetric detection of h - igg in a model reaction based on the specific binding affinity between the proteins . govorov , kotov , and co - workers prepared molecular spring assemblies of cdte nanowires and au nanoparticles , where the distance between the exciton and the plasmon can be reversibly varied . modulation of exciton plasmon interactions could serve as a wavelength - based biodetection tool . interfacial media provide a variety of possibilities for molecular recognition . at the air - water interface complicated binding sites can be constructed through self - assembly of rather simple molecular modules , where it is possible to generate well - defined recognition sites such as biomolecular receptors . recently , ariga et al . reported unusual shifts in dissociation constants of amino acid residues at the air - water interface that were accompanied by enzyme - like catalytic activities . thus , the air - water interface will be more important for developments in biomimetic chemistry in the future . on the other hand , solid surfaces should be useful for molecular sensor device preparation , for instance , in the preparation of sensing - site arrays which will be important for highly integrated molecular devices . recent developments in control of molecular arrangements on two - dimensional solid surfaces [ 101103 ] should result in sensor arrays with ultrahigh spatial resolution . in addition , high - surface materials currently under development , such as mesoporous materials , should provide supports useful for efficient molecular recognition . a combination of advanced surface technologies and material chemistry with well - established molecular recognition sciences is crucial to the future development of this field .

in biological systems , molecular recognition events occur mostly within interfacial environments such as at membrane surfaces , enzyme reaction sites , or at the interior of the dna double helix . investigation of molecular recognition at model interfaces provides great insights into biological phenomena . molecular recognition at interfaces not only has relevance to biological systems but is also important for modern applications such as high sensitivity sensors . selective binding of guest molecules in solution to host molecules located at solid surfaces is crucial for electronic or photonic detection of analyte substances . in response to these demands , molecular recognition at interfaces has been investigated extensively during the past two decades using langmuir monolayers , self - assembled monolayers , and lipid assemblies as recognition media . in this review , advances of molecular recognition at interfaces are briefly summarized .

1. Introduction 2. Molecular Recognition at the Air-Water Interface and Related Interfaces 3. Molecular Recognition at Other Interfaces 4. Future Perspectives

molecular recognition is one of the most important chemical events in biological systems and has been mimicked in supramolecular chemistry as , for example , artificial enzymes [ 13 ] . most of the supramolecular systems for molecular recognition are composed of molecularly dispersed states in homogeneous solutions . molecular recognition in living systems occurs mostly at interfacial environments such as membrane surfaces , enzyme reaction sites , or at the interior of the dna double helix . why have biological systems adopted interfacial environments for molecular recognition ? a part of the answer was provided by pioneering work by kunitake and co - workers who compared binding efficiency between guanidinium and phosphate in three different environments ( figure 1 ) . binding constants of adenosine monophosphate ( amp ) to guanidinium functionality in aqueous aggregates such as micelle or bilayer vesicles were evaluated at 1010 m. these values are significantly larger than those between molecularly - dispersed guanidinium and phosphate in water ( 1.4 m ) . surprisingly , a large enhancement in binding constant was reported for binding of amp to guanidinium groups when embedded at a water surface . these results clearly indicate that molecular recognition can be achieved much more efficiently at an appropriate interface . in order to further understand these findings , sakurai and co - workers considered theoretical aspects of molecular recognition at the air water interface using a quantum chemical approach including reaction field calculations combined with am1 molecular orbital methods [ 79 ] . these quantum chemical calculations suggested advantageous aspects of interfacial environments for efficient molecular recognition of substances in aqueous media . molecular recognition at interfaces not only has relevance to biological systems but also is important for modern applications such as high sensitivity sensors . selective binding of guest molecules in solution to host molecules located at a solid surface is crucial for electronic and photonic detection of target substances . in response to these demands , molecular recognition at interfaces has been researched extensively during the past two decades using langmuir monolayers , self - assembled monolayers , and lipid assemblies as recognition media . in this review , a molecular monolayer on a water surface provides an attractive medium for an aqueous guest to interact with host sites at the surface of the functionalized monolayer , while langmuir monolayers can be also regarded as media appropriate for research on molecular recognition between membrane components . binding of aqueous ionic species to host monolayer surfaces is one of the most popular targets in this research area , as summarized in a review by kruppa and kning . okahata and co - workers investigated binding of calcium ions to phospholipid monolayers through the combined study of surface pressure ( ) - molecular area ( a ) isotherm measurement , electrochemical response , and quartz crystal microbalance ( qcm ) . reported selective detection of iodide in admixture with other halogen anions by using the change in aggregation mode within a monolayer of n - confused tetraphenylporphyrin , which induces drastic variation in the absorption spectrum of the porphyrin . matsunaga and co - workers used an lb film composed of 4-n - dodecyl-6-(2-thiazolylazo)resorcinol for naked - eye detection of submicromolar levels of cadmium ions . binding of neutral substances to langmuir monolayers has been also extensively investigated , as seen in the rather ambiguous interaction with poly(vinylpyrrolidone ) reported by ariga et al . some of the most attractive guest molecules among neutral guests are the nucleic acid bases and related molecules . pioneering work on molecular recognition of aqueous nucleic acid base was performed by kitano and ringsdorf who demonstrated changes in the -a isotherm of an adenine - functionalized monolayer upon addition of thymidine to the subphase . subsequently , direct evidence for hydrogen bond formation in a wide variety of systems including recognition of nucleotides , nucleic acid bases , and their analogues , was obtained by kunitake and co - workers using analytical techniques such as fourier - transform ir ( ft - ir ) and x - ray photoelectron spectroscopy ( xps ) . these target guests often have multiple binding sites so that translational freedom of molecules embedded at the air - water interface is advantageous for recognition of such guest molecules . in an example of a binary system , recognition of flavin mononucleotide ( fmn ) by mixed monolayers of dialkylmelamine and a ternary recognition system has been similarly designed where one flavin adenine dinucleotide ( fad ) molecule has the potential to bind two guanidinium molecules at phosphate groups , one orotate molecule at adenine sites , and a diaminotriazine at an isoalloxiazine ring . formation of multi - point recognition sites in the mixed monolayer does not require great synthetic effort for the preparation of complicated covalently - linked host compounds , which is usually obstacle for research of molecular recognition . a combination of surface pressure studies with brewster angle microscopy ( bam ) imaging and grazing incidence x - ray diffraction ( gixd ) measurements proved to be optimal for the characterization of the change in structure and phase behavior during the interfacial recognition process . liang and co - workers also made considerable effort to analyse molecular recognition of nucleic acid base related molecules at the air - water interface . some proteins , especially antibodies , are capable of performing highly selective molecular recognition tasks . similarly assembled binding sites can be generated within monolayers at the air - water interface , as demonstrated by kunitake and co - workers in their systematic research on recognition of aqueous peptides by peptide - amphiphile monolayers ( figure 2 ) . an appropriate interval between host sites is the crucial point for successful recognition of the guest peptide molecules . systematic investigation by -a isotherm measurements revealed that the presence of phe and trp may be crucial for interaction with paraoxon . the same research group further investigated this recognition system using infrared reflection - absorption spectroscopy ( ir - ras ) of the peptide - amphiphile langmuir monolayer on subphases containing paraoxon . it was revealed that the molecular recognition in this system involved - interactions between the nitrobenzene group of paraoxon and aromatic groups in the peptide - amphiphile . izhaky and addadi also demonstrated the importance of spontaneous assembly of binding sites through stereoselective recognition by monoclonal antibodies of two - dimensional monolayers of cholesterol spread at the air - water interface . the recognition process requires a self - assembly of multiple calixarene units over the protein surface , which bind the protein in a cooperative fashion . oliviera and co - workers researched chitosan and phospholipid interactions using langmuir and lb films as cell membrane models . saccharide species are also important bioactive components . for example , recognition of saccharide is crucial in processes such as virus infection , and research on recognition of saccharide species has been recently investigated at the air - water interface . recognition of saccharides at the air - water interface was initiated in the late 1980s and 1990s and is exemplified in the cyclic resorcinol tetramer host reported by kurihara et al . this was followed by several examples with synthetic hosts such as phenylboronic acid or a macrocyclic sugar cluster . recently , miyahara and kurihara reported rational design of lb films that are capable of electrochemical detection of redox - active saccharides ( figure 3 ) . the selectivity of the electrode for sugar derivatives was achieved by attaching the molecular recognition site to the conjugated molecule . the conducting molecule with binding site , that is , the molecular wire with the connecting terminal , as designed in this study should open the possibility of designing well - defined complex molecular devices based on the available library of molecular assembly chemistry . binding of an aqueous guest to multiple amphiphiles in a monolayer should lead to formation of specific patterns in two - dimension . in the first example , aqueous dicarboxylate can bind to two dialkylguanidinium molecules , and a spacer between the two carboxylates of the aqueous template affects the crystallinity of the guanidinium amphiphiles . a further increase in the spacer length [ glutarate with ( ch2)3 spacer and adipate with ( ch2)4 spacer ] again induced the amorphous phase of the monolayer . an afm molecular image of the didodecylmelamine monolayer bound to the barbituric template could be easily observed , although the corresponding monolayer transferred from pure water was too fragile for afm observation . afm images of the formed complex transferred onto a mica surface showed the periodic brighter and darker portions that correspond to higher and lower regions of the monolayer surface , respectively . the binding of fad with the guanidinium / orotate mixed monolayer should dispose two functional units at the same level through binding to the template fad molecule where the flavin unit in fad may be buried under the phosphate group owing to the conformational flexibility of the fad molecule , and resulting in a height difference between the two terminal methyl groups . therefore , regulation of molecular recognition based on conformational changes of host amphiphiles is one of the distinguishing characteristics of molecular recognition . reported ph control of molecular recognition at the air - water interface . the specific binding of l - alanine to the monolayer was completely dependent on the structure of the monolayer , which can be regulated by variation of ph . realized mechanically controlled molecular recognition through dynamic formation of a cavity structure using host molecules , known as steroid cyclophanes [ 5660 ] . the steroid cyclophane molecule used in this research contains a 1,6,20,25-tetraaza[6.1.6.1]-paracyclophane cyclic core connected to four steroid moieties through a flexible l - lysine spacer with cholic acid ( see figure 5 ) . as a result reversible binding ( capture and release ) of an aqueous fluorescent guest ( 6-(p - toluidino)naphthalene-2-sulfonate ( tns ) was demonstrated by a repeated compression and expansion of the steroid cyclophane monolayer . according to repeated pressure application during the compression - expansion cycle , a periodic change in the fluorescence intensity was detected , because tns emits fluorescence strongly in a hydrophobic host interior . the concept of dynamic function at the air - water interface has been recently similarly reported by other research groups . resorcinarene cavitands with four quinoxaline bridges are a family of macrocycles that adopt a contracted , vase - type conformation , capable of guest inclusion , whereas they switch to an expanded , kite - type conformation with a large flat surface under other conditions . stoddart , ho , and co - workers demonstrated that redoxcontrollable molecular shuttles , in the shape of amphiphilic , bistable rotaxanes , switch mechanically under chemical stimulus even when contained in closely packed langmuir films . helicity is influenced by the chirality of the side arms especially when ordered or aggregated at the supramolecular level . application of lateral pressure should affect the helix structure and , consequently , the diastereomeric stability of complexes with guest molecules . apart from the air - water interface , various interfacial environments provide media useful for molecular recognition and its related functions . in particular , self - assembled monolayers ( sam ) are often used for sensor applications because the sam structures facilitate contact with artificial devices such as electrodes and field effect transistors , as has been reviewed by reinhoudt and co - workers . geiger and co - workers reported use of an enhanced surface second harmonic generation ( shg ) signal for detection of the toxic metal pollutant chromium(vi ) at custom - made amino acid functionalized fused quartz / water interfaces . reported a method to manipulate conductance using molecular recognition at a sam surface . electroactive functionalization of the monolayer was then achieved through binding of the complementary ferrocene - terminated uracil to the binder molecule . current - voltage properties of the patterned region were monitored by using an stm tip . noncovalent self - assembly provides a potential method to install and subsequently remove electroactive functionality in molecular electronics systems . kitano and taira used sam structures of cyclodextrin derivatives for detection of bisphenol - type substances . on the basis of the inhibitory effect of bisphenols on the inclusion of hydroquinone by the surface - confined cyclodextrin , use of -cyclodextrin and hexasodium calixarene hexasulfonic acid as host structures were also reported by the same research group . willner and co - workers demonstrated photochemical imprinting of molecular recognition sites for phenoxynaphthacene quinone in sam assembled on au surfaces . the primary step of their approach includes the assembly of the trans - phenoxynaphthacenequinone monolayer , followed by the rigidification of the monolayer with long chain alkanethiols generating a densely packed quinone monolayer . the second process involves the photoisomerization of the monolayer to the ana - quinone state , followed by the nucleophilic displacement of the quinone with butylamine . the binding of phenoxynaphthacenequinone to the imprinted recognition sites reveals selectivity , and structurally related substrates did not associate with the imprinted sites . combining recognition events with appropriate analytical methods can yield sensor devices suitable for biological applications . zeng , wang , and co - workers demonstrated that an unlabeled carbohydrate mass sensor in combination with lectin - bacterial o - antigen recognition can be used for detection of high molecular weight bacterial targets with remarkably high sensitivity and enhanced specificity . a functional mannose self - assembled monolayer in combination with lectin concanavalin a ( con a ) was used as the molecular recognition element for detection of escherichia coli w1485 using qcm as a transducer . whitesides and co - workers reported the synthesis of bifunctional polyacrylamides containing pendant vancomycin and fluorescein groups , and the use of these polymers to direct antibodies against fluorescein to sam presenting d - alanine - d - alanine groups . in this system a modified polyamidoamine carbazole / cu dendrimer , which is electrochemically cross - linked on a self - assembled monolayer ( sam ) modified au substrate , acted as an active sensing element for trapping the nerve agent analogs . bunker , stoddart , and co - workers developed supramolecular machines using sam structures in which molecular configurations can be reversibly programmed using electrochemical stimuli . interactions between the tethered tetracationic cyclophane host cyclobis(paraquat - p - phenylene ) and dissolved -electron - rich guest molecules , such as tetrathiafulvalene , were reversibly switched by oxidative electrochemistry . immiscible liquids produce a dynamic interface such as the water - oil interface that have been also used as media for specific molecular recognition . kitamura and co - workers investigated molecular recognition mediated by hydrogen - bonding interactions at a water - ccl4 interface by means of time - resolved total internal reflection ( tir ) fluorescence spectroscopy . watarai and co - workers reported a molecular recognition system of the interfacial aggregation of monocationic palladium(ii)-2-(5-bromo-2-pyridylazo)-5-diethylaminophenol complex with neutral diazine or purine bases at the toluene - water system . alkali metal ion recognition with [ 2-hydroxy-5-(4-nitrophenylazo)phenyl]-methyl-15-crown-5 at the heptane - water interface was investigated by teramae and co - workers , using in situ second harmonic generation ( shg ) spectroscopy . it was revealed experimentally that the na and k complexes were flatter while the li complex exhibited a lift - up orientation at the heptane - water interface . because of its relevance to biological systems , molecular recognition at the surface of aqueous lipid bilayers has also been investigated [ 8691 ] . recognition and binding of lead ions at the membrane surface resulted in a rapid and prominent reorganization of the receptor - lipids in the membrane that was measurable at both the macro- and nanoscales . removal of the lead ions , through the addition of edta , resulted in recovery of the original fluorescence and the reaggregation of structures in the membrane . darcy , ravoo , and co - workers synthesized amphiphilic cyclodextrins through 6-s - alkylation of the primary side and introduction of a poly(ethylene glycol ) chain to the secondary side of - , - , and -cyclodextrins , which formed nonionic bilayer vesicles in aqueous solution . molecular recognition of a hydrophobic anion ( adamantane carboxylate ) by the formed cyclodextrin vesicles was investigated by using capillary electrophoresis . the colorimetric transitions observed in the assembly , corresponding to perturbation of the polydiacetylene framework , are induced only upon recognition of the displayed epitope by its specific antibody present in the aqueous solution . this system could be utilized for studying antigen - antibody interactions and peptide - protein recognition , epitope mapping , and rapid screening of biological and chemical libraries . the subsequent covalent bioconjugation of amino - functionalized gold nanorod films with goat anti - human - immunoglobulin g ( anti - h - igg ) was successfully employed for the colorimetric detection of h - igg in a model reaction based on the specific binding affinity between the proteins . govorov , kotov , and co - workers prepared molecular spring assemblies of cdte nanowires and au nanoparticles , where the distance between the exciton and the plasmon can be reversibly varied . interfacial media provide a variety of possibilities for molecular recognition . at the air - water interface complicated binding sites can be constructed through self - assembly of rather simple molecular modules , where it is possible to generate well - defined recognition sites such as biomolecular receptors . reported unusual shifts in dissociation constants of amino acid residues at the air - water interface that were accompanied by enzyme - like catalytic activities . thus , the air - water interface will be more important for developments in biomimetic chemistry in the future . on the other hand , solid surfaces should be useful for molecular sensor device preparation , for instance , in the preparation of sensing - site arrays which will be important for highly integrated molecular devices . recent developments in control of molecular arrangements on two - dimensional solid surfaces [ 101103 ] should result in sensor arrays with ultrahigh spatial resolution . in addition , high - surface materials currently under development , such as mesoporous materials , should provide supports useful for efficient molecular recognition . a combination of advanced surface technologies and material chemistry with well - established molecular recognition sciences is crucial to the future development of this field .

los registros sanitarios electrnicos ( rse ) proporcionan una visin clnica de la salud del paciente . se est empezando a disponer de los rse mediante grandes conjuntos de datos , lo que posibilita una investigacin que transformar el panorama de la investigacin sanitaria . se necesitan mtodos para incorporar las dimensiones y puntos fuertes del bienestar en los grandes conjuntos de datos . el propsito de este estudio era examinar la posible convergencia del modelo de bienestar con un estndar de terminologa con conexin clnica , el sistema omaha , para documentar las evaluaciones del bienestar . planificar el sistema omaha y el modelo de bienestar para su uso en una evaluacin del bienestar de rse clnicos y evaluar su viabilidad en la descripcin de los puntos fuertes y las necesidades de los adultos que surjan de esta evaluacin . el modelo de bienestar y el sistema omaha se elaboraron mediante tcnicas de asignacin de conceptos . basndose en esta elaboracin , se desarroll una evaluacin del bienestar y se implement en un rse clnico . a partir de las evaluaciones del bienestar se extrajeron datos de indicadores de los puntos fuertes y de los signos o sntomas de 5 ancianos que vivan en una comunidad residencial y se analizaron empleando estadsticas descriptivas y tcnicas de visualizacin de patrones estndar . el acuerdo de la asignacin inicial fue del 93,5 % con las diferencias resueltas por consenso . el anlisis de los datos de bienestar mostr que los ancianos tenan una media de 34,8 ( intervalo = 22 - 49 ) indicadores de puntos fuertes para 22,8 conceptos . tuvieron una media de 6,4 ( intervalo = 4 - 8 ) signos / sntomas para una media de 3,2 ( intervalo = 2 - 5 ) conceptos . la relacin entre indicadores de puntos fuertes y signos / sntomas fue de 6:1 ( intervalo = 2,8 - 9,6 ) . juntos , el modelo de bienestar y el sistema omaha tienen potencial para poder realizar una perspectiva individual completa y mejorar el potencial de una perspectiva del bienestar en la investigacin de grandes volmenes de datos sanitarios . there have been major humanitarian and sociologic failings in medicine , but almost all of them can be attributed to our poor behavior as scientists as we have dealt with problems out of context and ignored data relevant to good medical care . big data refers to massive streams of digital data originating from diverse and ubiquitous sources , including clinical data . communities , health systems , and governments aspire toward the goal of a seamless flow of big data at the population level . liquid data refers to granular , interoperable data about people , places , and things that can be pooled for transformation into useful knowledge through rapid computing techniques . in healthcare and health systems , liquid big data are necessary in real time to support healthcare quality and enable population health research . having and using big data expands the potential for discovery of new and hidden patterns that may inform new directions in healthcare research . in the context of health and healthcare , discourse typically refers to big data as repositories of data generated by use of electronic health records ( ehrs ) to document a clinical view of patient health . some research also suggests that patients may one day contribute to health data repositories through opting to upload personal data from wearable devices or by entering data into personal health records ( phrs ) that are linked to ehrs . given the focus on ehrs , big data , and the potential of big data research to improve health and healthcare quality , it is essential to critically consider the context and paradigm of clinical data especially in regard to emerging concepts such as wellbeing , strengths , and a whole - person perspective . the disease and treatment paradigm of clinical healthcare may be seen as bounded by physical or mental disease or disability . this paradigm fails to acknowledge and address social dimensions of health or other holistic notions such as strengths or wellbeing . such dimensions of health may play a role in reducing disability and improving functioning and quality of life . the clinical ehr based on a disease and treatment paradigm translates to a narrow data set that consists of observational variables that describe what is wrong , what is done , and what is billed . this manifests within the ehr as problem , intervention , and cost concepts , which are entered as structured information in the form of assessments , physiological measures , laboratory values , medication records , claims data , and other clinical and administrative components . thus , use of data elements that originate in ehrs for big data research will produce results that are biased by the limited paradigm of disease and treatment clinical health - care and fail to demonstrate whole - person health or wellbeing , especially for patients living with chronic health conditions . looking for wellbeing in such a data set is akin to the streetlight effect : searching for an item under a streetlight where there is good lighting wellbeing encompasses a holistic view of physical , mental , and social dimensions and reflects personal meaning , strengths , and interactions of individuals , families , and communities . numerous models of wellbeing have been proposed to underpin psychological and social functioning . in the context of global and environmental health , kreitzer proposed that wellbeing is possible at individual , family , organization or system , and community levels . in kreitzer 's model , the concept of wellbeing incorporates health and extends further to the conditions that enable health and healing , in which people and systems are able to optimize their potential and flourish . the wellbeing model describes 6 dimensions that impact wellbeing and the ability to flourish at and across every level from individual to global . the dimensions are environment , health , relationships , security , purpose , and community ( figure 1 ) . environment is defined as access to nature as well as clean air , water , and toxin free ; health is defined as physical , emotional , mental , and spiritual health ; relationships encompasses social connections , networks , and the quality of relationships ; security relates to basic human needs , stable employment , sufficient finances , and personal safety ; purpose is defined as an aim and direction , a direct expression of spirituality that gives life and work meaning ; and community is defined as resources and infrastructure and the extent to which people are engaged and empowered . the model explains these factors as determinants of wellbeing and provides examples showing their interaction and their impact on health . consistent with the notion of wellbeing is a strengths perspective that goes beyond determining what is wrong and seeks to understand personal , family , and community assets that may be leveraged to address problems . use of the strengths - based approach has been shown to be effective in improving health for individuals with chronic illness . key to the strengths - based approach in relationship to the kreitzer wellbeing model dimensions are nonjudgmental interview assessments of patient motivation ( purpose ) , supports ( relationships , community ) , resources ( security , environment ) , and wellbeing ( health , including strengths as well as needs ) . to avoid looking at problems out of context and to enhance the value of clinical data for strengths - based care , it is essential to identify and implement new models of assessment and data collection that can seamlessly incorporate relevant data in ehrs and phrs . likewise , to reduce bias and enhance the value of clinical data for big data research in wellbeing , variables are needed that will expand the perspective of clinical data sets beyond disease , treatment , and cost to move from the narrow focus of what is wrong , what is done , and what is billed toward a wellbeing , strengths , whole - person perspective . the institute of medicine ( iom ) and others have begun to advocate for expanded documentation of psychosocial variables including the social determinants of health , acknowledging the need for a larger worldview for clinicians and researchers alike . however , there is no comprehensive and systematic method of data collection regarding health broadly defined as wellbeing across all determinants and settings . thus , methods are needed to seamlessly incorporate wellbeing dimensions and strengths indicators within in large data sets together with usual clinical documentation in order to enable meaningful whole - person big data research in health . previous research has examined standardized interface terminologies related to health and healthcare that describe the whole person and the variables that will enable examination of health in a comprehensive context . one such terminology , the omaha system , is a multidisciplinary clinical terminology that has the capability of expanding beyond a disease and treatment paradigm to include a strengths - based assessment and approach to care . the omaha system is a taxonomic health care terminology and measure that enables the management of comprehensive , holistic clinical information in healthcare . it has been widely used in community settings globally , especially in public health and home care . there is a growing literature base of healthcare quality research using big data methods with omaha system data . studies have demonstrated the feasibility of using the omaha system to describe strengths of patients with chronic illness and to describe interventions of the strengths - based approach and fit within a phr for use by patients . these preliminary studies suggest there may be potential to operationalize the wellbeing model using the omaha system to enable an expanded perspective within clinical data . the long - term goal of our research is to use the omaha system to discover novel patterns in large data set research that include wellbeing and strengths . the purpose of this study was to examine the potential alignment of the wellbeing model with the omaha system for documenting wellbeing assessments ( appendix ; available at www.gahmj.com ) . our objectives were to map the omaha system and wellbeing model for use in a clinical ehr for wellbeing assessment and to evaluate the feasibility of describing strengths and needs of seniors generated through this assessment . this mapping and feasibility study was exempted from review by the institutional review board of the university of minnesota , minneapolis . there were 55 residents ( 39 females , 16 males ) with an average age of 85 years . the administrators released 5 wellbeing assessments that were randomly sampled from the ehr , printed , and de - identified for this analysis . the de - identification process resulted in a sample for which no demographics were available . the omaha system in addition to being a standardized interface terminology for ehrs , is a comprehensive , holistic ontology for health . using the omaha system , health information is structured within 42 general concepts ( called problems ) that are organized within 4 domains : environmental , psychosocial , physiological , and health - related behaviors . these 4 domains represent an ecological perspective of physiological and psychosocial health within the environment , with personal health choices expressed by health - related behaviors . the omaha system has 3 components that enable holistic assessment , care planning and documentation , and outcomes measurement . these components are respectively the problem classification scheme , intervention scheme , and problem rating scale for outcomes . each of the components includes the 42 problem concepts that together describe health and wellbeing . the problem classification scheme is used to classify health assessments and is the component of the omaha system that was used in this study . each of the 42 omaha system problem concepts has a neutral definition ( table 1 ) and unique set of signs / symptoms . for example the signs / symptoms for the hearing problem concept are difficulty hearing normal speech tones , difficulty hearing speech in large group settings , difficulty hearing high frequency sounds , absent / abnormal response to sound , and abnormal results of hearing screening test . wellbeing mapped to the omaha system domains and 42 problem concepts for aim 1 , the wellbeing model was mapped to the omaha system using concept mapping techniques . first , the wellbeing model concepts were mapped to omaha system concepts independently by two content experts . these two mappings were combined in a single document and reviewed by a wellbeing model expert and an omaha system expert . differences were resolved by consensus . based on the final mapping , an omaha system wellbeing model assessment was adopted by nursing leaders for use in a senior residential community and incorporated within the existing clinical ehr . the comprehensive assessment included 37 omaha system problem concepts that operationalized the 6 dimensions of the wellbeing model specifically for seniors living in a residential community . five omaha system problem concepts were not selected for the assessment , including pregnancy , postpartum , reproductive function , family planning , and growth and development . wellbeing assessments were completed by a registered nurse . upon community entry , each resident was offered an assessment to establish a wellbeing baseline . wellbeing assessments were repeated with any change of condition , reflecting strength or limitation change , and every 60 to 90 days per state regulatory guidelines . aim 2 used assessment data that were recorded by registered nurses with bachelor's- , master's- , or doctoral - level preparation who conducted a comprehensive interview assessment with seniors joining the residential community . structured data entry for strengths indicators and signs / symptoms of 34 problem concepts were documented by nurses in the process of routine documentation if relevant to the resident ( the earliest version of the wellbeing assessment included 34 of the 37 selected omaha system problem concepts ) . printed copies of 5 assessments were de - identified and provided to the research team . the strengths indicators and signs / symptoms data were entered by the researchers into an excel spreadsheet with omaha system problem concepts as the organizing framework . data were analyzed to examine the feasibility of assessing the overall wellbeing and the relationships between strengths and needs of community - dwelling seniors , using microsoft excel 2013 ( microsoft corp , redmond , washington)for standard descriptive statistics and pattern visualization techniques . there were 55 residents ( 39 females , 16 males ) with an average age of 85 years . the administrators released 5 wellbeing assessments that were randomly sampled from the ehr , printed , and de - identified for this analysis . the de - identification process resulted in a sample for which no demographics were available . the omaha system in addition to being a standardized interface terminology for ehrs , is a comprehensive , holistic ontology for health . using the omaha system , health information is structured within 42 general concepts ( called problems ) that are organized within 4 domains : environmental , psychosocial , physiological , and health - related behaviors . these 4 domains represent an ecological perspective of physiological and psychosocial health within the environment , with personal health choices expressed by health - related behaviors . the omaha system has 3 components that enable holistic assessment , care planning and documentation , and outcomes measurement . these components are respectively the problem classification scheme , intervention scheme , and problem rating scale for outcomes . each of the components includes the 42 problem concepts that together describe health and wellbeing . the problem classification scheme is used to classify health assessments and is the component of the omaha system that was used in this study . each of the 42 omaha system problem concepts has a neutral definition ( table 1 ) and unique set of signs / symptoms . for example , the hearing problem concept definition is perception of sound by the ears . the signs / symptoms for the hearing problem concept are difficulty hearing normal speech tones , difficulty hearing speech in large group settings , difficulty hearing high frequency sounds , absent / abnormal response to sound , and abnormal results of hearing screening test . for aim 1 , the wellbeing model was mapped to the omaha system using concept mapping techniques . first , the wellbeing model concepts were mapped to omaha system concepts independently by two content experts . these two mappings were combined in a single document and reviewed by a wellbeing model expert and an omaha system expert . differences were resolved by consensus . based on the final mapping , an omaha system wellbeing model assessment was adopted by nursing leaders for use in a senior residential community and incorporated within the existing clinical ehr . the comprehensive assessment included 37 omaha system problem concepts that operationalized the 6 dimensions of the wellbeing model specifically for seniors living in a residential community . five omaha system problem concepts were not selected for the assessment , including pregnancy , postpartum , reproductive function , family planning , and growth and development . wellbeing assessments were repeated with any change of condition , reflecting strength or limitation change , and every 60 to 90 days per state regulatory guidelines . aim 2 used assessment data that were recorded by registered nurses with bachelor's- , master's- , or doctoral - level preparation who conducted a comprehensive interview assessment with seniors joining the residential community . structured data entry for strengths indicators and signs / symptoms of 34 problem concepts were documented by nurses in the process of routine documentation if relevant to the resident ( the earliest version of the wellbeing assessment included 34 of the 37 selected omaha system problem concepts ) . printed copies of 5 assessments were de - identified and provided to the research team . the strengths indicators and signs / symptoms data were entered by the researchers into an excel spreadsheet with omaha system problem concepts as the organizing framework . data were analyzed to examine the feasibility of assessing the overall wellbeing and the relationships between strengths and needs of community - dwelling seniors , using microsoft excel 2013 ( microsoft corp , redmond , washington)for standard descriptive statistics and pattern visualization techniques . omaha system problem concepts mapped successfully to the wellbeing model ( tables 1 and 2 ) . there was a range of 2 to 5 concepts per wellbeing dimension , with the exception of the health dimension , which had 25 concepts . three problem concepts mapped to the wellbeing model environment dimension were from the omaha system environmental domain . eight concepts mapped to the wellbeing model relationships and purpose dimensions were from the omaha system psychosocial domain . two problem concepts mapped to the wellbeing model community dimensions were from the omaha system psychosocial and health - related behaviors domains , and 4 problem concepts mapped to the wellbeing security dimension were from the omaha system environmental , psychosocial , and physiological domains . finally , 25 concepts mapped to the wellbeing model health dimension were from the omaha system psychosocial , physiological , and health - related behaviors domains . the mapping is depicted visually in figure 2 to show alignment of the models , with colors representing the wellbeing model dimensions and rings representing the omaha system domains . number of omaha system problem concepts by omaha system domain and wellbeing model dimension one wellbeing model dimension mapped to one omaha system domain one wellbeing model dimension mapped to more than one omaha system domain mapping of the omaha system and wellbeing model by domain ( rings ) . to apply the omaha system within a whole - person assessment , the nurses at the residential community developed a set of neutral assessment questions and one or more strengths indicators for each problem concept . these were incorporated within the assessment protocol in addition to the problem - specific signs / symptoms . an example of the wellbeing whole - person assessment for the social contact problem concept ( relationship dimension psychosocial domain ) defined as interaction between the individual and others outside the immediate living area includes the prompt , tell me about your social activity / friendships , significant people in your life , and things you enjoy doing and the strengths indicator extensive family engagement . signs / symptoms of the social contact problem are limited social contact , uses healthcare provider for social contact , and minimal outside stimulation / leisure time activities . an example of the wellbeing whole - person assessment as it appears in a paper form for the skin problem ( health dimension physiological domain ) is shown in figure 3 . wellbeing assessment skin ( health dimension physiological domain ) . a proprietary software system already in use at the residential community was retrofitted in the resident assessment documentation section to include omaha system terms as described above , including the problem concept specific assessment questions , strengths indicators , and signs / symptoms . despite providing screens for structured data entry , the software system did not build in new functionality to export , aggregate , or report this wellbeing assessment data nor link to plan of care functionality . seniors had an average of 34.8 ( range=22 - 49 ) strengths indicators for 22.8 ( range=16 - 29 ) problem concepts ( table 3 ) . they had an average of 6.4 ( range=4 - 8 ) signs / symptoms for 3.2 ( range=2 - 5 ) problem concepts . the ratio of strengths indicators to signs / symptoms was 6:1 ( range 2.8:1 - 9.6:1 ) . patterns in the data reflected inverse relationships between strengths and signs / symptoms by problem concept ( figure 4 ) . problem concepts with the most signs / symptoms were vision , neuro - musculo - skeletal function , circulation , and hearing ( figure 4 ) . signs / symptoms were most frequent in the omaha system physiological domain and wellbeing health dimension , and strengths indicators were most frequent in the omaha system psychosocial domain and the wellbeing relationship dimension . number of omaha system problem concepts in wellbeing assessment data of seniors living in residential communities by omaha system domain and wellbeing model dimension strengths and signs / symptoms by omaha system concept , wellbeing model dimension , and omaha system domain among seniors living in residential communities . results demonstrate the feasibility of using the wellbeing model , the omaha system , and the strengths approach to achieve the goal of a whole - person perspective in clinical data . furthermore , it was feasible to describe and document both strengths and signs / symptoms of seniors in residential communities for 34 omaha system problem concepts . thus , there is potential to generate large clinical data sets from phrs and ehrs that can be mined to discover new patterns in wellbeing and healthcare using a clinical terminology standard . the wellbeing model is a theoretical framework that aids in exploring the meaning of wellbeing for individuals , families , communities , and systems . the omaha system is an information model that enables comprehensive holistic assessment , care planning , documentation , and outcome measurement . the finding of very high level of agreement across content expert mappings is unusual and may indicate that the wellbeing model and omaha system share a basis in sound holistic health science . further research is needed to evaluate and validate this mapping and its associated wellbeing assessment tools , including strengths indicators . the notion that the omaha system , a recognized international clinical terminology standard , can operationalize the wellbeing model theoretical framework is key to accurate and comprehensive knowledge representation of the wellbeing model in clinical ehrs and phrs . such implementation within ehrs and phrs is in turn necessary for data capture in large data sets . for example , phrs are thought to be a mechanism for engaging patients in their care and exchanging data with clinicians and perhaps augmenting clinical data by adding patient - reported information , including patient - reported strengths . alignment between the environment dimension and the environmental domain and likewise between the relationships and purpose dimensions and the psychosocial domain show the conceptual alignment between the 2 models . in the case of the environment dimension , the definition suggests that a strengths indicator may be access to nature ; likewise , the corollary nature deficit could be recommended for a future revision of the omaha system as a sign / symptom for a problem concept in the environmental domain , such as neighborhood / workplace safety . alignment between community and the psychosocial and health - related behaviors domains suggests that there are both behavioral and psychological dimensions of wellbeing in community . the finding that security mapped to problem concepts in the environmental , psychosocial , and physiological domains demonstrates the multifaceted aspects of security that broadly underlie health . the finding that the health dimension mapped to problem concepts from psychosocial , physiological , and health - related behaviors domains is consistent with the primary focus of the omaha system as a structured ontology for health and healthcare . the fact that all wellbeing dimensions and omaha system problem concepts were mapped suggests that data from wellbeing assessments using the omaha system would comprehensively depict wellbeing , capture strengths and needs in a whole - person perspective , and enable large data set research to discover patterns in wellbeing . the omaha system has been successfully implemented in a number of clinical software applications that are widely used in community settings in the united states and internationally . the limited functionality of the software used for the wellbeing assessment in this study demonstrated challenges faced by clinicians who desire to improve documentation practices within established ehrs . the fact that the wellbeing assessment data could not be accessed from the ehr in this study points to the need for leaders to understand basic principles of data management in order to make informed decisions about clinical software . furthermore , the inability to link assessments to care plans and outcome measures points to the importance of improved software development based on sound taxonomic principles that can comprehensibly link data in a taxonomic ontological structure , as suggested by weed , martin , cimino , and others . further research is needed to understand best practices in software development that support holistic practice , improve clinical workflow , and enable structured wellbeing documentation , thus enabling capture of structured robust , relational data for clinical use , evaluation , and research . due to the software limitations , manual data extraction from printed wellbeing assessments was necessary and limited our analysis . nevertheless , the finding that strengths indicators were most likely in the psychosocial domain is consistent with previous research evaluating strengths of adults with multiple chronic conditions . these preliminary findings showed that the ratio of strengths to signs / symptoms was consistently high in this sample and that problem concepts with more signs / symptoms had fewer strengths . such patterns are of interest and may provide a glimpse of possible big data research in wellbeing and healthcare . further research is needed to examine these and other whole - person patterns in large data sets . the research agenda that is emerging from this structured whole person assessment approach is extensive . future research can incorporate variables from a perspective that describe overall wellbeing including strengths indicators , which may be associated with health outcomes . this study demonstrated the feasibility of using the omaha system in ehrs to operationalize wellbeing as described by kreitzer . the wellbeing assessment included both strengths indicators and signs / symptoms for 34 concepts and enabled a whole - person assessment of strengths and needs of seniors in a residential community . wellbeing assessment data revealed an inverse relationship between strengths and needs among seniors living in a residential community . together , the wellbeing model , a strengths - based assessment , and the omaha system have potential to fill the gap in big data and illuminate whole - person big data research .

background : electronic health records ( ehrs ) provide a clinical view of patient health . ehr data are becoming available in large data sets and enabling research that will transform the landscape of healthcare research . methods are needed to incorporate wellbeing dimensions and strengths in large data sets . the purpose of this study was to examine the potential alignment of the wellbeing model with a clinical interface terminology standard , the omaha system , for documenting wellbeing assessments.objective:to map the omaha system and wellbeing model for use in a clinical ehr wellbeing assessment and to evaluate the feasibility of describing strengths and needs of seniors generated through this assessment.methods:the wellbeing model and omaha system were mapped using concept mapping techniques . based on this mapping , a wellbeing assessment was developed and implemented within a clinical ehr . strengths indicators and signs / symptoms data for 5 seniors living in a residential community were abstracted from wellbeing assessments and analyzed using standard descriptive statistics and pattern visualization techniques.results:initial mapping agreement was 93.5% , with differences resolved by consensus . wellbeing data analysis showed seniors had an average of 34.8 ( range=22 - 49 ) strengths indicators for 22.8 concepts . they had an average of 6.4 ( range=4 - 8 ) signs / symptoms for an average of 3.2 ( range=2 - 5 ) concepts . the ratio of strengths indicators to signs / symptoms was 6:1 ( range 2.8 - 9.6 ) . problem concepts with more signs / symptoms had fewer strengths.conclusion:together , the wellbeing model and the omaha system have potential to enable a whole - person perspective and enhance the potential for a wellbeing perspective in big data research in healthcare .

Antecedentes: Objetivo: Mtodos: Resultados: Conclusin: BACKGROUND METHODS Sample Instrument Mapping Procedure Wellbeing Data Collection and Analysis RESULTS DISCUSSION CONCLUSION

el anlisis de los datos de bienestar mostr que los ancianos tenan una media de 34,8 ( intervalo = 22 - 49 ) indicadores de puntos fuertes para 22,8 conceptos . tuvieron una media de 6,4 ( intervalo = 4 - 8 ) signos / sntomas para una media de 3,2 ( intervalo = 2 - 5 ) conceptos . communities , health systems , and governments aspire toward the goal of a seamless flow of big data at the population level . in healthcare and health systems , liquid big data are necessary in real time to support healthcare quality and enable population health research . having and using big data expands the potential for discovery of new and hidden patterns that may inform new directions in healthcare research . in the context of health and healthcare , discourse typically refers to big data as repositories of data generated by use of electronic health records ( ehrs ) to document a clinical view of patient health . some research also suggests that patients may one day contribute to health data repositories through opting to upload personal data from wearable devices or by entering data into personal health records ( phrs ) that are linked to ehrs . given the focus on ehrs , big data , and the potential of big data research to improve health and healthcare quality , it is essential to critically consider the context and paradigm of clinical data especially in regard to emerging concepts such as wellbeing , strengths , and a whole - person perspective . the clinical ehr based on a disease and treatment paradigm translates to a narrow data set that consists of observational variables that describe what is wrong , what is done , and what is billed . thus , use of data elements that originate in ehrs for big data research will produce results that are biased by the limited paradigm of disease and treatment clinical health - care and fail to demonstrate whole - person health or wellbeing , especially for patients living with chronic health conditions . looking for wellbeing in such a data set is akin to the streetlight effect : searching for an item under a streetlight where there is good lighting wellbeing encompasses a holistic view of physical , mental , and social dimensions and reflects personal meaning , strengths , and interactions of individuals , families , and communities . in kreitzer 's model , the concept of wellbeing incorporates health and extends further to the conditions that enable health and healing , in which people and systems are able to optimize their potential and flourish . the wellbeing model describes 6 dimensions that impact wellbeing and the ability to flourish at and across every level from individual to global . environment is defined as access to nature as well as clean air , water , and toxin free ; health is defined as physical , emotional , mental , and spiritual health ; relationships encompasses social connections , networks , and the quality of relationships ; security relates to basic human needs , stable employment , sufficient finances , and personal safety ; purpose is defined as an aim and direction , a direct expression of spirituality that gives life and work meaning ; and community is defined as resources and infrastructure and the extent to which people are engaged and empowered . use of the strengths - based approach has been shown to be effective in improving health for individuals with chronic illness . key to the strengths - based approach in relationship to the kreitzer wellbeing model dimensions are nonjudgmental interview assessments of patient motivation ( purpose ) , supports ( relationships , community ) , resources ( security , environment ) , and wellbeing ( health , including strengths as well as needs ) . to avoid looking at problems out of context and to enhance the value of clinical data for strengths - based care , it is essential to identify and implement new models of assessment and data collection that can seamlessly incorporate relevant data in ehrs and phrs . likewise , to reduce bias and enhance the value of clinical data for big data research in wellbeing , variables are needed that will expand the perspective of clinical data sets beyond disease , treatment , and cost to move from the narrow focus of what is wrong , what is done , and what is billed toward a wellbeing , strengths , whole - person perspective . thus , methods are needed to seamlessly incorporate wellbeing dimensions and strengths indicators within in large data sets together with usual clinical documentation in order to enable meaningful whole - person big data research in health . previous research has examined standardized interface terminologies related to health and healthcare that describe the whole person and the variables that will enable examination of health in a comprehensive context . one such terminology , the omaha system , is a multidisciplinary clinical terminology that has the capability of expanding beyond a disease and treatment paradigm to include a strengths - based assessment and approach to care . the omaha system is a taxonomic health care terminology and measure that enables the management of comprehensive , holistic clinical information in healthcare . there is a growing literature base of healthcare quality research using big data methods with omaha system data . studies have demonstrated the feasibility of using the omaha system to describe strengths of patients with chronic illness and to describe interventions of the strengths - based approach and fit within a phr for use by patients . these preliminary studies suggest there may be potential to operationalize the wellbeing model using the omaha system to enable an expanded perspective within clinical data . the long - term goal of our research is to use the omaha system to discover novel patterns in large data set research that include wellbeing and strengths . the purpose of this study was to examine the potential alignment of the wellbeing model with the omaha system for documenting wellbeing assessments ( appendix ; available at www.gahmj.com ) . our objectives were to map the omaha system and wellbeing model for use in a clinical ehr for wellbeing assessment and to evaluate the feasibility of describing strengths and needs of seniors generated through this assessment . this mapping and feasibility study was exempted from review by the institutional review board of the university of minnesota , minneapolis . there were 55 residents ( 39 females , 16 males ) with an average age of 85 years . the omaha system in addition to being a standardized interface terminology for ehrs , is a comprehensive , holistic ontology for health . using the omaha system , health information is structured within 42 general concepts ( called problems ) that are organized within 4 domains : environmental , psychosocial , physiological , and health - related behaviors . these 4 domains represent an ecological perspective of physiological and psychosocial health within the environment , with personal health choices expressed by health - related behaviors . the omaha system has 3 components that enable holistic assessment , care planning and documentation , and outcomes measurement . each of the components includes the 42 problem concepts that together describe health and wellbeing . the problem classification scheme is used to classify health assessments and is the component of the omaha system that was used in this study . each of the 42 omaha system problem concepts has a neutral definition ( table 1 ) and unique set of signs / symptoms . for example the signs / symptoms for the hearing problem concept are difficulty hearing normal speech tones , difficulty hearing speech in large group settings , difficulty hearing high frequency sounds , absent / abnormal response to sound , and abnormal results of hearing screening test . wellbeing mapped to the omaha system domains and 42 problem concepts for aim 1 , the wellbeing model was mapped to the omaha system using concept mapping techniques . first , the wellbeing model concepts were mapped to omaha system concepts independently by two content experts . these two mappings were combined in a single document and reviewed by a wellbeing model expert and an omaha system expert . differences were resolved by consensus . based on the final mapping , an omaha system wellbeing model assessment was adopted by nursing leaders for use in a senior residential community and incorporated within the existing clinical ehr . the comprehensive assessment included 37 omaha system problem concepts that operationalized the 6 dimensions of the wellbeing model specifically for seniors living in a residential community . five omaha system problem concepts were not selected for the assessment , including pregnancy , postpartum , reproductive function , family planning , and growth and development . upon community entry , each resident was offered an assessment to establish a wellbeing baseline . wellbeing assessments were repeated with any change of condition , reflecting strength or limitation change , and every 60 to 90 days per state regulatory guidelines . structured data entry for strengths indicators and signs / symptoms of 34 problem concepts were documented by nurses in the process of routine documentation if relevant to the resident ( the earliest version of the wellbeing assessment included 34 of the 37 selected omaha system problem concepts ) . the strengths indicators and signs / symptoms data were entered by the researchers into an excel spreadsheet with omaha system problem concepts as the organizing framework . data were analyzed to examine the feasibility of assessing the overall wellbeing and the relationships between strengths and needs of community - dwelling seniors , using microsoft excel 2013 ( microsoft corp , redmond , washington)for standard descriptive statistics and pattern visualization techniques . the de - identification process resulted in a sample for which no demographics were available . the omaha system in addition to being a standardized interface terminology for ehrs , is a comprehensive , holistic ontology for health . using the omaha system , health information is structured within 42 general concepts ( called problems ) that are organized within 4 domains : environmental , psychosocial , physiological , and health - related behaviors . these 4 domains represent an ecological perspective of physiological and psychosocial health within the environment , with personal health choices expressed by health - related behaviors . the omaha system has 3 components that enable holistic assessment , care planning and documentation , and outcomes measurement . each of the components includes the 42 problem concepts that together describe health and wellbeing . the problem classification scheme is used to classify health assessments and is the component of the omaha system that was used in this study . each of the 42 omaha system problem concepts has a neutral definition ( table 1 ) and unique set of signs / symptoms . the signs / symptoms for the hearing problem concept are difficulty hearing normal speech tones , difficulty hearing speech in large group settings , difficulty hearing high frequency sounds , absent / abnormal response to sound , and abnormal results of hearing screening test . for aim 1 , the wellbeing model was mapped to the omaha system using concept mapping techniques . first , the wellbeing model concepts were mapped to omaha system concepts independently by two content experts . these two mappings were combined in a single document and reviewed by a wellbeing model expert and an omaha system expert . differences were resolved by consensus . based on the final mapping , an omaha system wellbeing model assessment was adopted by nursing leaders for use in a senior residential community and incorporated within the existing clinical ehr . the comprehensive assessment included 37 omaha system problem concepts that operationalized the 6 dimensions of the wellbeing model specifically for seniors living in a residential community . five omaha system problem concepts were not selected for the assessment , including pregnancy , postpartum , reproductive function , family planning , and growth and development . wellbeing assessments were repeated with any change of condition , reflecting strength or limitation change , and every 60 to 90 days per state regulatory guidelines . aim 2 used assessment data that were recorded by registered nurses with bachelor's- , master's- , or doctoral - level preparation who conducted a comprehensive interview assessment with seniors joining the residential community . structured data entry for strengths indicators and signs / symptoms of 34 problem concepts were documented by nurses in the process of routine documentation if relevant to the resident ( the earliest version of the wellbeing assessment included 34 of the 37 selected omaha system problem concepts ) . the strengths indicators and signs / symptoms data were entered by the researchers into an excel spreadsheet with omaha system problem concepts as the organizing framework . data were analyzed to examine the feasibility of assessing the overall wellbeing and the relationships between strengths and needs of community - dwelling seniors , using microsoft excel 2013 ( microsoft corp , redmond , washington)for standard descriptive statistics and pattern visualization techniques . omaha system problem concepts mapped successfully to the wellbeing model ( tables 1 and 2 ) . there was a range of 2 to 5 concepts per wellbeing dimension , with the exception of the health dimension , which had 25 concepts . three problem concepts mapped to the wellbeing model environment dimension were from the omaha system environmental domain . eight concepts mapped to the wellbeing model relationships and purpose dimensions were from the omaha system psychosocial domain . two problem concepts mapped to the wellbeing model community dimensions were from the omaha system psychosocial and health - related behaviors domains , and 4 problem concepts mapped to the wellbeing security dimension were from the omaha system environmental , psychosocial , and physiological domains . finally , 25 concepts mapped to the wellbeing model health dimension were from the omaha system psychosocial , physiological , and health - related behaviors domains . the mapping is depicted visually in figure 2 to show alignment of the models , with colors representing the wellbeing model dimensions and rings representing the omaha system domains . number of omaha system problem concepts by omaha system domain and wellbeing model dimension one wellbeing model dimension mapped to one omaha system domain one wellbeing model dimension mapped to more than one omaha system domain mapping of the omaha system and wellbeing model by domain ( rings ) . to apply the omaha system within a whole - person assessment , the nurses at the residential community developed a set of neutral assessment questions and one or more strengths indicators for each problem concept . these were incorporated within the assessment protocol in addition to the problem - specific signs / symptoms . an example of the wellbeing whole - person assessment for the social contact problem concept ( relationship dimension psychosocial domain ) defined as interaction between the individual and others outside the immediate living area includes the prompt , tell me about your social activity / friendships , significant people in your life , and things you enjoy doing and the strengths indicator extensive family engagement . signs / symptoms of the social contact problem are limited social contact , uses healthcare provider for social contact , and minimal outside stimulation / leisure time activities . an example of the wellbeing whole - person assessment as it appears in a paper form for the skin problem ( health dimension physiological domain ) is shown in figure 3 . a proprietary software system already in use at the residential community was retrofitted in the resident assessment documentation section to include omaha system terms as described above , including the problem concept specific assessment questions , strengths indicators , and signs / symptoms . despite providing screens for structured data entry , the software system did not build in new functionality to export , aggregate , or report this wellbeing assessment data nor link to plan of care functionality . seniors had an average of 34.8 ( range=22 - 49 ) strengths indicators for 22.8 ( range=16 - 29 ) problem concepts ( table 3 ) . they had an average of 6.4 ( range=4 - 8 ) signs / symptoms for 3.2 ( range=2 - 5 ) problem concepts . the ratio of strengths indicators to signs / symptoms was 6:1 ( range 2.8:1 - 9.6:1 ) . patterns in the data reflected inverse relationships between strengths and signs / symptoms by problem concept ( figure 4 ) . problem concepts with the most signs / symptoms were vision , neuro - musculo - skeletal function , circulation , and hearing ( figure 4 ) . signs / symptoms were most frequent in the omaha system physiological domain and wellbeing health dimension , and strengths indicators were most frequent in the omaha system psychosocial domain and the wellbeing relationship dimension . number of omaha system problem concepts in wellbeing assessment data of seniors living in residential communities by omaha system domain and wellbeing model dimension strengths and signs / symptoms by omaha system concept , wellbeing model dimension , and omaha system domain among seniors living in residential communities . results demonstrate the feasibility of using the wellbeing model , the omaha system , and the strengths approach to achieve the goal of a whole - person perspective in clinical data . furthermore , it was feasible to describe and document both strengths and signs / symptoms of seniors in residential communities for 34 omaha system problem concepts . thus , there is potential to generate large clinical data sets from phrs and ehrs that can be mined to discover new patterns in wellbeing and healthcare using a clinical terminology standard . the wellbeing model is a theoretical framework that aids in exploring the meaning of wellbeing for individuals , families , communities , and systems . the omaha system is an information model that enables comprehensive holistic assessment , care planning , documentation , and outcome measurement . the finding of very high level of agreement across content expert mappings is unusual and may indicate that the wellbeing model and omaha system share a basis in sound holistic health science . further research is needed to evaluate and validate this mapping and its associated wellbeing assessment tools , including strengths indicators . the notion that the omaha system , a recognized international clinical terminology standard , can operationalize the wellbeing model theoretical framework is key to accurate and comprehensive knowledge representation of the wellbeing model in clinical ehrs and phrs . such implementation within ehrs and phrs is in turn necessary for data capture in large data sets . alignment between the environment dimension and the environmental domain and likewise between the relationships and purpose dimensions and the psychosocial domain show the conceptual alignment between the 2 models . in the case of the environment dimension , the definition suggests that a strengths indicator may be access to nature ; likewise , the corollary nature deficit could be recommended for a future revision of the omaha system as a sign / symptom for a problem concept in the environmental domain , such as neighborhood / workplace safety . the finding that security mapped to problem concepts in the environmental , psychosocial , and physiological domains demonstrates the multifaceted aspects of security that broadly underlie health . the finding that the health dimension mapped to problem concepts from psychosocial , physiological , and health - related behaviors domains is consistent with the primary focus of the omaha system as a structured ontology for health and healthcare . the fact that all wellbeing dimensions and omaha system problem concepts were mapped suggests that data from wellbeing assessments using the omaha system would comprehensively depict wellbeing , capture strengths and needs in a whole - person perspective , and enable large data set research to discover patterns in wellbeing . the omaha system has been successfully implemented in a number of clinical software applications that are widely used in community settings in the united states and internationally . the limited functionality of the software used for the wellbeing assessment in this study demonstrated challenges faced by clinicians who desire to improve documentation practices within established ehrs . the fact that the wellbeing assessment data could not be accessed from the ehr in this study points to the need for leaders to understand basic principles of data management in order to make informed decisions about clinical software . furthermore , the inability to link assessments to care plans and outcome measures points to the importance of improved software development based on sound taxonomic principles that can comprehensibly link data in a taxonomic ontological structure , as suggested by weed , martin , cimino , and others . further research is needed to understand best practices in software development that support holistic practice , improve clinical workflow , and enable structured wellbeing documentation , thus enabling capture of structured robust , relational data for clinical use , evaluation , and research . due to the software limitations , manual data extraction from printed wellbeing assessments was necessary and limited our analysis . nevertheless , the finding that strengths indicators were most likely in the psychosocial domain is consistent with previous research evaluating strengths of adults with multiple chronic conditions . these preliminary findings showed that the ratio of strengths to signs / symptoms was consistently high in this sample and that problem concepts with more signs / symptoms had fewer strengths . such patterns are of interest and may provide a glimpse of possible big data research in wellbeing and healthcare . further research is needed to examine these and other whole - person patterns in large data sets . this study demonstrated the feasibility of using the omaha system in ehrs to operationalize wellbeing as described by kreitzer . the wellbeing assessment included both strengths indicators and signs / symptoms for 34 concepts and enabled a whole - person assessment of strengths and needs of seniors in a residential community . wellbeing assessment data revealed an inverse relationship between strengths and needs among seniors living in a residential community . together , the wellbeing model , a strengths - based assessment , and the omaha system have potential to fill the gap in big data and illuminate whole - person big data research .

hypothalamic neural circuits are involved in the control of a large number of functions , the regulation of endocrine axes and energy balance being of particular importance ( yeo and heisler , 2012 , schneeberger et al . , 2014 , substantial interest and efforts have been devoted to the analysis of the arcuate nucleus of the hypothalamus ( arc ) , which , in fact , has been considered as the master hypothalamic center for energy homeostasis ( yeo and heisler , 2012 , schneeberger et al . , 2014 , 2016 ) . despite this evidence , other hypothalamic sites , such as the ventromedial nucleus of the hypothalamus ( vmh ) , formerly characterized as the brain satiety center , and the lateral hypothalamic area ( lha ) , formerly characterized as the brain hunger center ( hecherington and ranson , 1942 , anand and brobeck , 1951 ) , also play major and probably more integrative roles than the arc in the modulation of energy balance . these regions receive information from arc neurons and also project their axons to other hypothalamic and extra - hypothalamic sites ( schneeberger et al . , 2014 , 2016 ) to control not only feeding but also several peripheral processes , such as adipose and hepatic metabolism , as well as brown adipose tissue ( bat ) thermogenesis through the sympathetic ( sns ) and parasympathetic nervous system ( psns ) ( nogueiras et al . , 2007 , fliers et al . , 2010 , stanley et al . , 2010 , imbernon et al . , 2013 , the molecular mediators within those areas have begun to be characterized , and over the last 5 years extensive attention has been focused on the role of amp - activated protein kinase ( ampk ) in the vmh and orexin ( ox ) in the lha as key modulators of brown fat . thus , inhibition of ampk function in the vmh ( lpez et al . , 2010b , lpez et al . , 2016 , martnez de morentin et al . , 2012 , martnez de morentin et al . , 2014 , whittle et al . , 2012 , , 2013 , beiroa et al . , 2014 ) or increased orexigenic tone in the lha ( teske et al . , 2006 , , 2012 , madden et al . , 2012 ) activates the bat thermogenic program , leading to elevated temperature , increased energy expenditure ( ee ) , and weight loss . in addition , ox neurons in the lha are required for bat development and differentiation ( sellayah et al . , 2011 , sellayah and sikder , 2012 , sellayah and sikder , 2014 ) . in spite of this evidence , no functional data have linked those neuronal populations , and it is currently unknown whether ampk in the vmh and ox neurons in the lha are part of the same circuit modulating bat function or , alternatively , they function separately . bone morphogenetic proteins ( bmps ) belong to the transforming growth factor ( tgf- ) superfamily . bmps regulate a wide range of processes from embryonic development to tissue homeostasis ( schulz and tseng , 2009 , modica and wolfrum , 2013 ) . among these functions , bmps bmp2 and -4 and bmp7 and -9 were identified as important elements in white and brown adipocyte differentiation , respectively ( tang et al . , 2004 , bowers et al . , 2006 , huang et al . , 2009 , kuo et al . , 2014 ) . in addition , a more recent report has shown that bmp4 induces brown adipocyte characteristics in mature white adipocytes ( qian et al . , 2013 ) . other recent findings , including those from our group , surfaced a role for bmps in energy balance . thus , bmp7 and bmp8b were demonstrated to regulate bat thermogenic function and increase ee ( tseng et al . , 2008 , townsend et al . , 2012 , , 2013 , whittle et al . , 2012 ) . given that bmp8b acts centrally , its thermogenic effect is dependent upon the activation status of ampk in the vmh , and it increases neuronal activation in the lha ( whittle et al . , 2012 ) , we aimed to uncover the neuronal circuitry linking energy sensors and neuropeptides involved in the thermogenic effect of central bmp8b . recent evidence has demonstrated that central bmp8b exerts a robust thermogenic effect in females ( whittle et al . , 2012 ) . our data showed that after 2 hr of an intracerebroventricular ( i.c.v . ) injection of bmp8b , male rats did not show an increase in either body ( figure 1a ) or bat temperature ( figure 1b ) . next we analyzed the effect of central bmp8b in ovariectomized ( ovx ) female rats with and without estradiol ( e2 ) replacement and their sham - operated controls ( martnez de morentin et al . ovx rats gained significantly more weight ( sham : 23.1 2.38 g versus ovx : 71.3 28 g ; p < 0.001 ) and developed a marked hyperphagia ( sham : 14.87 0.31 g / day versus ovx : 17.70 0.49 g / day ; p < 0.01 ) , confirming the efficiency of the ovx procedure . while central administration of bmp8b to sham controls induced a clear thermogenic action , ovx totally abolished it at both body and bat temperature levels . of note , ovx rats with e2 replacement responded normally to bmp8b ( figures 1c and 1d ) . overall , these data indicated that the thermogenic action of bmp8b shows a sexual dimorphism and depends on physiological levels of ovarian estrogens . we aimed to investigate whether the thermogenic central actions of bmp8b were affected by temperature . therefore , we administered central bmp8b to female rats kept at 23c or 4c during 10 hr prior to the i.c.v . treatment and for a total of 12 hr . our data showed that bmp8b protected against the loss of both core ( figure 1e ) and bat ( figure 1f ) temperature when given at 4c . to investigate the effect of chronic nucleus - specific bmp8b treatment , stereotaxic cannulae were placed in the vmh and the lha . the correct position of the cannulae was verified by histological examination of coronal sections of the brains ( figures s1a and s1b ) . noteworthy , the expression of bmp type i receptors alk4 , alk5 , and alk7 in those nuclei was demonstrated by real - time rt - pcr analysis ( figure s2a ) of dissected vmh and lha , whose specificity was controlled by analysis of the specific markers steroidogenic factor 1 ( sf1 ) and ox , respectively ( figure s2b ) . when given in the vmh , bmp8b promoted a feeding - independent weight loss ( figure 2a ; initial body weight vehicle vmh : 240.0 4.4 g versus bmp8b vmh : 237.5 4.0 g ; non - significant ) ; no effect was observed when bmp8b was injected in the lha ( figure 2b ; initial body weight vehicle lha : 233.0 3.1 g versus bmp8b lha : 235.1 1.8 g ; non - significant ) . the action of bmp8b in the vmh was associated with a trend ( p = 0.07 ) to reduce adiposity without changes in the lean mass ( figure 2c ) . bmp8b in the lha promoted no changes either in fat or lean mass ( figure 2d ) . treatment with bmp8b within the vmh induced a marked elevation in body temperature , which was consistent during the whole period of administration ( figure 2e ) . no effect on body temperature moreover , bmp8b in the vmh , but not in the lha , increased ee ( figures 2 g and 2h ) and reduced the respiratory quotient ( rq ; figures 2i and 2j ) . no changes in locomotor activity ( la ) were found when bmp8b was administered either in the vmh or the lha ( figures 2k and 2l ) , although a non - significant trend ( p = 0.07 ) for increased la was detected in vmh bmp8b - treated rats . we aimed to investigate whether hypothalamic nucleus - specific bmp8b actions result in changes in ampk signaling within the vmh . we found that vmh administration of bmp8b decreased the phosphorylated levels of ampk ( pampk ) and its downstream target acetyl - coa carboxylase alpha ( pacc ) ; no changes were found in total levels of ampk1 , ampk2 , acc , and fatty acid synthase ( fas ) ( figure 3a ) . bmp8b within the vmh also increased ucp1 protein levels ( figure 3b ) and the mrna expression of thermogenic markers ( figure 3c ) in the brown fat . when administered in the lha , bmp8b did not affect either ampk signaling in the vmh ( figure 3d ) or ucp1 protein levels ( figure 3e ) or the mrna expression of thermogenic markers ( figure 3f ) in the bat . this evidence suggests that bmp8b s actions on the vmh are direct and not mediated by a previous effect on the lha . in relation to the white adipose tissue ( wat ) , our mrna data showed that browning markers had a tendency to be increased in the wat of rats receiving bmp8b in the vmh , but not in the lha ( figures 3 g and 3i ) . histological analysis of wat showed that bmp8b - treated rats in the vmh displayed a brown - like pattern , associated with increased ucp1 immunostaining and decreased adipocyte area ( figure 3h ) . no changes were found when bmp8b was given in the lha ( figure 3j ) . overall , these data indicated that the effects of bmp8b on browning are specific for the vmh . recent data have demonstrated a key role for ox on bat thermogenic activity ( tupone et al . , 2011 , sellayah et al . , 2011 , morrison et al . , 2012 , , 2012 , sellayah and sikder , 2012 , sellayah and sikder , 2014 ) . furthermore , it has been reported that central administration of bmp8b increased neuronal activity in the lha ( whittle et al . , 2012 ) , the main hypothalamic area expressing ox . thus , we investigated the possible effect of bmp8b on ox expression in the lha . 0.07c ; p < 0.001 ) and ucp1 protein levels in bat ( vehicle i.c.v . : 100% 14.6% versus bmp8b i.c.v . : 137.8% 10.1% ; p < 0.05 ) , in association with elevated ox mrna expression in the lha ( figure s3a ) . to gain further insight into bmp8b effects on ox expression our data showed that , when given in that nucleus , bmp8b also increased the mrna levels of ox in the lha ( figure 4a ) . these results suggested that bmp8b in the vmh acts upstream in the lha to promote ox expression . to elucidate the contribution of ampk activity in the vmh to the thermogenic effect of bmp8b , adenoviruses encoding constitutively active ampk ( ampk-ca ) together with gfp or control adenovirus expressing gfp alone ( lpez et al . , 2010b , whittle et al . , 2012 , martnez de morentin et al . , 2012 , martnez de morentin et al . , 2014 , infection efficiency was assessed by the expression of gfp in the vmh ( figure 4b ) . the administration of ampk-ca adenoviruses alone did not have any impact either in body temperature change ( vehicle gfp : 0.37c 0.14c versus vehicle ampk-ca : 0.3c 0.11c , non - significant ) or bat temperature ( vehicle gfp : 0.35c 0.21c versus vehicle ampk-ca : 0.29c 0.15c , non - significant ) . when given to bmp8b - treated rats , constitutive activation of ampk in the vmh reversed the thermogenic effect of bmp8b at both whole body ( figure 4c ) and bat levels ( figure 4d ) . this effect also was associated with a reduction in the expression of ox in the lha ( figure 4e ) . these results suggest that central bmp8b action on ox expression is mediated by the inhibition of ampk in the vmh . it has been reported recently that ox1r mediates ox actions on bat function ( sellayah and sikder , 2012 ) . thus , rats received the ox1r antagonist sb-334867 ( jia et al . , 2012 ) sb-334867 alone had no effect on body temperature at any examined time ( 2 hr vehicle vehicle : 0.19c 0.08c versus vehicle sb-334867 : 0.20 0.07 , non - significant ; 4 hr vehicle vehicle : 0.40c 0.14c versus vehicle sb-334867 : 0.39 0.17 , non - significant ; and 6 hr vehicle vehicle : 0.26c 0.13c versus vehicle sb-334867 : 0.23 0.19 , non - significant ) . however , sb-334867 significantly blunted the thermogenic effect of bmp8b at 4 and 6 hr after bmp8b treatment but only partially at 2 hr ( figure 4f ) , probably due to early effects independent of ox1r or a delayed effect of the antagonist . of note , sb-334867 did not have any impact on the bmp8b - induced decrease in ampk signaling in the vmh ( figure 4 g ) , suggesting that ampk in that nucleus operates upstream of ox1r signaling in this pathway . afterward , we examined the effect of bmp8b on ox - null mice and their wild - type ( wt ) littermates . when given centrally , bmp8b increased body temperature ( figure 5a ) and bat temperature ( figure 5b ) in wt , but not in ox knockout ( ko ) mice . notably , ampk signaling was decreased in the vmh of both wt ( figure 5c ) and ox ko mice ( figure 5d ) , suggesting again that ampk in the vmh is upstream of ox in the lha . we also investigated whether the effect of ox on bmp8b - induced thermogenesis was dependent on temperature . our data showed that null mice were cold intolerant when exposed to 4c for just 12 hr ( figure 6a ) . our data demonstrated that , while wt mice kept at 4c had a full thermogenic response to central bmp8b ( similarly to rats ; figures 1e and 1f ) , ox ko mice failed to respond ( figures 6b and 6c ) . those effects were associated with a similar effect in the vmh in both genotypes , i.e. , reduced pampk levels , after the i.c.v . however , accordingly with the temperature data , while wt mice displayed elevated ucp1 protein levels in bat ( figure 6f ) , ko mice did not ( figure 6 g ) . again , these data reinforced the ideas that the vmh - lha circuit is essential for the central actions of bmp8b on bat and that ox acts downstream of ampk in the vmh . finally , we aimed to investigate the cellular mechanism through which bmp8b mediates increased ox expression in the lha . it is known that glutamate and glutamate transporters , particularity glutamate vesicular transporter 2 ( vglut2 ) , play a main role in hypothalamic regulation of energy balance ( tong et al . , 2007 ) . therefore , we investigated whether virogenetic targeting of vglut2 within the lha , by using lentiviruses harboring a small hairpin rna ( shrna ) against vglut2 ( or their gfp controls ) , might reverse the effects of bmp8b on ox expression . infection efficiency was assessed by decreased vglut2 levels in that area ( figure 7a ) . our data showed that shvglut2 lentiviruses totally blunted central bmp8b effects on both core and bat temperatures ( figures 7b and 7c ) . those functional readouts were associated with a normalization in the bmp8b - induced increase in ox mrna levels in the lha ( figure 7d ) and ucp1 protein concentration in the bat ( figures 7e and 7f ) . of note , pampk protein levels were reduced in both the gfp- and the shvglut2-treated groups ( vehicle gfp : 100 18.6 versus bmp8b gfp : 42.6 12.3 , p < 0.05 ; and vehicle shvglut2 : 100 10.7 versus bmp8b shvglut2 : 62.9 12.5 , p < 0.05 ) , indicating that glutamatergic signaling was acting downstream of ampk . here , we report a functional link between ampk in the vmh and ox neurons in the lha . moreover , we show that the thermogenic effect of bmp8b is mediated by the inhibition of ampk in the vmh , the glutamatergic signaling in the lha , and the subsequent increase in ox signaling through ox1r . bat is a specialized tissue responsible for heat production through non - shivering thermogenesis ( nst ) ( cannon and nedergaard , 2004 , morrison et al . , 2014 , until recently , bat was considered to be important only in small or hibernating mammals and in newborn humans . recent studies have challenged that view by using positron emission tomography - computed tomography ( pet - ct ) scans to identify functional bat in adult humans ( nedergaard et al . 2009 ) , and brown fat is now considered as a potential target organ in the treatment of obesity ( villarroya and vidal - puig , 2013 , contreras et al . , 2015 ) bat is activated by increased firing of the sympathetic nerves , leading to the release of noradrenaline and activation on 3-adrenergic receptors ( cannon and nedergaard , 2004 , contreras et al . , 2015 , lpez et al . , 2016 ) . recent data have demonstrated that central bmp8b elicits bat thermogenesis in females through modulation of hypothalamic ampk ( whittle et al . , 2012 ) . of note , this mechanism is shared by e2 , which also displays a potent central thermogenic action ( martnez de morentin et al . , 2014 , martnez de morentin et al . , 2015 ) . here we show that male rats did not respond to central bmp8b administration , which might indicate dependence on ovarian steroids , a possibility that was further confirmed by using ovx rats with and without e2 replacement . this is of relevance because it indicates that bmp8b actions are sexually dimorphic . in this sense , it has been reported that bmp8b expression in bat is regulated by estrogens ( grefhorst et al . , 2015 ) . further work analyzing e2 effects in bmp8b ko mice ( whittle et al . , 2012 ) will help to clarify this issue . within the hypothalamus , the vmh was the first hypothalamic site to be identified as important in bat thermogenic action ( perkins et al . , 1981 ) . anatomical data also have demonstrated that vmh neurons project to autonomic centers in the hypothalamus and the hindbrain linked to the regulation of bat ( lindberg et al . , 2013 ) . recent evidence has revealed that particular sets of cells within the vmh , such as sf1 neurons ( kim et al . , 2011 , xu et al . , 2011 ) , and specific energy sensors , such as ampk ( lpez et al . , 2010b , lpez et al . , 2016 , martnez de morentin et al . , 2012 , 2014 ) , control the sympathetic firing on bat to regulate thermogenesis and browning of wat ( beiroa et al . , 2014 ) . activation of the lha promotes bat function ( cerri and morrison , 2005 ) , and compelling evidence has shown that this effect is mediated by ox acting on ox1r and involving direct projections from the lha to the raphe pallidus ( rpa ) , a key area modulating the sympathetic tone to brown fat ( yasuda et al . , 2005 , , 2011 , tupone et al . , 2011 , madden et al . , 2012 , sellayah and sikder , 2012 , sellayah and sikder , 2014 ) . in spite of this evidence , it was totally unclear whether ampk in the vmh and ox in the lha are part of the same functional link or they exert their thermogenic actions separately . anatomical data supported the hypothesis of an interconnected pathway , because it was known that vmh efferents project to the lha and provide passing innervation on their way to their target areas ( lindberg et al . , 2013 ) . to address that possibility , we investigated the central thermogenic effect of bmp8b , which is associated with ( 1 ) decreased ampk function in the vmh ; ( 2 ) increased activity in rpa , inferior olive ( io ) , and sympathetic tone ; and ( 3 ) neuronal activation within the lha ( whittle et al . , 2012 ) . our data showed that administration of bmp8b within the vmh recapitulated the effect of its central ( i.c.v . ) administration ( whittle et al . , 2012 ) , namely feeding - independent weight loss , increased ee and bat thermogenesis , and reduced rq , in association with reduced ampk signaling in the vmh and elevated bat ucp1 protein content as well as browning of wat . none of these outcomes was observed when bmp8b was specifically delivered into the lha , which excluded its direct effect at this level . interestingly , the thermogenic effect of both central and vmh - specific bmp8b administration correlated with increased ox expression in the lha . considering that ox also is known to stimulate thermogenesis through the rpa - sns route ( tupone et al . , 2011 ) , we investigated its potential link with the bmp8b - ampk pathway . we observed that both the thermogenic effect of bmp8b and the elevation of ox mrna expression were totally absent when bmp8b was given in combination with adenoviral particles encoding ampk-ca , indicating that ampk in the vmh was acting upstream of ox neurons in the lha . importantly , the above effects were reversed by pharmacological blockage of ox1r and were totally absent when bmp8b was centrally delivered in ox ko mice , even in a cold environment , which confirmed again ampk s upstream position in this network . to gain further insight into this central pathway it is known that glutamate transporters , particularity vglut2 , are highly expressed in the hypothalamus , including ox neurons ( ziegler et al . , 2002 , 2003 ) , and play a main role in the regulation of energy balance ( tong et al . , 2007 ) . therefore , we hypothesized a possible link with bmp8b effects on ox neurons in the lha . to check that possibility , we knocked down vglut2 in the lha using lentiviruses harboring an shrna . notably , vglut2 inhibition totally blunted bmp8b s effects on temperature , ox expression in the lha , and ucp1 expression in bat . however , it has been revealed recently , by using conditional viral tracing , that sf1 neurons , a major cell population inside the vmh , project and terminate in the lha ( lindberg et al . , 2013 ) , which may provide an anatomical basis for the interaction of ampk(vmh)-glutamate - ox(lha ) . first , they uncover a physiological hypothalamic network governing energy homeostasis , which might be of relevance for therapeutic approaches . in this sense , it is important to highlight that the original theories explaining the central control of energy balance were based on a dual centre hypothesis ( hecherington and ranson , 1942 , anand and brobeck , 1951 ) . in that model , feeding was controlled by two hypothalamic areas : the lha hunger center and the vmh satiety center . conversely , lesions of the vmh ( and the neighboring mediobasal area ) resulted in obesity . the present results do not show an impact on feeding , but this vmh ( ampk ) , glutamate and lha ( ox ) pathway provides the molecular basis for the interplay and balance between these two major areas in the dynamic control of other key aspect of the energy balance equation , namely ee . second , the sexual dependency of the thermogenic effect of bmp8b , and in particular the relevance of endogenous ovarian steroids in the modulation of bmp8b thermogenic actions , might be of clinical relevance , considering the different thermogenic adaptation observed in women and men ( palmer and clegg , 2015 , mauvais - jarvis , 2015 ) . third , oxs are highly pleiotropic peptides ( tsujino and sakurai , 2009 , lpez et al . , 2010a , gao and horvath , 2014 ) , one of their most important functions being the regulation of arousal and sleep - wake cycle ; actually , the lack of oxs or impaired oxr signaling leads to narcolepsy in several animal models ( such as mice , rats , and dogs ) and humans ( chemelli et al . , 1999 , lin et al . , 1999 , nishino et al . , 2000 , the fact that bmp8b increased ox expression opens up the possibility that it may have a potential role in the modulation of sleep patterns . we did not address that possibility specifically , but our data show that chronic treatment with bmp8b , specifically within the vmh , tended to increase la , which may be related to an increase in the arousal state of the animals . in summary , this study demonstrates that bmp8b modulates energy homeostasis and , more precisely , thermogenesis , via a well - defined hypothalamic pathway involving a vmh - lha interplay , and it uncovers a functional connection among ampk in the vmh , glutamatergic signaling , and ox in the lha . our results show that two important brain mediators of bat thermogenesis act in a coordinate network . whether this mechanism is exclusive for bmp8b or it mediates the effect of other thermogenic factors , such as leptin , thyroid hormones , estradiol , glucagon - like peptide-1 , or nicotine , is currently unknown . however , considering that ( 1 ) all these factors share the same vmh ampk - sns - bat axis ( lpez et al . , 2010b , lpez et al . , 2016 , martnez de morentin et al . , 2012 , martnez de morentin et al , 2012 , tanida et al . , 2013 , beiroa et al . , 2014 , contreras et al . , 2015 ) , ( 2 ) some of them also modulate the neuronal activity in rpa and io ( lpez et al . , 2010b , martnez de morentin et al . , 2014 ) , and ( 3 ) some regulate ox expression ( lpez et al . , 2000 , kane et al . , 2000 , porkka - heiskanen et al . , 2004 , pasumarthi et al . , 2006 ) , it is tempting to speculate that the ampk(vmh)-glutamate - ox(lha)-sns - bat axis may be a canonical mechanism to regulate bat metabolism , with pathophysiological implications in obesity and metabolic disorders . adult female and male sprague - dawley rats ( 200250 g , animalario general usc ) , adult female null ox / hcrt mice ( orexin / hypocretin ; b6.129s6-hcrttm1ywa / j , 810 weeks old , the jackson laboratory ) , and their wt littermates were used . the animals were housed with an artificial 12-hr light ( 8:00 a.m. to 8:00 p.m.)/12-hr dark cycle , under controlled temperature and humidity conditions , and they were allowed free access to standard laboratory chow and tap water . during all experimental approaches , the animals were monitored daily for food intake and body weight changes . the experiments were performed in agreement with the international law on animal experimentation and were approved by the usc ethical committee ( project i d 15010/14/006 ) . nucleus - specific treatments , and stereotaxic microinjection of adenoviral and lentiviral vectors were performed as described previously ( nogueiras et al . , 2010b , whittle et al . , 2012 , martnez de morentin et al . , 2012 , martnez de morentin et al . , 2014 , martnez de morentin et al . , 2015 , lvarez - crespo et al . , 2013 , , 2013 , contreras et al . , 2014 , beiroa et al . , 2014 , sample processing , ee , la , rq , and nuclear magnetic resonance ( nmr ) analyses ; temperature measurements ; in situ hybridization ; western blotting ; real - time pcr ; and immunohistochemistry were performed as described previously ( lpez et al . , 2008 , , 2010b , whittle et al . , 2012 , martnez de morentin et al . , 2012 , martnez de morentin et al . , 2014 , , 2012 , lvarez - crespo et al . , 2013 , contreras et al . , 2014 , the mrna and protein data were expressed in relation ( % ) to control ( vehicle - treated ) rats . statistical significance was determined by student s t test when two groups were compared or anova and post hoc two - tailed bonferroni test when more than two groups were compared ( p < 0.05 was considered significant ) . n.m .- s . performed the in vivo experiments ( hormonal , drug , and viral treatments ) ; the analytical methods ( in situ hybridization , western blotting , real - time qpcr , and hormone measurements ) ; and the analysis of temperature , ee , rq , la , and nmr , as well as analyzed and collected the data . m.l . developed the hypothesis , coordinated and directed the project , and wrote the manuscript . all authors reviewed and edited the manuscript and had final approval of the submitted manuscript .

summaryamp - activated protein kinase ( ampk ) in the ventromedial nucleus of the hypothalamus ( vmh ) and orexin ( ox ) in the lateral hypothalamic area ( lha ) modulate brown adipose tissue ( bat ) thermogenesis . however , whether these two molecular mechanisms act jointly or independently is unclear . here , we show that the thermogenic effect of bone morphogenetic protein 8b ( bmp8b ) is mediated by the inhibition of ampk in the vmh and the subsequent increase in ox signaling via the ox receptor 1 ( ox1r ) . accordingly , the thermogenic effect of bmp8b is totally absent in ox - null mice . bmp8b also induces browning of white adipose tissue ( wat ) , its thermogenic effect is sexually dimorphic ( only observed in females ) , and its impact on ox expression and thermogenesis is abolished by the knockdown of glutamate vesicular transporter 2 ( vglut2 ) , implicating glutamatergic signaling . overall , our data uncover a central network controlling energy homeostasis that may be of considerable relevance for obesity and metabolic disorders .

Introduction Results Discussion Experimental Procedures Author Contributions

hypothalamic neural circuits are involved in the control of a large number of functions , the regulation of endocrine axes and energy balance being of particular importance ( yeo and heisler , 2012 , schneeberger et al . , 2014 , substantial interest and efforts have been devoted to the analysis of the arcuate nucleus of the hypothalamus ( arc ) , which , in fact , has been considered as the master hypothalamic center for energy homeostasis ( yeo and heisler , 2012 , schneeberger et al . despite this evidence , other hypothalamic sites , such as the ventromedial nucleus of the hypothalamus ( vmh ) , formerly characterized as the brain satiety center , and the lateral hypothalamic area ( lha ) , formerly characterized as the brain hunger center ( hecherington and ranson , 1942 , anand and brobeck , 1951 ) , also play major and probably more integrative roles than the arc in the modulation of energy balance . , 2014 , 2016 ) to control not only feeding but also several peripheral processes , such as adipose and hepatic metabolism , as well as brown adipose tissue ( bat ) thermogenesis through the sympathetic ( sns ) and parasympathetic nervous system ( psns ) ( nogueiras et al . , 2013 , the molecular mediators within those areas have begun to be characterized , and over the last 5 years extensive attention has been focused on the role of amp - activated protein kinase ( ampk ) in the vmh and orexin ( ox ) in the lha as key modulators of brown fat . thus , inhibition of ampk function in the vmh ( lpez et al . , 2012 ) activates the bat thermogenic program , leading to elevated temperature , increased energy expenditure ( ee ) , and weight loss . in spite of this evidence , no functional data have linked those neuronal populations , and it is currently unknown whether ampk in the vmh and ox neurons in the lha are part of the same circuit modulating bat function or , alternatively , they function separately . given that bmp8b acts centrally , its thermogenic effect is dependent upon the activation status of ampk in the vmh , and it increases neuronal activation in the lha ( whittle et al . , 2012 ) , we aimed to uncover the neuronal circuitry linking energy sensors and neuropeptides involved in the thermogenic effect of central bmp8b . recent evidence has demonstrated that central bmp8b exerts a robust thermogenic effect in females ( whittle et al . injection of bmp8b , male rats did not show an increase in either body ( figure 1a ) or bat temperature ( figure 1b ) . ovx rats gained significantly more weight ( sham : 23.1 2.38 g versus ovx : 71.3 28 g ; p < 0.001 ) and developed a marked hyperphagia ( sham : 14.87 0.31 g / day versus ovx : 17.70 0.49 g / day ; p < 0.01 ) , confirming the efficiency of the ovx procedure . overall , these data indicated that the thermogenic action of bmp8b shows a sexual dimorphism and depends on physiological levels of ovarian estrogens . we aimed to investigate whether the thermogenic central actions of bmp8b were affected by temperature . our data showed that bmp8b protected against the loss of both core ( figure 1e ) and bat ( figure 1f ) temperature when given at 4c . to investigate the effect of chronic nucleus - specific bmp8b treatment , stereotaxic cannulae were placed in the vmh and the lha . noteworthy , the expression of bmp type i receptors alk4 , alk5 , and alk7 in those nuclei was demonstrated by real - time rt - pcr analysis ( figure s2a ) of dissected vmh and lha , whose specificity was controlled by analysis of the specific markers steroidogenic factor 1 ( sf1 ) and ox , respectively ( figure s2b ) . when given in the vmh , bmp8b promoted a feeding - independent weight loss ( figure 2a ; initial body weight vehicle vmh : 240.0 4.4 g versus bmp8b vmh : 237.5 4.0 g ; non - significant ) ; no effect was observed when bmp8b was injected in the lha ( figure 2b ; initial body weight vehicle lha : 233.0 3.1 g versus bmp8b lha : 235.1 1.8 g ; non - significant ) . the action of bmp8b in the vmh was associated with a trend ( p = 0.07 ) to reduce adiposity without changes in the lean mass ( figure 2c ) . no effect on body temperature moreover , bmp8b in the vmh , but not in the lha , increased ee ( figures 2 g and 2h ) and reduced the respiratory quotient ( rq ; figures 2i and 2j ) . no changes in locomotor activity ( la ) were found when bmp8b was administered either in the vmh or the lha ( figures 2k and 2l ) , although a non - significant trend ( p = 0.07 ) for increased la was detected in vmh bmp8b - treated rats . we found that vmh administration of bmp8b decreased the phosphorylated levels of ampk ( pampk ) and its downstream target acetyl - coa carboxylase alpha ( pacc ) ; no changes were found in total levels of ampk1 , ampk2 , acc , and fatty acid synthase ( fas ) ( figure 3a ) . bmp8b within the vmh also increased ucp1 protein levels ( figure 3b ) and the mrna expression of thermogenic markers ( figure 3c ) in the brown fat . when administered in the lha , bmp8b did not affect either ampk signaling in the vmh ( figure 3d ) or ucp1 protein levels ( figure 3e ) or the mrna expression of thermogenic markers ( figure 3f ) in the bat . this evidence suggests that bmp8b s actions on the vmh are direct and not mediated by a previous effect on the lha . in relation to the white adipose tissue ( wat ) , our mrna data showed that browning markers had a tendency to be increased in the wat of rats receiving bmp8b in the vmh , but not in the lha ( figures 3 g and 3i ) . histological analysis of wat showed that bmp8b - treated rats in the vmh displayed a brown - like pattern , associated with increased ucp1 immunostaining and decreased adipocyte area ( figure 3h ) . overall , these data indicated that the effects of bmp8b on browning are specific for the vmh . furthermore , it has been reported that central administration of bmp8b increased neuronal activity in the lha ( whittle et al . , 2012 ) , the main hypothalamic area expressing ox . thus , we investigated the possible effect of bmp8b on ox expression in the lha . : 137.8% 10.1% ; p < 0.05 ) , in association with elevated ox mrna expression in the lha ( figure s3a ) . to gain further insight into bmp8b effects on ox expression our data showed that , when given in that nucleus , bmp8b also increased the mrna levels of ox in the lha ( figure 4a ) . these results suggested that bmp8b in the vmh acts upstream in the lha to promote ox expression . to elucidate the contribution of ampk activity in the vmh to the thermogenic effect of bmp8b , adenoviruses encoding constitutively active ampk ( ampk-ca ) together with gfp or control adenovirus expressing gfp alone ( lpez et al . , 2014 , infection efficiency was assessed by the expression of gfp in the vmh ( figure 4b ) . when given to bmp8b - treated rats , constitutive activation of ampk in the vmh reversed the thermogenic effect of bmp8b at both whole body ( figure 4c ) and bat levels ( figure 4d ) . these results suggest that central bmp8b action on ox expression is mediated by the inhibition of ampk in the vmh . however , sb-334867 significantly blunted the thermogenic effect of bmp8b at 4 and 6 hr after bmp8b treatment but only partially at 2 hr ( figure 4f ) , probably due to early effects independent of ox1r or a delayed effect of the antagonist . of note , sb-334867 did not have any impact on the bmp8b - induced decrease in ampk signaling in the vmh ( figure 4 g ) , suggesting that ampk in that nucleus operates upstream of ox1r signaling in this pathway . afterward , we examined the effect of bmp8b on ox - null mice and their wild - type ( wt ) littermates . when given centrally , bmp8b increased body temperature ( figure 5a ) and bat temperature ( figure 5b ) in wt , but not in ox knockout ( ko ) mice . notably , ampk signaling was decreased in the vmh of both wt ( figure 5c ) and ox ko mice ( figure 5d ) , suggesting again that ampk in the vmh is upstream of ox in the lha . our data showed that null mice were cold intolerant when exposed to 4c for just 12 hr ( figure 6a ) . our data demonstrated that , while wt mice kept at 4c had a full thermogenic response to central bmp8b ( similarly to rats ; figures 1e and 1f ) , ox ko mice failed to respond ( figures 6b and 6c ) . those effects were associated with a similar effect in the vmh in both genotypes , i.e. however , accordingly with the temperature data , while wt mice displayed elevated ucp1 protein levels in bat ( figure 6f ) , ko mice did not ( figure 6 g ) . again , these data reinforced the ideas that the vmh - lha circuit is essential for the central actions of bmp8b on bat and that ox acts downstream of ampk in the vmh . finally , we aimed to investigate the cellular mechanism through which bmp8b mediates increased ox expression in the lha . it is known that glutamate and glutamate transporters , particularity glutamate vesicular transporter 2 ( vglut2 ) , play a main role in hypothalamic regulation of energy balance ( tong et al . therefore , we investigated whether virogenetic targeting of vglut2 within the lha , by using lentiviruses harboring a small hairpin rna ( shrna ) against vglut2 ( or their gfp controls ) , might reverse the effects of bmp8b on ox expression . those functional readouts were associated with a normalization in the bmp8b - induced increase in ox mrna levels in the lha ( figure 7d ) and ucp1 protein concentration in the bat ( figures 7e and 7f ) . of note , pampk protein levels were reduced in both the gfp- and the shvglut2-treated groups ( vehicle gfp : 100 18.6 versus bmp8b gfp : 42.6 12.3 , p < 0.05 ; and vehicle shvglut2 : 100 10.7 versus bmp8b shvglut2 : 62.9 12.5 , p < 0.05 ) , indicating that glutamatergic signaling was acting downstream of ampk . here , we report a functional link between ampk in the vmh and ox neurons in the lha . moreover , we show that the thermogenic effect of bmp8b is mediated by the inhibition of ampk in the vmh , the glutamatergic signaling in the lha , and the subsequent increase in ox signaling through ox1r . 2009 ) , and brown fat is now considered as a potential target organ in the treatment of obesity ( villarroya and vidal - puig , 2013 , contreras et al . here we show that male rats did not respond to central bmp8b administration , which might indicate dependence on ovarian steroids , a possibility that was further confirmed by using ovx rats with and without e2 replacement . within the hypothalamus , the vmh was the first hypothalamic site to be identified as important in bat thermogenic action ( perkins et al . anatomical data also have demonstrated that vmh neurons project to autonomic centers in the hypothalamus and the hindbrain linked to the regulation of bat ( lindberg et al . , 2011 ) , and specific energy sensors , such as ampk ( lpez et al . , 2012 , 2014 ) , control the sympathetic firing on bat to regulate thermogenesis and browning of wat ( beiroa et al . activation of the lha promotes bat function ( cerri and morrison , 2005 ) , and compelling evidence has shown that this effect is mediated by ox acting on ox1r and involving direct projections from the lha to the raphe pallidus ( rpa ) , a key area modulating the sympathetic tone to brown fat ( yasuda et al . in spite of this evidence , it was totally unclear whether ampk in the vmh and ox in the lha are part of the same functional link or they exert their thermogenic actions separately . to address that possibility , we investigated the central thermogenic effect of bmp8b , which is associated with ( 1 ) decreased ampk function in the vmh ; ( 2 ) increased activity in rpa , inferior olive ( io ) , and sympathetic tone ; and ( 3 ) neuronal activation within the lha ( whittle et al . our data showed that administration of bmp8b within the vmh recapitulated the effect of its central ( i.c.v . ) , 2012 ) , namely feeding - independent weight loss , increased ee and bat thermogenesis , and reduced rq , in association with reduced ampk signaling in the vmh and elevated bat ucp1 protein content as well as browning of wat . interestingly , the thermogenic effect of both central and vmh - specific bmp8b administration correlated with increased ox expression in the lha . , 2011 ) , we investigated its potential link with the bmp8b - ampk pathway . we observed that both the thermogenic effect of bmp8b and the elevation of ox mrna expression were totally absent when bmp8b was given in combination with adenoviral particles encoding ampk-ca , indicating that ampk in the vmh was acting upstream of ox neurons in the lha . importantly , the above effects were reversed by pharmacological blockage of ox1r and were totally absent when bmp8b was centrally delivered in ox ko mice , even in a cold environment , which confirmed again ampk s upstream position in this network . to gain further insight into this central pathway it is known that glutamate transporters , particularity vglut2 , are highly expressed in the hypothalamus , including ox neurons ( ziegler et al . , 2002 , 2003 ) , and play a main role in the regulation of energy balance ( tong et al . therefore , we hypothesized a possible link with bmp8b effects on ox neurons in the lha . to check that possibility , we knocked down vglut2 in the lha using lentiviruses harboring an shrna . notably , vglut2 inhibition totally blunted bmp8b s effects on temperature , ox expression in the lha , and ucp1 expression in bat . however , it has been revealed recently , by using conditional viral tracing , that sf1 neurons , a major cell population inside the vmh , project and terminate in the lha ( lindberg et al . first , they uncover a physiological hypothalamic network governing energy homeostasis , which might be of relevance for therapeutic approaches . in that model , feeding was controlled by two hypothalamic areas : the lha hunger center and the vmh satiety center . conversely , lesions of the vmh ( and the neighboring mediobasal area ) resulted in obesity . the present results do not show an impact on feeding , but this vmh ( ampk ) , glutamate and lha ( ox ) pathway provides the molecular basis for the interplay and balance between these two major areas in the dynamic control of other key aspect of the energy balance equation , namely ee . second , the sexual dependency of the thermogenic effect of bmp8b , and in particular the relevance of endogenous ovarian steroids in the modulation of bmp8b thermogenic actions , might be of clinical relevance , considering the different thermogenic adaptation observed in women and men ( palmer and clegg , 2015 , mauvais - jarvis , 2015 ) . , 2010a , gao and horvath , 2014 ) , one of their most important functions being the regulation of arousal and sleep - wake cycle ; actually , the lack of oxs or impaired oxr signaling leads to narcolepsy in several animal models ( such as mice , rats , and dogs ) and humans ( chemelli et al . , 2000 , the fact that bmp8b increased ox expression opens up the possibility that it may have a potential role in the modulation of sleep patterns . we did not address that possibility specifically , but our data show that chronic treatment with bmp8b , specifically within the vmh , tended to increase la , which may be related to an increase in the arousal state of the animals . in summary , this study demonstrates that bmp8b modulates energy homeostasis and , more precisely , thermogenesis , via a well - defined hypothalamic pathway involving a vmh - lha interplay , and it uncovers a functional connection among ampk in the vmh , glutamatergic signaling , and ox in the lha . , 2014 ) , and ( 3 ) some regulate ox expression ( lpez et al . , 2006 ) , it is tempting to speculate that the ampk(vmh)-glutamate - ox(lha)-sns - bat axis may be a canonical mechanism to regulate bat metabolism , with pathophysiological implications in obesity and metabolic disorders . adult female and male sprague - dawley rats ( 200250 g , animalario general usc ) , adult female null ox / hcrt mice ( orexin / hypocretin ; b6.129s6-hcrttm1ywa / j , 810 weeks old , the jackson laboratory ) , and their wt littermates were used . performed the in vivo experiments ( hormonal , drug , and viral treatments ) ; the analytical methods ( in situ hybridization , western blotting , real - time qpcr , and hormone measurements ) ; and the analysis of temperature , ee , rq , la , and nmr , as well as analyzed and collected the data .

bacteria from genus borrelia are mico - aerophilic and slow - growing pathogens known for their persistency 1 - 7 . out of 36 currently recognized species , 13 of them are either acknowledged or suspected to cause lyme disease ( ld ) which is the most common tick - born infection in europe and north america 8 - 11 . they are known as borrelia burgdorferi sensu lato and include species such as borrelia burgdroferi sensu stricto ( predominantly causing this illness in north america ) as well as borrelia afzelii and borrelia garinii ( predominantly causing this illness in europe ) 12 . are spirochetes that are motile and can survive viscous conditions in human and animal blood , and as well they are capable of entering their cells 13 - 16 . when hostile conditions are introduced these bacteria can adopt different latent forms such as rounded bodies ( cysts , granular forms , l - forms ) and aggregates ( biofilm - like structures ) 17 - 19 . especially the ability of borrelia burgdorferi sensu lato to convert and re - convert to cystic form was observed , which may be one of the reasons why this infection can sometimes become persistent and/or re - surface after being silent for a long time . . moreover , genomic analysis revealed that borrelia burgdorferi sensu lato has a gene for efflux mechanism . this might be responsible for developing antibiotic resistance , although more in depth studies are warranted to prove such hypothesis 22 . these aspects all highlight the need for either new or improved treatments against borrelia sp . 21 . based on diagnostic test results , the most recent estimates , indicate that the number of ld cases in just the united states reaches 300,000 each year ; however , there might be unreported cases that are not reflected in the statistics 10 , 19 , 23 , 24 . this has made this disease the most common vector - born disease in the northern hemisphere 10 - 11 , 19 . beta lactams are the most frequently applied and include doxycycline , amoxicillin , and cefuroxime axetil . macrolides , such as azithromycin , clarithromycin , and erythromycin , are the second class and appear to be less effective than beta lactams 25 . currently one of the most often prescribed antibiotics against borrelia burgdorferi sensu lato infection is doxycycline 25 - 29 . its use began in the late sixties last century against infections other than ld 30 , 31 . since then , the application of doxycycline extended and has been on the world health organization 's list of essential medicines as one of the most important medications needed in a basic health system 31 , 32 . doxycycline is a broad - spectrum antibiotic belonging to the class of tetracyclines . like other agents in this class , it is an anti - bacterial and anti - parasite agent targeting protein production in general 31 - 33 . its side effects are similar to those of other members of this antibiotic class , including the development of an erythematous rash after exposure to sun . moreover , doxycycline is classified as a class d drug and is thus restricted for administration to pregnant women and children under the age of eight 34 . although , doxycycline is one of the most frequently prescribed antibiotics , especially for the early stages of ld , its continued treatment is not recommended since the long - term effectiveness has not been proven 4 , 35 . in vitro studies revealed that doxycycline is very effective against active ( vegetative ) form of borrelia sp . , with moderate action against their biofilms , and ineffective against the latent rounded forms 2 , 36 . observed persistency and/or relapse of ld symptoms in the absence of ongoing antibiotic treatment was observed as well 19 , 21 , 37 . this would suggest that doxycycline may either facilitate generation of latent forms or is inefficient in their elimination 5 , 17 , 38 - 40 . while , it was shown that doxycycline has no useful activity against latent rounded forms of borrelia sp . , its general activity could potentially be improved if applied in combination with other antimicrobial agents that express similar or complementary action . antimicrobials derived from natural sources such as plants , herbs , and fruits , and essential oils , have shown activity against a plethora of bacteria and fungi , but are poorly explored against borrelia sp . 41 - 43 . despite a rather small pool of available data on this subject , bronson and bronson study exhibited grapefruit seed extract as a powerful in vitro agent against spirochetes and their cystic forms of borrelia afzelii 44 . dipsacus sylvestris extract against borrelia burgdorferi sensu stricto revealed growth inhibiting activity as well 45 , whereas sapi , et al . , reported significant efficacy of leaf extracts from stevia rebaudiana on all forms of borrelia burgdorferi sensu stricto 46 . our recent study documented that other naturally occurring compounds like baicalein and luteolin , belonging to the flavones , are also potent against borrelia sp . in addition , fatty acids , including monolaurin and cis-2-decenoic acid , and iodine exhibited significant bacteriostatic and bactericidal activities 47 . our and other studies indicate that naturally derived compounds and antibiotics that display essential anti - borreliae activities might be an option for achieving optimal bacteriostatic and bactericidal activity at lower mics and mbcs , which could potentially reduce their side effects and treatment costs . in this study , we examined reciprocal cooperation of selected phytochemicals and micronutrients with documented anti - borrelae activities , with commercial antibiotic doxycycline against two borrelia species . to our knowledge , this is the first scientific report about interaction between doxycycline and phytochemicals and micronutrients against active and latent forms of borrelia sp . , vitamin c , cis-2-decenoic acid ( 10-had ) and doxycycline , with the purity between 90%-98% according to the manufacturer , were obtained from sigma ( st . louis , mo ) . luteolin and rosmarinic acid , with the purity between 97%-99% according to the manufacturer , were purchased from tocris bioscience ( bristol , united kingdom ) . kelp with standardized iodine content ( i.e. , 150 g / ml as 100 % of daily value ) was from world organic ltd . monolaurin ( lauricidin ) , as a pure sn-1 monolaurin ( glycerol monolaurate ) derived from coconut oil , was from med - chem laboratories , inc . , ( goodyear , az ) ( 50 - 100 mg / ml ) of each compound ( depending on solubility of the substance ) was prepared by suspending individual test compounds in absolute ethanol and sterilized by 0.22 m syringe filtration . all stock solutions were stored in aluminum foil - wrapped tubes at - 20c . due to bactericidal effect of a high percentage of ethanol , its added amount to the growth medium a preliminary experiment determined that ethanol content should not exceed 0.5% ( v / v ) ( data not shown ) . the appropriate amount of each stock solution was then added to 1.8 ml sterile screw - cap test tubes containing 1 ml of bsk - h complete medium to yield final concentrations of 5 - 500 g / ml for all compounds . as a negative control , ethanol at 0.1 - 0.4% ( v / v ) two borrelia species such as borrelia burgdorferi sensu stricto and borrelia garinii were tested in their three morphological forms : spirochetes , rounded forms , and biofilm . low passage isolates of the b31 strain of borrelia burgdorferi and cip103362 strain of borrelia garinii were obtained from the american type culture collection ( manassas , va ) . b31 strain is an isolate from ixodes dammini whereas the cip103362 strain is an isolate from ixodes ricinus . the stocks of both species were cultured in commonly used conditions , i.e. , barbour - stoner - kelly h ( bsk - h ) medium supplemented with 6% rabbit serum ( sigma , st . louis , mo ) without antibiotics at 33c in 5% co2 , in sterile screw - cap 15 ml polypropylene test tubes with or without gentle shaking . , the strains were activated from original cryobank vials and inoculated into 10 ml bsk - h complete medium , and maintained at 33c in 5% co2 . generation of homogeneous cultures ( i.e. , having only spirochete form ) of tested borrelia sp . were obtained by maintaining the inoculums in a shaking incubator at 33c and 250 rpm where there is no biofilm formation , respectively 2 . generation of biofilm - like colonies of tested borrelia sp . was attained by incubation of inoculums in 4-well chambers ( bd biosciences , sparks , md ) coated with collagen type i from rat tail for up to one week without shaking at 33c in 5% co2 , respectively . interaction of doxycycline with studied phytochemicals was assessed against spirochetes and rounded forms in 1.8 ml sterile screw - capped test tubes as well as against biofilm in 4-well chambers coated with collagen i from rat tail , according to checkerboard format . samples were inoculated with inoculums of test organisms , respectively , and set up at the appropriate times with increasing concentrations of doxycycline ( dox ) ( 0 to 500 g / ml ) and the active phytochemical ( ph ) ( 0 to 500 g / ml ) or micronutrient ( 0 to 88 g / ml ) , according to checkerboard assay . these ranges were selected based on our earlier studies and other research groups ( applied as a single dose , not in combination with a partner agent ) 2 , 47 . all tubes and chambers with mature biofilm fractional inhibitory concentration indexes ( ficis ) , fractional bactericidal concentration indexes ( fbcis ) , and fractional eradication ( of biofilm ) concentration indexes ( fecis ) were calculated and interpreted as follows : synergy = fici / fbci / feci of 0.5 ; antagonism = fici / fbci / feci > 4.0 ; additive = 0.5 < fici / fbci / feci < 1.0 , and indifferent ( no interaction ) = 1.0 < fici / fbci / feci < 4.0 48 - 50 . discrepant results with fici / fbci / feci 1 were confirmed by performing an additional duplicate test . evaluation of reciprocal activities of tested combinations of agents against spirochetes of borrelia sp . reciprocity of test compounds against spirochetes of studied borrelia sp . was performed using a macro - dilution method according to checkerboard format . briefly , 1.8 ml sterile screw - capped test tubes containing 1 ml bsk - h complete medium , supplemented with the tested combinations of agents were inoculated with 2 x 10 spirochetes / ml of the homogenous bacterial suspension . the tubes were then incubated at 33c in 5% co2 and growth and viability was monitored at regular intervals for up to 72 h using a bacterial petroff - hausser counting chamber with dark field microscopy and/or live / dead baclight bacterial viability staining with fluorescent microscopy as a standard procedure . control cultures were treated with ethanol ( i.e. , 0.1 - 0.4 v / v ) alone . ficis were calculated as follows : ficdox = micdox+ph / micdox + ficph = micph+dox / micph . fbcdox = mbc90dox+ph / mbc90dox + fbcph = mbc90ph+dox / mbc90ph , as standard calculations 50 - 52 . evaluation of reciprocal activities of tested combinations of agents against rounded forms of borrelia sp . efficacy of test compound combinations against rounded forms was examined using a live / dead baclight bacterial viability staining method , where green fluorescence determines the presence of live rounded forms and red fluorescence dead rounded forms , using a macro - dilution method according to checkerboard format . briefly , 2 x 10 spirochetes / ml of the homogenous bacterial suspension was inoculated into each 1.8 ml sterile screw - capped test tubes containing 1 ml bsk - h complete medium , supplemented with the tested combinations of agents . the tubes were then incubated at 33c in 5% co2 and viability was monitored at regular intervals for up to 72 h using fluorescent microscopy ( nikon , eclipse e600 ) as standard procedure . control cultures were treated with ethanol ( 0.1 - 0.4 v / v ) alone . fbcis were calculated as follows : fbcdox = mbc50dox+ph / mbc50dox + fbcph = mbc50ph+dox / mbc50ph , where mbc50 is a minimal concentration causing 50% of killing . evaluation of reciprocal activities of tested combinations of agents against biofilms of borrelia sp . efficacy of test compound combinations against biofilm of tested borrelia sp . was evaluated using the qualitative method based on crystal violet staining 2 . briefly , 1 x 10 spirochetes / ml of the homogeneous culture were inoculated into four - well chambers coated with collagen type i and incubated at 33c in 5% co2 for up to one week . earlier studies in our laboratory have documented a lack of antifungal carryover using this procedure 47 . once the biofilm was established , all chambers were supplemented with the tested combinations of agents and incubated at 33c in 5% co2 for up to 72 h. control wells were treated with ethanol ( 0.1 - 0.4 v / v ) alone . all wells were fixed with 500 l of cold methanol - formalin ( 1:1 ) for 30 minutes and stained with 1 ml of crystal violet ( 0.1% ) for 10 minutes . the biofilms were then washed carefully three times with 1 x pbs ( phosphate - buffered saline ) , and 1 ml of methanol was added to each well to extract a dye which was measured at 595 nm using a spectrophotometer ( molecular device , spectra max 340 ) . reciprocal interaction was evaluated according to checkerboard format and the percentage of biofilm eradication ( be% ) was calculated as be% = [ 1-(od959 of cells treated with doxycycline and phytochemical ( micronutrient)/od959 of untreated control ] x 100% . also , fractional eradication concentration indexes fecis ( adapted from the fici / fbci equation reported by elion , et al . ) were calculated as follows : fecdox = ec50dox+ph / ec50dox + fecph = ec50ph+dox / ec50ph , where ec50 is an effective concentration causing at least 50% of biofilm eradication 53 . the experiment was repeated three times for each species and each compounds combination . for quantitative analysis , all chambers were fixed with 500 l of cold formalin - acetic acid mixture for 20 min . followed by staining with 200 l of baclight staining mixture for 15 min . in the dark , according to the manufacturer 's recommendation . pictures were immediately taken from untreated ( control ) and treated mounted slides using a fluorescence microscope ( nikon , eclipse e600 ) . the anova and/or student 's two - tailed t test was used to determine statistically significant differences set at 0.05 levels . reciprocal cooperation between doxycycline and tested phytochemicals against spirochetes of borrelia sp . the examined combinations of doxycycline with tested phytochemicals and micronutrients against spirochetes of test microorganisms with the mics , fics and ficis values are presented in table 1a . screening of tested combinations indicates additive effects ( 0.5 < fici < 1.0 ) of doxycycline with 6 partner agents such as baicalein , luteolin , iodine , rosmarinic acid , vitamin d3 , and vitamin c. indifference ( no interaction ) was seen between doxycycline and fatty acids like monolaurin and 10-had . adding phytochemicals and micronutrients allowed for lowering of doxycycline mic value from 25 g / ml to 12.5 g / ml ( valid for both tested borrelia sp . ) . adding phytochemicals and micronutrients allowed for lowering of doxycycline mbc90 value from 200 g / ml to 100 g / ml ( for b. burgdorferi ) and from 250 g / ml to 125 g / ml ( for b. garinii ) . the presence of sub - inhibitory concentration of doxycycline ( 12.5 g / ml ) reduced the mics of the partner phytochemical agents that fluctuated between 62.5 g / ml and 150 g / ml and the partner micronutrient agents between 0.00025 g / ml and 44 g / ml . fics and ficis for all tested agent combinations corresponded to each other for both tested borrelia sp . the presence of sub - bactericidal concentration of doxycycline ( 100 g / ml for b. burgdorferi ; 125 g / ml b. garinii ) reduced the mbc90 values of the phytochemical agents that ranged between 125 g / ml and 225 g / ml and the partner micronutrient agents between 0.0005 g / ml and 44 g / ml . fbcs and fbcis for all tested agent combinations corresponded to each other for both tested borrelia sp . reciprocal cooperation between doxycycline and tested phytochemicals against rounded forms of borrelia sp . the effects of doxycycline in combination with tested phytochemicals and micronutrients against rounded forms of both studied borrelia sp . are presented in table 2 , with their mbc50s , fbcs and fbcis . our previous results indicated that among several tested compounds , a few ( baicalein , luteolin , monolaurin , 10-had , and iodine ) showed anti - rounded forms efficacy that satisfied the mbc50 requirement . the other agents ( rosmarinic acid , vitamin d3 , vitamin c , and doxycycline ) did not affect rounded forms to this extent 47 . evaluation of all the phytochemicals and micronutrients in combination with doxycycline did not indicate synergy or antagonistic interaction in any of the cases . however , we noticed an additive effect which was restricted to combinations of doxycycline with baicalein or its close related agent luteolin , in both studied borrelia sp . their fbcs and fbcis corresponded to each other for both tested species of borrelia . combinations of doxycycline with flavones were able to induce the death of latent rounded forms up to 50% at lower concentrations of each partner agent . as such , effective concentrations of baicalein decreased from 350 g / ml to 275 g / ml , luteolin decreased from 200 g / ml to 150 g / ml , and doxycycline decreased from 500 g / ml to 100 g / ml . indifference ( no interaction ) was seen between doxycycline and fatty acids such as monolaurin and 10-had as well as iodine . mbc50 mark was achieved at their values identical to their single dose such as 300 g / ml for monolaurin , 500 g / ml for 10-had and 20 g / ml for iodine . however , at the same time , their addition allowed for reducing effective doxycycline concentration from 500 g / ml to 100 g / ml . the combinations with the rest of the tested agents did not achieve mbc50 mark , which did not allow performing correct calculations . none of the added tested agents alone or in combinations could eliminate rounded forms in 90 - 99% , even at their maximal tested concentrations . its efficacy was shown to be ~ 5 - 10% at 500 g / ml 2 , 47 , 54 . kinetic evaluation of bactericidal effect against spirochetes and rounded forms of borrelia sp . kinetic evaluation of bactericidal effect of baicalein and luteolin was conducted as these compounds were able to eliminate both spirochete and rounded forms of tested borrelia sp . the results of this experiment show that doxycycline in combination with these phytochemicals , used at concentrations fulfilling the 0.5 < fbci < 1.0 requirement , could significantly eliminate spirochetes of borrelia burgdorferi and borrelia garinii in a dose- and time - dependent manner , reaching mic50 mark after 24 h and mic90 mark after 72 h ( figure 1a and b ) . similarly , dose - and time - dependent killing effect on their rounded forms was noticed with mbc50 mark reached after 72 h ( figure 2a - c ) at concentrations fulfilling the 0.5 < fbci < 1.0 requirement . reciprocal cooperation between doxycycline and tested phytochemicals against biofilm of borrelia sp.the combinations of doxycycline with test phytochemicals and micronutrients against biofilm formed by test microorganisms with the ec50s , fecs , and fecis are presented in table 3 . in our previous study we observed that several of these compounds had anti - biofilm effects that satisfied the ec50 requirement . these were baicalein , luteolin , monolaurin , 10-had , and iodine for b. burgdorferi , as well as baicalein and monolaurin for b. garinii . the other tested agents such as rosmarinic acid , vitamin d3 , and vitamin c did not display such effect 47 . the additive anti - biofilm effect was achieved when doxycycline was co - administrated with baicalein , luteolin , and iodine , respectively ( valid for both tested borrelia sp . ) , significantly reducing established biofilms of test microorganisms by ~ 50 - 60% , and satisfying the ec50 requirement ( figure 3a and b ) . combinations of doxycycline with flavones were able to enhance eradication of biofilm up to 50% at their lower effective concentrations which decreased from 750 g / ml to 500 g / ml for baicalein , and from 350 g / ml to 175 g / ml for luteolin , and from 40 g / ml to 20 g / ml for iodine . at the same time doxycycline concentration could also be decreased from 250 g / ml to 125 g / ml . their fecs and fecis corresponded to each other for both tested species of borrelia . indifference ( no interaction ) was seen for doxycycline combined with fatty acids such as monolaurin and 10-had . ec50 mark was achieved at their values identical to these of their single dose such as 375 g / ml for monolaurin , 800 g / ml for 10-had , and at the same time allowing for reducing doxycycline concentration from 250 g / ml to 125 g / ml . the combinations with the rest of the tested agents did not achieve ec50 mark , which did not allow performing correct calculations . none of the agents tested alone or in combinations could eradicate biofilms of both studied borrelia sp . in 90 - 99% , even when their maximal tested this is a very rigorous requirement for any antibiotic , including doxycycline , for which anti - biofilm efficacy was shown to be ~ 30 - 40% at 250 the emergence of the relapse of symptoms of this disease has been noticed 7 , 19 , 37 , 55 . the challenge in treating these bacteria has been complicated by several pathophysiological factors , including their ability to remain latent for decades 5 , 56 - 58 . the challenge is further exacerbated by findings indicating that borrelia biofilm may harbor a heterogeneous population of spirochetes and rounded forms with distinct genetic and protein profiles 59 - 61 . in addition , the role of an antibiotic efflux gene needs to be explored 22 . improved prospects in targeting borrelia sp . may result from applications of agents that cooperate in eliminating spirochetes as well as their dormant forms . therefore , it is important to investigate the interaction of such agents with the most common clinically used antibiotics against ld , like doxycycline . our previous study indicated that phytochemicals may provide a new reservoir of active antimicrobial agents , and serve as a resource for the development of novel anti - borreliae approaches 47 . that study showed that the most promising compounds effective against spirochete and/or latent forms of borrelia sp . were flavones such as baicalein and luteolin , fatty acids including monolaurin and 10-had , and iodine . even 10 - 20 times higher than its mic value did not show efficacy towards rounded forms 2 , 47 . that might lead to a subsequent loss of doxycycline efficacy in vitro and in vivo when applied long - term . taking all these aspects into consideration , we were interested in evaluating the reciprocal interactions of these phytochemicals and micronutrients with doxycycline against active and latent forms of borrelia sp . in the present study , we tested eight combinations of phytochemicals with doxycycline against all morphological forms of borrelia sp . additive effects were confined to a small number of combinations against latent forms ( doxycycline with baicalein or luteolin ) , while against spirochetes these activities were noticeable with more combinations , such as doxycycline with baicalein , luteolin , iodine , rosmarinic acid , and vitamin d3 or vitamin c. indifference was demonstrated for a combination of doxycycline with fatty acids such as monolaurin and 10-had . time - dependent bactericidal effects against spirochetes and latent rounded forms of borrelia burgdorferi and borrelia garinii were observed when doxycycline was combined with the flavones baicalein and luteolin , respectively , at concentrations fulfilling the 0.5 < fbci < 1.0 requirement . interestingly , the addition of iodine did not improve doxycycline 's efficacy towards rounded forms , but demonstrated additive effects against spirochete and biofilm forms of both tested borrelia sp . whether similar in vitro effects there are only a few published reports about reciprocal interactions between doxycycline and other antibiotics , and none of them used borrelia sp . as a subject matter . as such , bruhn , et al . this observation prompted them to examine whether spectinamides sensitize mycobacterium tuberculosis to antibiotics not traditionally used in the treatment of tuberculosis . further in vivo study of the efficacy of these combinations documented additional bacterial killing effect in a mouse model of acute tuberculosis infection , but not in a chronic infection model 62 . in the study of gonzalo , et al . , amikacin in combination with doxycycline tested against clinical strains of mycobacterium tuberculosis showed a synergistic effect in 18 of the 29 strains , and indifference in 11 strains 63 . , suggest that addition of amikacin does not improve the results obtained by imipenem monotherapy , but adding doxycycline to amikacin is an alternative to imipenem in the therapy of acinetobacter baumannii pneumonia 64 . even fewer studies have investigated the interactions of doxycycline with phytochemicals and none with micronutrients . among these , research by valcourt , et al . , showed synergy between doxycycline and essential oils ( eos ) such as carvacrol ( oregano oil ) , eugenol ( clove oil ) and cinnamaldehyde ( cinnamon oil ) against gram - negative strains of acinetobacter baumannii , klebsiella pneumoniae , escherichia coli , and pseudomonas aeruginosa . no growth inhibiting interactions were found between eos and doxycycline , however , synergistic bactericidal effects were observed , supported by the results visualizing the holes - type destructive changes in the bacterial membrane with the leakage of cellular contents 65 . there is one report demonstrating synergy of baicalein with tetracycline and beta - lactams against methicillin - resistant staphylococcus aureus 66 . in addition , synergistic effect was demonstrated when luteolin was co - administrated with ceftazidime against streptococcus pyogenes that concluded that luteolin has high potential to be developed as an adjunct to ceftazidime for the treatment of beta - lactamase - producing bacteria and streptococcus pyogenes infections 67 . moreover , isenberg , et al . , found that half - strength ( 5% ) povidone - iodine solution significantly reduced bacterial flora of the conjunctiva . later , they compared this concentration of povidone - iodine on the conjunctiva with a prophylactic antibiotic ( neosporin ophthalmic solution ) in 35 patients undergoing ocular surgery . the results showed that the antibiotic and povidone - iodine solutions , used individually , caused a similar and substantial decrease in the number of colonies ( biofilm ) and species of bacteria cultured . however , when both drugs were used together the decrease was even more striking , making 83% of the conjunctivae sterile . thus , their recommendation was to use a broad - spectrum topical antibiotic with the half - strength povidone - iodine solution as a part of the preoperative preparation 68 . synergistic or additive interactions are not universal which makes it difficult to compare and generalize the results . in addition , the use of different testing methods further complicates interpretation of the results . we find it promising that our results obtained with borrelia sp . corroborate with the findings of other research groups . it is important to note that while bactericidal concentrations of doxycycline may induce persistent phenotypic changes , combining it with selected phytochemicals or micronutrients may not only allow for reducing its concentration to more safe values , but also expand the spectrum of bacterial forms affected and perhaps offer additional benefits such as anti - inflammatory properties . in summary , this study documents in vitro efficacy of doxycycline co - administrated with several phytochemicals and micronutrients against active and latent persistent forms of borrelia burgdorferi sensu stricto and borrelia garinii . these findings indicate an additive effect of doxycycline co - administrated with flavones like baicalein or luteolin against all morphological forms of these species in vitro . therefore , combinations of these phytochemicals with an antibiotic present a promising alternative towards combating borrelia sp . the study provides additional support for phytochemicals as valuable adjuvants in antimicrobial chemotherapy , although further in vivo and human studies are warranted to draw a final conclusion .

phytochemicals and micronutrients represent a growing theme in antimicrobial defense ; however , little is known about their anti - borreliae effects of reciprocal cooperation with antibiotics . a better understanding of this aspect could advance our knowledge and help improve the efficacy of current approaches towards borrelia sp . in this study , phytochemicals and micronutrients such as baicalein , luteolin , 10-had , iodine , rosmarinic acid , and monolaurin , as well as , vitamins d3 and c were tested in a combinations with doxycycline for their in vitro effectiveness against vegetative ( spirochetes ) and latent ( rounded bodies , biofilm ) forms of borrelia burgdorferi and borrelia garinii . anti - borreliae effects were evaluated according to checkerboard assays and supported by statistical analysis . the results showed that combination of doxycycline with flavones such as baicalein and luteolin exhibited additive effects against all morphological forms of studied borrelia sp . doxycycline combined with iodine demonstrated additive effects against spirochetes and biofilm , whereas with fatty acids such as monolaurin and 10-had it produced ficis of indifference . additive anti - spirochetal effects were also observed when doxycycline was used with rosmarinic acid and both vitamins d3 and c. antagonism was not observed in any of the cases . this data revealed the intrinsic anti - borreliae activity of doxycycline with tested phytochemicals and micronutrients indicating that their addition may enhance efficacy of this antibiotic in combating borrelia sp . especially the addition of flavones balcalein and luteolin to a doxycycline regimen could be explored further in defining more effective treatments against these bacteria .

Introduction Materials and Methods Results Discussion

they are known as borrelia burgdorferi sensu lato and include species such as borrelia burgdroferi sensu stricto ( predominantly causing this illness in north america ) as well as borrelia afzelii and borrelia garinii ( predominantly causing this illness in europe ) 12 . when hostile conditions are introduced these bacteria can adopt different latent forms such as rounded bodies ( cysts , granular forms , l - forms ) and aggregates ( biofilm - like structures ) 17 - 19 . especially the ability of borrelia burgdorferi sensu lato to convert and re - convert to cystic form was observed , which may be one of the reasons why this infection can sometimes become persistent and/or re - surface after being silent for a long time . these aspects all highlight the need for either new or improved treatments against borrelia sp . based on diagnostic test results , the most recent estimates , indicate that the number of ld cases in just the united states reaches 300,000 each year ; however , there might be unreported cases that are not reflected in the statistics 10 , 19 , 23 , 24 . macrolides , such as azithromycin , clarithromycin , and erythromycin , are the second class and appear to be less effective than beta lactams 25 . currently one of the most often prescribed antibiotics against borrelia burgdorferi sensu lato infection is doxycycline 25 - 29 . since then , the application of doxycycline extended and has been on the world health organization 's list of essential medicines as one of the most important medications needed in a basic health system 31 , 32 . like other agents in this class , it is an anti - bacterial and anti - parasite agent targeting protein production in general 31 - 33 . its side effects are similar to those of other members of this antibiotic class , including the development of an erythematous rash after exposure to sun . in vitro studies revealed that doxycycline is very effective against active ( vegetative ) form of borrelia sp . while , it was shown that doxycycline has no useful activity against latent rounded forms of borrelia sp . antimicrobials derived from natural sources such as plants , herbs , and fruits , and essential oils , have shown activity against a plethora of bacteria and fungi , but are poorly explored against borrelia sp . despite a rather small pool of available data on this subject , bronson and bronson study exhibited grapefruit seed extract as a powerful in vitro agent against spirochetes and their cystic forms of borrelia afzelii 44 . dipsacus sylvestris extract against borrelia burgdorferi sensu stricto revealed growth inhibiting activity as well 45 , whereas sapi , et al . , reported significant efficacy of leaf extracts from stevia rebaudiana on all forms of borrelia burgdorferi sensu stricto 46 . our recent study documented that other naturally occurring compounds like baicalein and luteolin , belonging to the flavones , are also potent against borrelia sp . in addition , fatty acids , including monolaurin and cis-2-decenoic acid , and iodine exhibited significant bacteriostatic and bactericidal activities 47 . our and other studies indicate that naturally derived compounds and antibiotics that display essential anti - borreliae activities might be an option for achieving optimal bacteriostatic and bactericidal activity at lower mics and mbcs , which could potentially reduce their side effects and treatment costs . in this study , we examined reciprocal cooperation of selected phytochemicals and micronutrients with documented anti - borrelae activities , with commercial antibiotic doxycycline against two borrelia species . to our knowledge , this is the first scientific report about interaction between doxycycline and phytochemicals and micronutrients against active and latent forms of borrelia sp . , vitamin c , cis-2-decenoic acid ( 10-had ) and doxycycline , with the purity between 90%-98% according to the manufacturer , were obtained from sigma ( st . luteolin and rosmarinic acid , with the purity between 97%-99% according to the manufacturer , were purchased from tocris bioscience ( bristol , united kingdom ) . as a negative control , ethanol at 0.1 - 0.4% ( v / v ) two borrelia species such as borrelia burgdorferi sensu stricto and borrelia garinii were tested in their three morphological forms : spirochetes , rounded forms , and biofilm . low passage isolates of the b31 strain of borrelia burgdorferi and cip103362 strain of borrelia garinii were obtained from the american type culture collection ( manassas , va ) . interaction of doxycycline with studied phytochemicals was assessed against spirochetes and rounded forms in 1.8 ml sterile screw - capped test tubes as well as against biofilm in 4-well chambers coated with collagen i from rat tail , according to checkerboard format . samples were inoculated with inoculums of test organisms , respectively , and set up at the appropriate times with increasing concentrations of doxycycline ( dox ) ( 0 to 500 g / ml ) and the active phytochemical ( ph ) ( 0 to 500 g / ml ) or micronutrient ( 0 to 88 g / ml ) , according to checkerboard assay . all tubes and chambers with mature biofilm fractional inhibitory concentration indexes ( ficis ) , fractional bactericidal concentration indexes ( fbcis ) , and fractional eradication ( of biofilm ) concentration indexes ( fecis ) were calculated and interpreted as follows : synergy = fici / fbci / feci of 0.5 ; antagonism = fici / fbci / feci > 4.0 ; additive = 0.5 < fici / fbci / feci < 1.0 , and indifferent ( no interaction ) = 1.0 < fici / fbci / feci < 4.0 48 - 50 . evaluation of reciprocal activities of tested combinations of agents against spirochetes of borrelia sp . reciprocity of test compounds against spirochetes of studied borrelia sp . was performed using a macro - dilution method according to checkerboard format . evaluation of reciprocal activities of tested combinations of agents against rounded forms of borrelia sp . efficacy of test compound combinations against rounded forms was examined using a live / dead baclight bacterial viability staining method , where green fluorescence determines the presence of live rounded forms and red fluorescence dead rounded forms , using a macro - dilution method according to checkerboard format . evaluation of reciprocal activities of tested combinations of agents against biofilms of borrelia sp . efficacy of test compound combinations against biofilm of tested borrelia sp . reciprocal interaction was evaluated according to checkerboard format and the percentage of biofilm eradication ( be% ) was calculated as be% = [ 1-(od959 of cells treated with doxycycline and phytochemical ( micronutrient)/od959 of untreated control ] x 100% . reciprocal cooperation between doxycycline and tested phytochemicals against spirochetes of borrelia sp . the examined combinations of doxycycline with tested phytochemicals and micronutrients against spirochetes of test microorganisms with the mics , fics and ficis values are presented in table 1a . screening of tested combinations indicates additive effects ( 0.5 < fici < 1.0 ) of doxycycline with 6 partner agents such as baicalein , luteolin , iodine , rosmarinic acid , vitamin d3 , and vitamin c. indifference ( no interaction ) was seen between doxycycline and fatty acids like monolaurin and 10-had . adding phytochemicals and micronutrients allowed for lowering of doxycycline mic value from 25 g / ml to 12.5 g / ml ( valid for both tested borrelia sp . ) adding phytochemicals and micronutrients allowed for lowering of doxycycline mbc90 value from 200 g / ml to 100 g / ml ( for b. burgdorferi ) and from 250 g / ml to 125 g / ml ( for b. garinii ) . the presence of sub - inhibitory concentration of doxycycline ( 12.5 g / ml ) reduced the mics of the partner phytochemical agents that fluctuated between 62.5 g / ml and 150 g / ml and the partner micronutrient agents between 0.00025 g / ml and 44 g / ml . the presence of sub - bactericidal concentration of doxycycline ( 100 g / ml for b. burgdorferi ; 125 g / ml b. garinii ) reduced the mbc90 values of the phytochemical agents that ranged between 125 g / ml and 225 g / ml and the partner micronutrient agents between 0.0005 g / ml and 44 g / ml . reciprocal cooperation between doxycycline and tested phytochemicals against rounded forms of borrelia sp . the effects of doxycycline in combination with tested phytochemicals and micronutrients against rounded forms of both studied borrelia sp . our previous results indicated that among several tested compounds , a few ( baicalein , luteolin , monolaurin , 10-had , and iodine ) showed anti - rounded forms efficacy that satisfied the mbc50 requirement . the other agents ( rosmarinic acid , vitamin d3 , vitamin c , and doxycycline ) did not affect rounded forms to this extent 47 . evaluation of all the phytochemicals and micronutrients in combination with doxycycline did not indicate synergy or antagonistic interaction in any of the cases . however , we noticed an additive effect which was restricted to combinations of doxycycline with baicalein or its close related agent luteolin , in both studied borrelia sp . combinations of doxycycline with flavones were able to induce the death of latent rounded forms up to 50% at lower concentrations of each partner agent . as such , effective concentrations of baicalein decreased from 350 g / ml to 275 g / ml , luteolin decreased from 200 g / ml to 150 g / ml , and doxycycline decreased from 500 g / ml to 100 g / ml . indifference ( no interaction ) was seen between doxycycline and fatty acids such as monolaurin and 10-had as well as iodine . mbc50 mark was achieved at their values identical to their single dose such as 300 g / ml for monolaurin , 500 g / ml for 10-had and 20 g / ml for iodine . however , at the same time , their addition allowed for reducing effective doxycycline concentration from 500 g / ml to 100 g / ml . the combinations with the rest of the tested agents did not achieve mbc50 mark , which did not allow performing correct calculations . kinetic evaluation of bactericidal effect against spirochetes and rounded forms of borrelia sp . kinetic evaluation of bactericidal effect of baicalein and luteolin was conducted as these compounds were able to eliminate both spirochete and rounded forms of tested borrelia sp . the results of this experiment show that doxycycline in combination with these phytochemicals , used at concentrations fulfilling the 0.5 < fbci < 1.0 requirement , could significantly eliminate spirochetes of borrelia burgdorferi and borrelia garinii in a dose- and time - dependent manner , reaching mic50 mark after 24 h and mic90 mark after 72 h ( figure 1a and b ) . reciprocal cooperation between doxycycline and tested phytochemicals against biofilm of borrelia sp.the combinations of doxycycline with test phytochemicals and micronutrients against biofilm formed by test microorganisms with the ec50s , fecs , and fecis are presented in table 3 . these were baicalein , luteolin , monolaurin , 10-had , and iodine for b. burgdorferi , as well as baicalein and monolaurin for b. garinii . the other tested agents such as rosmarinic acid , vitamin d3 , and vitamin c did not display such effect 47 . the additive anti - biofilm effect was achieved when doxycycline was co - administrated with baicalein , luteolin , and iodine , respectively ( valid for both tested borrelia sp . ) combinations of doxycycline with flavones were able to enhance eradication of biofilm up to 50% at their lower effective concentrations which decreased from 750 g / ml to 500 g / ml for baicalein , and from 350 g / ml to 175 g / ml for luteolin , and from 40 g / ml to 20 g / ml for iodine . indifference ( no interaction ) was seen for doxycycline combined with fatty acids such as monolaurin and 10-had . ec50 mark was achieved at their values identical to these of their single dose such as 375 g / ml for monolaurin , 800 g / ml for 10-had , and at the same time allowing for reducing doxycycline concentration from 250 g / ml to 125 g / ml . none of the agents tested alone or in combinations could eradicate biofilms of both studied borrelia sp . in 90 - 99% , even when their maximal tested this is a very rigorous requirement for any antibiotic , including doxycycline , for which anti - biofilm efficacy was shown to be ~ 30 - 40% at 250 the emergence of the relapse of symptoms of this disease has been noticed 7 , 19 , 37 , 55 . the challenge is further exacerbated by findings indicating that borrelia biofilm may harbor a heterogeneous population of spirochetes and rounded forms with distinct genetic and protein profiles 59 - 61 . may result from applications of agents that cooperate in eliminating spirochetes as well as their dormant forms . our previous study indicated that phytochemicals may provide a new reservoir of active antimicrobial agents , and serve as a resource for the development of novel anti - borreliae approaches 47 . that study showed that the most promising compounds effective against spirochete and/or latent forms of borrelia sp . were flavones such as baicalein and luteolin , fatty acids including monolaurin and 10-had , and iodine . that might lead to a subsequent loss of doxycycline efficacy in vitro and in vivo when applied long - term . taking all these aspects into consideration , we were interested in evaluating the reciprocal interactions of these phytochemicals and micronutrients with doxycycline against active and latent forms of borrelia sp . in the present study , we tested eight combinations of phytochemicals with doxycycline against all morphological forms of borrelia sp . additive effects were confined to a small number of combinations against latent forms ( doxycycline with baicalein or luteolin ) , while against spirochetes these activities were noticeable with more combinations , such as doxycycline with baicalein , luteolin , iodine , rosmarinic acid , and vitamin d3 or vitamin c. indifference was demonstrated for a combination of doxycycline with fatty acids such as monolaurin and 10-had . time - dependent bactericidal effects against spirochetes and latent rounded forms of borrelia burgdorferi and borrelia garinii were observed when doxycycline was combined with the flavones baicalein and luteolin , respectively , at concentrations fulfilling the 0.5 < fbci < 1.0 requirement . interestingly , the addition of iodine did not improve doxycycline 's efficacy towards rounded forms , but demonstrated additive effects against spirochete and biofilm forms of both tested borrelia sp . whether similar in vitro effects there are only a few published reports about reciprocal interactions between doxycycline and other antibiotics , and none of them used borrelia sp . further in vivo study of the efficacy of these combinations documented additional bacterial killing effect in a mouse model of acute tuberculosis infection , but not in a chronic infection model 62 . , amikacin in combination with doxycycline tested against clinical strains of mycobacterium tuberculosis showed a synergistic effect in 18 of the 29 strains , and indifference in 11 strains 63 . , suggest that addition of amikacin does not improve the results obtained by imipenem monotherapy , but adding doxycycline to amikacin is an alternative to imipenem in the therapy of acinetobacter baumannii pneumonia 64 . even fewer studies have investigated the interactions of doxycycline with phytochemicals and none with micronutrients . , showed synergy between doxycycline and essential oils ( eos ) such as carvacrol ( oregano oil ) , eugenol ( clove oil ) and cinnamaldehyde ( cinnamon oil ) against gram - negative strains of acinetobacter baumannii , klebsiella pneumoniae , escherichia coli , and pseudomonas aeruginosa . no growth inhibiting interactions were found between eos and doxycycline , however , synergistic bactericidal effects were observed , supported by the results visualizing the holes - type destructive changes in the bacterial membrane with the leakage of cellular contents 65 . the results showed that the antibiotic and povidone - iodine solutions , used individually , caused a similar and substantial decrease in the number of colonies ( biofilm ) and species of bacteria cultured . however , when both drugs were used together the decrease was even more striking , making 83% of the conjunctivae sterile . in addition , the use of different testing methods further complicates interpretation of the results . it is important to note that while bactericidal concentrations of doxycycline may induce persistent phenotypic changes , combining it with selected phytochemicals or micronutrients may not only allow for reducing its concentration to more safe values , but also expand the spectrum of bacterial forms affected and perhaps offer additional benefits such as anti - inflammatory properties . in summary , this study documents in vitro efficacy of doxycycline co - administrated with several phytochemicals and micronutrients against active and latent persistent forms of borrelia burgdorferi sensu stricto and borrelia garinii . these findings indicate an additive effect of doxycycline co - administrated with flavones like baicalein or luteolin against all morphological forms of these species in vitro . therefore , combinations of these phytochemicals with an antibiotic present a promising alternative towards combating borrelia sp . the study provides additional support for phytochemicals as valuable adjuvants in antimicrobial chemotherapy , although further in vivo and human studies are warranted to draw a final conclusion .

hypertrophic cardiomyopathy ( hcm ) has had a variety of names because of its diverse clinical manifestations since it was first described in detail by braunwald et al . in 1964.1)2 ) owing to these variations in the clinical course , the prognosis and natural course of hcm differs across patient cohorts . few definite risk factors have been identified for morbidity and mortality in hcm , except a family history of sudden cardiac death ( scd ) . recent studies have reported that the natural course of hcm , particularly in older age groups , is better than previously considered , irrespective of the presence of well - known risk factors.2)3 ) apical hypertrophic cardiomyopathy ( ahcm ) is an uncommon morphologic variant of hcm wherein the hypertrophy of the myocardium predominantly involves the apex of the left ventricle ( lv).4)5 ) this entity is very rare in non - asian populations , with most reports suggesting an overall prevalence of 1 - 3% in hcm patients.6)7)8 ) most ahcm patients present with mild or no symptoms and good prognosis.5)9 ) however , clinical presentations with wide qrs tachycardia , subendocardial myocardial infarction , atrial flutter , and lv aneurysm have been reported in some cases.10)11)12)13)14 ) despite these reports , the natural course of ahcm remains obscure as compared to that of classical asymmetric septal hcm . ahcm has been classified as pure and mixed types , based on the extent of myocardial involvement . mixed - type ahcm - which involves the myocardium from the apex to the interventricular septum - is supposed to have poor prognosis compared to pure ahcm ; however , there is no definite data to support this.5 ) symptoms of angina are a major clinical manifestation of many variants of hcm , including hcm with ventricular hypertrophy , myocardial bridging , and compression . a previous study revealed that coronary artery disease ( cad ) diagnosed by coronary angiography is associated with increased mortality in hcm patients . moreover , cad with hcm is associated with higher mortality than hcm with normal lv function.15 ) our study aimed to investigate the impact of cad on the natural course of ahcm . we also analyzed the general clinical course of ahcm and evaluated other risk factors for cardiovascular ( cv ) events and the clinical impact of the subtypes of ahcm . we enrolled 98 consecutive patients with ahcm 30 years and older who underwent coronary angiography or coronary computed tomography ( ct ) scans in yonsei cardiovascular hospital and eulji general hospital ( korea ) between january 2002 and march 2012 . the diagnostic criteria of ahcm was lv hypertrophy which was mainly confined to the apex with wall thickness 15 mm and ratio of maximal apical thickness to posterior wall 1.5 . a complete medical history of all patients was recorded , including diabetes , hypertension , smoking history , atrial fibrillation , previous cerebrovascular accidents , the presence and severity of symptoms such as angina and dyspnea , and medication for any of these conditions . two - dimensional and doppler echocardiography was used to assess left atrial diameter , lv end - systolic and end - diastolic dimensions , lv wall thickness , and presence of obstruction in the intraventricular space or lv outflow tract . classification as to pure or mixed type was done in 90/98 patients . in the remaining 8/98 patients , the pure type was defined as hypertrophy limited to the apical portion of the lv below the papillary muscle level , whereas the mixed type was defined as apical hypertrophy with coexistent hypertrophy in other lv segments . for ahcm in the " mixed " type , the greatest wall thickness had to be located in the apical segments.5 ) significant cad was defined as presence of luminal narrowing > 50% in at least one vessel of the coronary arteries . apart from the diagnosis of cad , the dominancy , blood supply to the apex , and presence of myocardial bridging ( only in coronary angiography ) were recorded . using chart reviews , past clinical history , including cv risk factors , was assessed for all the patients . the index of clinical outcome was adverse cv events , including death , non - fatal myocardial infarction or stroke , heart failure ( hf ) , coronary artery revascularization , and hospital admission due to cv disease . patients were grouped according to the degree of cad ( absent cad vs. cad ) and the type of ahcm ( pure vs. mixed ) . we compared continuous variations with independent t - test and categorical variables by pearson 's chi - square test . differences in the duration of cv event - free survival were evaluated using the kaplan - meier survival analysis ( log rank test ) and cox proportional - hazards regression model . all statistical analyses were performed with statistical package for the social sciences ( spss ) version 18.0 ( spss inc . , we enrolled 98 consecutive patients with ahcm 30 years and older who underwent coronary angiography or coronary computed tomography ( ct ) scans in yonsei cardiovascular hospital and eulji general hospital ( korea ) between january 2002 and march 2012 . the diagnostic criteria of ahcm was lv hypertrophy which was mainly confined to the apex with wall thickness 15 mm and ratio of maximal apical thickness to posterior wall 1.5 . a complete medical history of all patients was recorded , including diabetes , hypertension , smoking history , atrial fibrillation , previous cerebrovascular accidents , the presence and severity of symptoms such as angina and dyspnea , and medication for any of these conditions . two - dimensional and doppler echocardiography was used to assess left atrial diameter , lv end - systolic and end - diastolic dimensions , lv wall thickness , and presence of obstruction in the intraventricular space or lv outflow tract . classification as to pure or mixed type was done in 90/98 patients . in the remaining 8/98 patients , the pure type was defined as hypertrophy limited to the apical portion of the lv below the papillary muscle level , whereas the mixed type was defined as apical hypertrophy with coexistent hypertrophy in other lv segments . for ahcm in the " mixed " type , the greatest wall thickness had to be located in the apical segments.5 ) significant cad was defined as presence of luminal narrowing > 50% in at least one vessel of the coronary arteries . apart from the diagnosis of cad , the dominancy , blood supply to the apex , and presence of myocardial bridging ( only in coronary angiography ) were recorded . using chart reviews , past clinical history , including cv risk factors , was assessed for all the patients . the index of clinical outcome was adverse cv events , including death , non - fatal myocardial infarction or stroke , heart failure ( hf ) , coronary artery revascularization , and hospital admission due to cv disease . patients were grouped according to the degree of cad ( absent cad vs. cad ) and the type of ahcm ( pure vs. mixed ) . we compared continuous variations with independent t - test and categorical variables by pearson 's chi - square test . differences in the duration of cv event - free survival were evaluated using the kaplan - meier survival analysis ( log rank test ) and cox proportional - hazards regression model . all statistical analyses were performed with statistical package for the social sciences ( spss ) version 18.0 ( spss inc . , the mean age at the time of enrollment was 61.459.78 years , and 38.6% of the patients were female . the proportions of mixed and pure types of ahcm were 34.4% ( n=31 ) and 65.6% ( n=59 ) , respectively . in eight patients , the prevalence of hypertension , diabetes , and smoking were 62.2% , 25.5% , and 37.8% , respectively . beta - blockers and calcium - channel blockers ( ccb ) were prescribed for 45.9% and 30.6% of patients , respectively . overall , 74.5% of patients were symptomatic , with chest discomfort ( 57.1% ) being the most common symptom ( table 1 ) . coronary angiography and ct scans were performed in 72.4% ( n=71 ) and 27.6% ( n=27 ) of patients , respectively . significant cad was found in 31 ( 31.6% ) patients ( 1- , 2- , 3-vessel disease , and left main disease in 15.3% , 9.2% , 6.1% , and 1.0% , respectively ) . myocardial bridging was found in 20.4% ( n=20/71 ) of patients . in the 27 patients who underwent ct scans , determining of myocardial bridging was impossible . the mean follow - up period was 53.160.7 ( median 45.6 ) months , and cv events occurred in 22.4% ( 22/98 ) patients . mortality occurred in five ( 5.1% ) patients ; the cause of death was hf in two cases and cancer in one case . in two cases we could not find the definite cause of death because it occurred outside of the study hospital , but the patients had already been diagnosed as having the cv event ( myocardial infarction , revascularization ) 14 - 15 months earlier ( table 2 ) . the patients with cv events were older than those without cv events ( mean age , 66.87.5 years vs. 59.99.9 years ) . further , in the cv event group , the smoking rate and lv ejection fraction were higher and the lv end systolic diameter ( lvesd ) was smaller . prevalence of mixed - type ahcm was higher in the cv event group , although this difference was not statistically significant ( mixed vs. pure ahcm , 50% vs. 30% , p=0.097 ) . prescription rates of drugs acting on the sympatho - renal axis , such as beta - blockers , angiotensin converting enzyme ( ace ) inhibitors , and angiotensin ii receptor blockers ( arb ) reduced the prevalence of cv events ( table 3 ) . cv events were more frequent in patients with cad , being more apparent in patients with multi - vessel disease . myocardial bridging , dominancy , and difference in blood supply to the apex did not affect the clinical outcome ( table 4 ) . at baseline , 12 ( 12.2% ) patients had atrial fibrillation , and during the follow - up period , new onset atrial fibrillation occurred in 10 ( 10.0% ) patients . there was no significant difference in the prevalence of new onset atrial fibrillation according to the presence of cad { cad- 11.9% ( n=8 ) vs. cad+ 6.5% ( n=2 ) , p=0.497}. in cox univariate regression analysis , the cv event - free survival rate was significantly worse in the cad group { hazard ratio ( hr)= 3.18 , 95% confidence interval ( ci ) : 1.36 - 7.45 , p=0.008}. higher age ( hr=2.12 per 10 years increase , p=0.005 ) and smaller lv systolic diameter ( hr=0.84 with lvesd increase , p=0.006 ) were related with poorer prognosis . myocardial bridging did not affect the cv eventfree survival in ahcm patients ( hr=0.61 , 95% ci : 0.20 - 1.75 , p= 0.362 ) . pure - type ahcm showed a tendency toward better survival without statistical significance ( hr=1.96 , 95% ci : 0.81 - 4.72 , p= 0.134 ) ( table 5 ) . coronary artery disease persisted as an independent risk factor for cv events ( hr=2.91 , 95% ci : 1.19 - 7.13 , p=0.021 ) after adjusting for age and lvesd , which were significantly related with shorter event - free survival in univariate analysis or adding other cv risk factors ( table 6 ) . kaplan - meier survival analysis also revealed better survival in patients without cad { p=0.005 by log rank test ( mantel - cox ) } ( fig . the mean age at the time of enrollment was 61.459.78 years , and 38.6% of the patients were female . the proportions of mixed and pure types of ahcm were 34.4% ( n=31 ) and 65.6% ( n=59 ) , respectively . in eight patients , the prevalence of hypertension , diabetes , and smoking were 62.2% , 25.5% , and 37.8% , respectively . beta - blockers and calcium - channel blockers ( ccb ) were prescribed for 45.9% and 30.6% of patients , respectively . overall , 74.5% of patients were symptomatic , with chest discomfort ( 57.1% ) being the most common symptom ( table 1 ) . coronary angiography and ct scans were performed in 72.4% ( n=71 ) and 27.6% ( n=27 ) of patients , respectively . significant cad was found in 31 ( 31.6% ) patients ( 1- , 2- , 3-vessel disease , and left main disease in 15.3% , 9.2% , 6.1% , and 1.0% , respectively ) . myocardial bridging was found in 20.4% ( n=20/71 ) of patients . in the 27 patients who underwent ct scans , determining of myocardial bridging was impossible . the mean follow - up period was 53.160.7 ( median 45.6 ) months , and cv events occurred in 22.4% ( 22/98 ) patients . mortality occurred in five ( 5.1% ) patients ; the cause of death was hf in two cases and cancer in one case . in two cases we could not find the definite cause of death because it occurred outside of the study hospital , but the patients had already been diagnosed as having the cv event ( myocardial infarction , revascularization ) 14 - 15 months earlier ( table 2 ) . the patients with cv events were older than those without cv events ( mean age , 66.87.5 years vs. 59.99.9 years ) . further , in the cv event group , the smoking rate and lv ejection fraction were higher and the lv end systolic diameter ( lvesd ) was smaller . prevalence of mixed - type ahcm was higher in the cv event group , although this difference was not statistically significant ( mixed vs. pure ahcm , 50% vs. 30% , p=0.097 ) . prescription rates of drugs acting on the sympatho - renal axis , such as beta - blockers , angiotensin converting enzyme ( ace ) inhibitors , and angiotensin ii receptor blockers ( arb ) reduced the prevalence of cv events ( table 3 ) . cv events were more frequent in patients with cad , being more apparent in patients with multi - vessel disease . myocardial bridging , dominancy , and difference in blood supply to the apex did not affect the clinical outcome ( table 4 ) . at baseline , 12 ( 12.2% ) patients had atrial fibrillation , and during the follow - up period , new onset atrial fibrillation occurred in 10 ( 10.0% ) patients . there was no significant difference in the prevalence of new onset atrial fibrillation according to the presence of cad { cad- 11.9% ( n=8 ) vs. cad+ 6.5% ( n=2 ) , p=0.497}. in cox univariate regression analysis , the cv event - free survival rate was significantly worse in the cad group { hazard ratio ( hr)= 3.18 , 95% confidence interval ( ci ) : 1.36 - 7.45 , p=0.008}. higher age ( hr=2.12 per 10 years increase , p=0.005 ) and smaller lv systolic diameter ( hr=0.84 with lvesd increase , p=0.006 ) were related with poorer prognosis . myocardial bridging did not affect the cv eventfree survival in ahcm patients ( hr=0.61 , 95% ci : 0.20 - 1.75 , p= 0.362 ) . pure - type ahcm showed a tendency toward better survival without statistical significance ( hr=1.96 , 95% ci : 0.81 - 4.72 , p= 0.134 ) ( table 5 ) . coronary artery disease persisted as an independent risk factor for cv events ( hr=2.91 , 95% ci : 1.19 - 7.13 , p=0.021 ) after adjusting for age and lvesd , which were significantly related with shorter event - free survival in univariate analysis or adding other cv risk factors ( table 6 ) . kaplan - meier survival analysis also revealed better survival in patients without cad { p=0.005 by log rank test ( mantel - cox ) } ( fig . our findings demonstrated cad diagnosed by coronary angiography or coronary ct scan to be an important risk factor for cv events in ahcm patients , with increasing risk associated with the increasing severity of cad . age , smoking , increased lv contractility , and no prescription of beta - blockers , arb , and/or ace inhibitors were significantly related to cv events in univariate analysis . overall , mortality occurred in five patients ; however , no cases of definite scd were observed . ahcm without cad showed an excellent prognosis even in the advanced age group , with a survival rate of 97.0% and cv event - free survival rate of 86.6% for over 50 months of follow - up . the presence of mixed - type ahcm did not significantly increase the risk of cv events . apical hypertrophic cardiomyopathy is a rare disease that occurs only in small proportion of hcm patients . its natural course is known to be benign ; however , some controversial data has been reported in japanese and western ahcm populations , including the occurrence of scd and apical aneurysms.5)14)16 ) eriksson et al.5 ) reported the long - term clinical outcomes of ahcm in a comparatively large number of patients ( canada , n=105 ) for a mean follow - up period of 13.6 years ; they demonstrated a low mortality but an increased rate of cv events , including myocardial infarction ( 10% ) and atrial fibrillation ( 12% ) , as compared to the general population . thus , ahcm was shown to have a benign prognosis ; however , it was associated with an increased rate of cv events , including 11 cases of myocardial infarction . unfortunately , this study provided coronary artery anatomical data for only some of the patients ( 42/105 ) , which were obtained in the course of follow - up . a previous study concerning hcm ( not " ahcm " ) patients that included the results of coronary angiography , showed that the presence of severe cad increased mortality rates in these patients.15 ) another study reported on coexistent cad in hcm patients who suffered scd but did not have the risk factors typically associated with scd , suggesting that cad may be an unfavorable prognostic factor in hcm patients.17 ) to the best of our knowledge , our study is the first to investigate the impact of cad on the natural course of ahcm with coronary imaging data for all patients . the impact of cad in ahcm was greater than in hcm , which may be due to the relatively benign clinical course of ahcm . further , considering the higher mean age of the patients in the present study as compared to that in the previous study ( 61.45 years vs. 41.4 years ) , ahcm patients without cad had a benign natural course that was comparable to the results of the korean heart study which revealed that the 15 year follow - up cv hospitalization rate was 12.6% ( 54472/430920 ) , and mortality was 2.3% ( 9943/430920 ) for 430920 members of the general population aged 30 - 74 years.18 ) several pathophysiological features of hcm predispose individuals to the development of ischemia , which may be induced by an increased demand for myocardial oxygen or a reduction in the myocardial blood flow and oxygen supply.19 ) factors that increase the myocardial oxygen demand include myocyte hypertrophy and increased myocardial mass . factors that reduce myocardial blood flow include impaired vasodilation and compression of intramural vessels due to myocardial bridging.2)19 ) thus , various factors have been proposed to explain ischemia in hcm . as discussed above , the inherent abnormalities of hcm along with the concurrent presence of cad have a synergistic effect in the induction myocardial ischemia . therefore , cad could be an independent prognostic factor by aggravating the inherent ischemic features of ahcm . in the present study , myocardial bridging was evaluated only in the coronary angiography group ; however , it was not observed to affect the natural course of ahcm , similar to a previous study on hcm.20 ) mixed type ahcm , which involves part of lv other than the apex , is supposed to have worse clinical prognosis because of its wider involvement . a previous report showed mixed - type ahcm to be associated with the severity of symptoms but not the clinical outcome.5 ) the present findings also did not reveal any definitive effects of the ahcm type on the clinical outcomes . determining the precise extent of myocardial involvement with echocardiography is technically difficult ; moreover , in mixed - type ahcm , the extent of involvement is considerably diverse . if magnetic resonance imaging were used to differentiate between pure - type and mixed - type ahcm , the results may differ . however , extensive myocardial involvement apart from the apex does not lead to hemodynamic compromise , such as lv outflow tract obstruction or mitral regurgitation ; therefore , it is reasonable that the risk of cv events does not increase significantly in mixed - type ahcm . our results revealed that increased lv systolic function and reduced lv end - systolic diameter resulting in advanced hypertrophy were associated with an increased prevalence of cv events . the use of beta - blockers , ace inhibitors , and arb - but not ccb - had a protective effect against cv events . these drugs affect the activities of the sympathetic nervous system , suggesting that increased sympathetic nervous activity may adversely affect the prognosis of ahcm . however , in survival analysis , these factors did not significantly affect the event - free survival rate ; therefore , the relationship between sympathetic activity and clinical outcomes in ahcm remains unclear . as compared to general ahcm patients , the present ahcm cohort was at a comparatively higher risk for cad and cv events , since coronary imaging studies were not performed randomly , but rather for the evaluation of suspected coronary artery obstructive disease , with 74.5% of the patients being symptomatic and over 50% of the patients reporting chest discomfort . coronary artery disease diagnosed by coronary angiography or ct scan is one of the most important risk factors for cv events in ahcm patients . ahcm is frequently accompanied by myocardial ischemia and it is associated with a higher cv risk . cad might aggravate the ischemia of ahcm and lead to worse clinical outcomes . in the absence of cad , ahcm patients over 30 years of age have a good prognosis , comparable with that of the general population . the benign natural course of ahcm compared to cad also contributes to the major impact of cad on ahcm to some degree . to prevent cv events in ahcm with cad , active cv risk reduction intervention may be needed . as compared to general ahcm patients , the present ahcm cohort was at a comparatively higher risk for cad and cv events , since coronary imaging studies were not performed randomly , but rather for the evaluation of suspected coronary artery obstructive disease , with 74.5% of the patients being symptomatic and over 50% of the patients reporting chest discomfort . coronary artery disease diagnosed by coronary angiography or ct scan is one of the most important risk factors for cv events in ahcm patients . ahcm is frequently accompanied by myocardial ischemia and it is associated with a higher cv risk . cad might aggravate the ischemia of ahcm and lead to worse clinical outcomes . in the absence of cad , ahcm patients over 30 years of age have a good prognosis , comparable with that of the general population . the benign natural course of ahcm compared to cad also contributes to the major impact of cad on ahcm to some degree . to prevent cv events in ahcm with cad , active cv risk reduction intervention may be needed .

background and objectivesapical hypertrophic cardiomyopathy ( ahcm ) is an uncommon variant of hypertrophic cardiomyopathy with a relatively benign course . however , the prognostic factors of ahcm - particularly those associated with coronary artery disease ( cad ) and its anatomical subtypes - are not well known.subjects and methodswe enrolled 98 consecutive patients with ahcm who underwent coronary angiography or coronary computed tomography scanning at two general hospitals in korea from january 2002 to march 2012 . patient charts were reviewed for information regarding cardiovascular ( cv ) risk factors , symptoms , and occurrence of cv events and/or mortality . we also reviewed echocardiographic data and angiography records.resultsthe mean age at the time of enrollment was 61.459.78 years , with female patients comprising 38.6% . the proportions of mixed and pure types of ahcm were 34.4% and 65.6% , respectively . cad was found in 31 ( 31.6% ) patients . the mean follow - up period was 53.160.7 months . cv events occurred in 22.4% of patients , and the mortality rate was 5.1% . the mixed - type was more frequent in cv event group although this difference was not statistically significant ( 50% vs. 30% , p=0.097 ) . the presence of cad emerged as an independent risk factor for cv events in univariate and multivariate cox regression analysis after adjusting for other cv risk factors.conclusioncoronary artery disease is an independent risk factor for cv events in ahcm patients . however , ahcm without cad has a benign natural course , comparable with the general population .

Introduction Subjects and Methods Patients Clinical follow-up Statistical analysis Results Baseline characteristics Clinical follow-up data Comparison of cardiovascular event-free survival Discussion Limitations Conclusion

in 1964.1)2 ) owing to these variations in the clinical course , the prognosis and natural course of hcm differs across patient cohorts . recent studies have reported that the natural course of hcm , particularly in older age groups , is better than previously considered , irrespective of the presence of well - known risk factors.2)3 ) apical hypertrophic cardiomyopathy ( ahcm ) is an uncommon morphologic variant of hcm wherein the hypertrophy of the myocardium predominantly involves the apex of the left ventricle ( lv).4)5 ) this entity is very rare in non - asian populations , with most reports suggesting an overall prevalence of 1 - 3% in hcm patients.6)7)8 ) most ahcm patients present with mild or no symptoms and good prognosis.5)9 ) however , clinical presentations with wide qrs tachycardia , subendocardial myocardial infarction , atrial flutter , and lv aneurysm have been reported in some cases.10)11)12)13)14 ) despite these reports , the natural course of ahcm remains obscure as compared to that of classical asymmetric septal hcm . mixed - type ahcm - which involves the myocardium from the apex to the interventricular septum - is supposed to have poor prognosis compared to pure ahcm ; however , there is no definite data to support this.5 ) symptoms of angina are a major clinical manifestation of many variants of hcm , including hcm with ventricular hypertrophy , myocardial bridging , and compression . a previous study revealed that coronary artery disease ( cad ) diagnosed by coronary angiography is associated with increased mortality in hcm patients . moreover , cad with hcm is associated with higher mortality than hcm with normal lv function.15 ) our study aimed to investigate the impact of cad on the natural course of ahcm . we also analyzed the general clinical course of ahcm and evaluated other risk factors for cardiovascular ( cv ) events and the clinical impact of the subtypes of ahcm . we enrolled 98 consecutive patients with ahcm 30 years and older who underwent coronary angiography or coronary computed tomography ( ct ) scans in yonsei cardiovascular hospital and eulji general hospital ( korea ) between january 2002 and march 2012 . a complete medical history of all patients was recorded , including diabetes , hypertension , smoking history , atrial fibrillation , previous cerebrovascular accidents , the presence and severity of symptoms such as angina and dyspnea , and medication for any of these conditions . in the remaining 8/98 patients , the pure type was defined as hypertrophy limited to the apical portion of the lv below the papillary muscle level , whereas the mixed type was defined as apical hypertrophy with coexistent hypertrophy in other lv segments . for ahcm in the " mixed " type , the greatest wall thickness had to be located in the apical segments.5 ) significant cad was defined as presence of luminal narrowing > 50% in at least one vessel of the coronary arteries . apart from the diagnosis of cad , the dominancy , blood supply to the apex , and presence of myocardial bridging ( only in coronary angiography ) were recorded . using chart reviews , past clinical history , including cv risk factors , was assessed for all the patients . the index of clinical outcome was adverse cv events , including death , non - fatal myocardial infarction or stroke , heart failure ( hf ) , coronary artery revascularization , and hospital admission due to cv disease . patients were grouped according to the degree of cad ( absent cad vs. cad ) and the type of ahcm ( pure vs. mixed ) . differences in the duration of cv event - free survival were evaluated using the kaplan - meier survival analysis ( log rank test ) and cox proportional - hazards regression model . , we enrolled 98 consecutive patients with ahcm 30 years and older who underwent coronary angiography or coronary computed tomography ( ct ) scans in yonsei cardiovascular hospital and eulji general hospital ( korea ) between january 2002 and march 2012 . a complete medical history of all patients was recorded , including diabetes , hypertension , smoking history , atrial fibrillation , previous cerebrovascular accidents , the presence and severity of symptoms such as angina and dyspnea , and medication for any of these conditions . in the remaining 8/98 patients , the pure type was defined as hypertrophy limited to the apical portion of the lv below the papillary muscle level , whereas the mixed type was defined as apical hypertrophy with coexistent hypertrophy in other lv segments . for ahcm in the " mixed " type , the greatest wall thickness had to be located in the apical segments.5 ) significant cad was defined as presence of luminal narrowing > 50% in at least one vessel of the coronary arteries . apart from the diagnosis of cad , the dominancy , blood supply to the apex , and presence of myocardial bridging ( only in coronary angiography ) were recorded . using chart reviews , past clinical history , including cv risk factors , was assessed for all the patients . the index of clinical outcome was adverse cv events , including death , non - fatal myocardial infarction or stroke , heart failure ( hf ) , coronary artery revascularization , and hospital admission due to cv disease . patients were grouped according to the degree of cad ( absent cad vs. cad ) and the type of ahcm ( pure vs. mixed ) . differences in the duration of cv event - free survival were evaluated using the kaplan - meier survival analysis ( log rank test ) and cox proportional - hazards regression model . , the mean age at the time of enrollment was 61.459.78 years , and 38.6% of the patients were female . the proportions of mixed and pure types of ahcm were 34.4% ( n=31 ) and 65.6% ( n=59 ) , respectively . in eight patients , the prevalence of hypertension , diabetes , and smoking were 62.2% , 25.5% , and 37.8% , respectively . beta - blockers and calcium - channel blockers ( ccb ) were prescribed for 45.9% and 30.6% of patients , respectively . coronary angiography and ct scans were performed in 72.4% ( n=71 ) and 27.6% ( n=27 ) of patients , respectively . significant cad was found in 31 ( 31.6% ) patients ( 1- , 2- , 3-vessel disease , and left main disease in 15.3% , 9.2% , 6.1% , and 1.0% , respectively ) . myocardial bridging was found in 20.4% ( n=20/71 ) of patients . the mean follow - up period was 53.160.7 ( median 45.6 ) months , and cv events occurred in 22.4% ( 22/98 ) patients . mortality occurred in five ( 5.1% ) patients ; the cause of death was hf in two cases and cancer in one case . the patients with cv events were older than those without cv events ( mean age , 66.87.5 years vs. 59.99.9 years ) . further , in the cv event group , the smoking rate and lv ejection fraction were higher and the lv end systolic diameter ( lvesd ) was smaller . prevalence of mixed - type ahcm was higher in the cv event group , although this difference was not statistically significant ( mixed vs. pure ahcm , 50% vs. 30% , p=0.097 ) . prescription rates of drugs acting on the sympatho - renal axis , such as beta - blockers , angiotensin converting enzyme ( ace ) inhibitors , and angiotensin ii receptor blockers ( arb ) reduced the prevalence of cv events ( table 3 ) . cv events were more frequent in patients with cad , being more apparent in patients with multi - vessel disease . at baseline , 12 ( 12.2% ) patients had atrial fibrillation , and during the follow - up period , new onset atrial fibrillation occurred in 10 ( 10.0% ) patients . there was no significant difference in the prevalence of new onset atrial fibrillation according to the presence of cad { cad- 11.9% ( n=8 ) vs. cad+ 6.5% ( n=2 ) , p=0.497}. in cox univariate regression analysis , the cv event - free survival rate was significantly worse in the cad group { hazard ratio ( hr)= 3.18 , 95% confidence interval ( ci ) : 1.36 - 7.45 , p=0.008}. coronary artery disease persisted as an independent risk factor for cv events ( hr=2.91 , 95% ci : 1.19 - 7.13 , p=0.021 ) after adjusting for age and lvesd , which were significantly related with shorter event - free survival in univariate analysis or adding other cv risk factors ( table 6 ) . the mean age at the time of enrollment was 61.459.78 years , and 38.6% of the patients were female . the proportions of mixed and pure types of ahcm were 34.4% ( n=31 ) and 65.6% ( n=59 ) , respectively . in eight patients , the prevalence of hypertension , diabetes , and smoking were 62.2% , 25.5% , and 37.8% , respectively . beta - blockers and calcium - channel blockers ( ccb ) were prescribed for 45.9% and 30.6% of patients , respectively . coronary angiography and ct scans were performed in 72.4% ( n=71 ) and 27.6% ( n=27 ) of patients , respectively . significant cad was found in 31 ( 31.6% ) patients ( 1- , 2- , 3-vessel disease , and left main disease in 15.3% , 9.2% , 6.1% , and 1.0% , respectively ) . myocardial bridging was found in 20.4% ( n=20/71 ) of patients . the mean follow - up period was 53.160.7 ( median 45.6 ) months , and cv events occurred in 22.4% ( 22/98 ) patients . mortality occurred in five ( 5.1% ) patients ; the cause of death was hf in two cases and cancer in one case . the patients with cv events were older than those without cv events ( mean age , 66.87.5 years vs. 59.99.9 years ) . further , in the cv event group , the smoking rate and lv ejection fraction were higher and the lv end systolic diameter ( lvesd ) was smaller . prevalence of mixed - type ahcm was higher in the cv event group , although this difference was not statistically significant ( mixed vs. pure ahcm , 50% vs. 30% , p=0.097 ) . prescription rates of drugs acting on the sympatho - renal axis , such as beta - blockers , angiotensin converting enzyme ( ace ) inhibitors , and angiotensin ii receptor blockers ( arb ) reduced the prevalence of cv events ( table 3 ) . cv events were more frequent in patients with cad , being more apparent in patients with multi - vessel disease . at baseline , 12 ( 12.2% ) patients had atrial fibrillation , and during the follow - up period , new onset atrial fibrillation occurred in 10 ( 10.0% ) patients . there was no significant difference in the prevalence of new onset atrial fibrillation according to the presence of cad { cad- 11.9% ( n=8 ) vs. cad+ 6.5% ( n=2 ) , p=0.497}. in cox univariate regression analysis , the cv event - free survival rate was significantly worse in the cad group { hazard ratio ( hr)= 3.18 , 95% confidence interval ( ci ) : 1.36 - 7.45 , p=0.008}. coronary artery disease persisted as an independent risk factor for cv events ( hr=2.91 , 95% ci : 1.19 - 7.13 , p=0.021 ) after adjusting for age and lvesd , which were significantly related with shorter event - free survival in univariate analysis or adding other cv risk factors ( table 6 ) . our findings demonstrated cad diagnosed by coronary angiography or coronary ct scan to be an important risk factor for cv events in ahcm patients , with increasing risk associated with the increasing severity of cad . age , smoking , increased lv contractility , and no prescription of beta - blockers , arb , and/or ace inhibitors were significantly related to cv events in univariate analysis . ahcm without cad showed an excellent prognosis even in the advanced age group , with a survival rate of 97.0% and cv event - free survival rate of 86.6% for over 50 months of follow - up . the presence of mixed - type ahcm did not significantly increase the risk of cv events . its natural course is known to be benign ; however , some controversial data has been reported in japanese and western ahcm populations , including the occurrence of scd and apical aneurysms.5)14)16 ) eriksson et al.5 ) reported the long - term clinical outcomes of ahcm in a comparatively large number of patients ( canada , n=105 ) for a mean follow - up period of 13.6 years ; they demonstrated a low mortality but an increased rate of cv events , including myocardial infarction ( 10% ) and atrial fibrillation ( 12% ) , as compared to the general population . thus , ahcm was shown to have a benign prognosis ; however , it was associated with an increased rate of cv events , including 11 cases of myocardial infarction . unfortunately , this study provided coronary artery anatomical data for only some of the patients ( 42/105 ) , which were obtained in the course of follow - up . a previous study concerning hcm ( not " ahcm " ) patients that included the results of coronary angiography , showed that the presence of severe cad increased mortality rates in these patients.15 ) another study reported on coexistent cad in hcm patients who suffered scd but did not have the risk factors typically associated with scd , suggesting that cad may be an unfavorable prognostic factor in hcm patients.17 ) to the best of our knowledge , our study is the first to investigate the impact of cad on the natural course of ahcm with coronary imaging data for all patients . the impact of cad in ahcm was greater than in hcm , which may be due to the relatively benign clinical course of ahcm . further , considering the higher mean age of the patients in the present study as compared to that in the previous study ( 61.45 years vs. 41.4 years ) , ahcm patients without cad had a benign natural course that was comparable to the results of the korean heart study which revealed that the 15 year follow - up cv hospitalization rate was 12.6% ( 54472/430920 ) , and mortality was 2.3% ( 9943/430920 ) for 430920 members of the general population aged 30 - 74 years.18 ) several pathophysiological features of hcm predispose individuals to the development of ischemia , which may be induced by an increased demand for myocardial oxygen or a reduction in the myocardial blood flow and oxygen supply.19 ) factors that increase the myocardial oxygen demand include myocyte hypertrophy and increased myocardial mass . as discussed above , the inherent abnormalities of hcm along with the concurrent presence of cad have a synergistic effect in the induction myocardial ischemia . in the present study , myocardial bridging was evaluated only in the coronary angiography group ; however , it was not observed to affect the natural course of ahcm , similar to a previous study on hcm.20 ) mixed type ahcm , which involves part of lv other than the apex , is supposed to have worse clinical prognosis because of its wider involvement . a previous report showed mixed - type ahcm to be associated with the severity of symptoms but not the clinical outcome.5 ) the present findings also did not reveal any definitive effects of the ahcm type on the clinical outcomes . determining the precise extent of myocardial involvement with echocardiography is technically difficult ; moreover , in mixed - type ahcm , the extent of involvement is considerably diverse . if magnetic resonance imaging were used to differentiate between pure - type and mixed - type ahcm , the results may differ . however , extensive myocardial involvement apart from the apex does not lead to hemodynamic compromise , such as lv outflow tract obstruction or mitral regurgitation ; therefore , it is reasonable that the risk of cv events does not increase significantly in mixed - type ahcm . our results revealed that increased lv systolic function and reduced lv end - systolic diameter resulting in advanced hypertrophy were associated with an increased prevalence of cv events . however , in survival analysis , these factors did not significantly affect the event - free survival rate ; therefore , the relationship between sympathetic activity and clinical outcomes in ahcm remains unclear . as compared to general ahcm patients , the present ahcm cohort was at a comparatively higher risk for cad and cv events , since coronary imaging studies were not performed randomly , but rather for the evaluation of suspected coronary artery obstructive disease , with 74.5% of the patients being symptomatic and over 50% of the patients reporting chest discomfort . coronary artery disease diagnosed by coronary angiography or ct scan is one of the most important risk factors for cv events in ahcm patients . ahcm is frequently accompanied by myocardial ischemia and it is associated with a higher cv risk . in the absence of cad , ahcm patients over 30 years of age have a good prognosis , comparable with that of the general population . the benign natural course of ahcm compared to cad also contributes to the major impact of cad on ahcm to some degree . to prevent cv events in ahcm with cad , active cv risk reduction intervention may be needed . as compared to general ahcm patients , the present ahcm cohort was at a comparatively higher risk for cad and cv events , since coronary imaging studies were not performed randomly , but rather for the evaluation of suspected coronary artery obstructive disease , with 74.5% of the patients being symptomatic and over 50% of the patients reporting chest discomfort . coronary artery disease diagnosed by coronary angiography or ct scan is one of the most important risk factors for cv events in ahcm patients . ahcm is frequently accompanied by myocardial ischemia and it is associated with a higher cv risk . in the absence of cad , ahcm patients over 30 years of age have a good prognosis , comparable with that of the general population . the benign natural course of ahcm compared to cad also contributes to the major impact of cad on ahcm to some degree . to prevent cv events in ahcm with cad , active cv risk reduction intervention may be needed .

halogen and hydrogen bonding plays an essential role in chemistry and biochemistry [ 114 ] . recently , the halogen bonding , a xb ( x halogen atom , b lewis base , a electronegative atom ) , attracted considerable attention due to its strong , selective and directional character [ 414 ] . these features make them very important in supramolecular crystal engineering and in determination of biological structures [ 413 ] . therefore , many efforts are made to describe the halogen bonding by theoretical methods of quantum chemistry ; this in turn can help in rational design of new halogen bonded systems with desired molecular properties [ 414 ] . the pioneering experimental studies reporting on the existence of halogen bonding dates back to the mid nineteenth century [ 1519 ] . the first theoretical analysis of the resonance structures has been proposed by mulliken to explain the molecular spectra of halogen bonded complexes . one decade later hassel and co - workers expanded this area by developing new systems containing halogen bonds [ 1921 ] . in his nobel lecture hassel highlighted that the electrophilic part of halogen atom can be very crucial in molecular self - assembly phenomena . in addition , it has been stressed the importance of charge transfer in halogen bonded systems . the novel concept that explains the origin of halogen bonding was proposed by politzer and coworkers [ 69 , 13 ] . the authors noticed for the first time , based on the molecular electrostatic potential , that there exists an electron deficiency at the outer part of the halogen atom , so called -hole , what leads in turn to the electrostatic attraction with lewis bases . hence , the halogen bonding is driven mainly by the electrostatic term [ 69 ] . very recently hennemann and coworkers extended the -hole concept to analysis of polarized hydrogen bonds . in the present study we will characterize the halogen bonding in selected molecules in terms of quantitative role of the electronic ( the charge transfer and covalency ) and electrostatic factors . the recently developed ets - nocv scheme will be used , that originates from a combination of the extended transition state ( ets ) energy decomposition approach with the natural orbitals for chemical valence ( nocv ) analysis [ 2329 ] . it was shown that ets - nocv is able to extract and directly quantify the crucial components ( , , , etc . ) that constitute various types of chemical bonds including donor - acceptor [ 23 , 24 , 2629 ] , covalent , intra - molecular agostic [ 30 , 31 ] and inter - molecular hydrogen bonding [ 3235 ] . we will first apply the ets - nocv scheme in a description of relatively weak halogen bonds ( 1-nh3 and 1-cph3 , see panels a and b of fig . 1 ) . ammonia and divalent carbon ( 0 ) ligands [ c(ph3)2 ] will be used as electron donating species , whereas cf3i molecule as electron acceptor . the cl2 ligands ( where l is an electron donor ) have recently attracted considerable attention due to untypical oxidation state of carbon atom [ 3638 ] . ets - nocv approach will also be used for the first time to describe the -hole at iodine atom of cf3i , based on deformation density contributions obtained from the nocv analysis of the bond between the cf3 and i fragments . in the next step , we will focus our attention on the catalytic system [ c6h4(c3h2n2i)2][otf]2 ( 2 in fig . 1 ) . it has been proven experimentally that this halogen bond donor is a very strong electrophilic species that is able to break covalent c br bond in benzhydryl bromide ( ph2hc br ) . accordingly , as the second objective of our study we will perform pioneering theoretical analysis of the interaction between isopropyl bromide ( the methyl groups were used instead of phenyl rings ) and the model [ c6h4(c3h2n2i)2 ] system ( the counter anion otf has been omitted in the calculations ) , see 2-br in fig . 1 . further , we will demonstrate that very high electrophilicity of 2 can lead to the formation of dihydrogen complex ( 2-h2 in fig . 1 ) . finally , the halogen ( cl i ) and hydrogen bonds ( cl hn ) co - existing within the same molecule ( 3-cl in fig . this novel urea - based system has been recently developed experimentally by chudzinski and coworkers .fig . a thick black line represents the way of fragmentation used in a description of bonding halogen bonded systems studied in the present work . all the dft calculations presented here are based on the amsterdam density functional ( adf2009 ) program in which ets - nocv scheme was implemented [ 4145 ] . the becke - perdew exchange - correlation functional [ 46 , 47 ] was applied with an inclusion of the dispersion correction ( bp86-d ) . a standard triple - zeta sto basis containing two sets of polarization functions auxiliary s , p , d , f , and g sto functions , centered on all nuclei , were used to fit the electron density and to obtain accurate coulomb potentials in each scf cycle . the contours of deformation densities were plotted based on adf - gui interface . in our analysis of halogen bonding each of the systems are divided up into two individual fragments as shown schematically by thick black lines in fig . 1 . then we used the ets - nocv method to study the interaction between these subsystems . thus , our analysis is based on the bonding between the two close shell molecular fragments . bonding analysis presented in this study is based on the ets - nocv approach which is a combination of the extended transition state ( ets ) method with the natural orbitals for chemical valence ( nocv ) scheme [ 2329 ] . historically , the natural orbitals for chemical valence ( nocv ) have been derived from the nalewajski - mrozek valence theory [ 5056 ] as eigenvectors of the deformation density matrix . it was shown [ 24 , 26 ] that the natural orbitals for chemical valence pairs ( -k,,k ) decompose the differential density into nocv - contributions ( k):1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta \rho ( r ) = \sum\limits_{{k = 1}}^{{{{m } \left/ { 2 } \right . } } } { { v_k } [ - \psi _ { { - k}}^2(r ) + \psi_k^2 } ( r ) ] = \sum\limits_{{k = 1}}^{{{{m } \left/ { 2 } \right . } } } { \delta { \rho_k}(r ) } , $ $ \end{document}where k and m stand for the nocv eigenvalues and the number of basis functions , respectively . visual inspection of deformation density plots ( k ) helps to attribute symmetry and the direction of the charge flow . negative values of this function are marked by red color ( outflow of electrons due to bond formation ) , whereas positive values of k are in blue color ( charge accumulation ) . in addition , within ets - nocv scheme , the deformation - density based picture is enriched by the energetic estimations , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^k $ $ \end{document } , for each k . in the ets energy decomposition scheme , the interaction energy eint between the fragments ( exhibiting geometries as in the combined molecule ) is divided into three components:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta { e_{\text{int } } } = \delta { e_{\text{elstat } } } + \delta { e_{\text{pauli } } } + \delta { e_{\text{orb}}}. $ $ \end{document } the first term , eelstat , corresponds to the classical electrostatic interaction between the fragments as they are brought to their positions in the final molecule . the second term , epauli , accounts for the repulsive pauli interaction between occupied orbitals on the fragments in the combined molecule . the third stabilizing term , eorb , represents the interactions between the occupied molecular orbitals of one fragment with the unoccupied molecular orbitals of the other fragments as well as mixing of occupied and virtual orbitals within the same fragment ( inner - fragment polarization ) . this energy term may be linked to the electronic bonding effect coming from the formation of a chemical bond . in the combined ets - nocv scheme the orbital interaction term ( eorb ) is expressed in terms of nocv s eigenvalues ( vk ) as:3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta { e_{{orb } } } = \sum\limits_k { \delta e_{{orb}}^k = } \sum\limits_{{k = 1}}^{{{{m } \left/ { 2 } \right . } } } { { v_k } [ - f _ { { - k , - k}}^{{ts } } } + f_{{k , k}}^{{ts } } ] , $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f_{{i , i}}^{{ts } } $ $ \end{document } are diagonal kohn - sham matrix elements defined over nocv with respect to the transition state ( ts ) density ( at the midpoint between density of the molecule and the sum of fragment densities ) . the above components \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^k $ $ \end{document } provide energetic estimation of k , thus , they characterize the importance of a particular electron flow channel for the bonding between considered molecular fragments . as has been already mentioned , we will use the dispersion corrected bp86-d functional [ 4648 ] , hence , the dispersion correction ( edisp ) will be added to eint values . we will start our discussion from the nocv s based description of halogen bonding in 1-nh3 . 2a that the leading deformation density channel , 1 , with the corresponding electronic stabilization \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^1 = { { { - 8.2kcal } } \left/ { { mol } } \right . } $ $ \end{document } , constitutes the halogen bonding between ammonia and cf3i molecule . qualitatively , 1 is based on the donation from the lone electron pair of nitrogen to the empty *(i - c ) . this transfer leads to elongation of c i bond , by 0.015 , as compared to non - bonded cf3i species . it is important to note that of similar importance is the covalent contribution that originates from the electron transfer from both the nitrogen and iodine atoms to the i it is noteworthy that , due to formation of halogen bond , the accumulation of electron density is also observed at the carbon atom of cf3i , which is in line with an increase in s character of carbon ; this issue has been deeply analyzed recently by grabowski using nbo method . the charge transfer , 1 , characterized by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^1 = { { { - 8.2kcal } } \left/ { { mol } } \right . } $ $ \end{document } nearly covers the total orbital interaction term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left ( { \delta { e_{{orb } } } = { { { - 9.2kcal } } \left/ { { mol } } \right . } } \right ) $ $ \end{document } , see table 1 . the remaining part of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^{{rest } } = { { { - 1.0kcal } } \left/ { { mol } } \right . } $ $ \end{document } corresponds to cf3i nh3 back - donation ( ca . 0.5 kcal mol ) and the intra - fragment polarization ( ca . 0.5 kcal mol).fig . 2the contour of deformation density contribution 1 describing formation of the halogen bonding in 1-nh 3 ( part a ) and in 1-cph 3 ( part b ) . in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown . the numerically smallest contour values are 0.0005 a.u.table 1ets - energy decomposition ( in kcal mol ) characterizing the halogen bonded systems ( bp86-d / tz2p)systems e pauli e elstat e orb e disp e int 1-nh3 18.3 - 15.2 - 9.2 - 1.3 - 7.41-cph3 34.0 - 27.6 - 16.8 - 2.8 - 13.22-br22.8 - 20.3 - 19.6 - 2.8 - 19.92-h2 3.0 - 1.6 - 2.2 - 1.0 - 1.83-cl48.0 - 63.8 - 44.1 - 2.5 - 62.4 eint=epauli+eelstat+edisp+eorb the labeling corresponds to fig . 1 the contour of deformation density contribution 1 describing formation of the halogen bonding in 1-nh 3 ( part a ) and in 1-cph 3 ( part b ) . in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown . ets - energy decomposition ( in kcal mol ) characterizing the halogen bonded systems ( bp86-d / tz2p ) eint=epauli+eelstat+edisp+eorb the labeling corresponds to fig . 1 an analysis of the data presented in table 1 for 1-nh3 leads to the conclusion that the electrostatic contribution ( eelstat = -15.2 kcal mol ) is visibly more important than the orbital - interaction factor ( covalent + charge transfer , as discussed above , eorb = -9.2 kcal mol ) . thus , our ets - nocv result supports the role of electrostatic factor disclosed in the previous studies based on the other theoretical approaches ( electrostatic potential , symmetry adapted perturbation theory - based energy partitioning ) [ 69 , 57 , 58 ] . however , it should be emphasized that both , the electrostatic and orbital - interaction components are crucial for total energetic stabilization . the total interaction energy that includes all bonding contributions , is stabilizing : eint = -7.4 kcal mol . domination of the electrostatic component can be understood in terms of -hole concept that has been introduced by politzer and coworkers and extensively studied during recent years [ 59 ] . according to this concept halogen bonding is based on the electrostatic attraction between the lewis base and lewis acid parts ( -hole ) of the halogen atom [ 59 ] . such electron deficiency in the outer part of iodine atom in cf3i ( -hole ) is manifested by positive molecular electrostatic potential , as can be seen from fig . the contour of deformation density contribution 1 describing the formation of c i bond in cf3i molecule ( part b ) . the contour of deformation density contribution 1 describing the formation of c i bond in cf3i molecule ( part b ) . namely , whether we are able to visualize the formation of -hole using deformation density contributions originating from nocv . for this purpose we have analyzed the bonding between the iodium atom and the cf3 radical ( each carrying one unpaired electron with the opposite spin polarizations ) . 3b , the leading deformation density channel , 1 , with the corresponding energy \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^1 = { { { - 98.6kcal } } \left/ { { mol } } \right . } in addition , it is gratifying to see an outflow of electron density from the outer area of iodine atom , which clearly corresponds to the formation of -hole . it should further be noted that apart from the above components , the charge accumulation at iodine is observed due to formation of c i bond , which confirms significant charge anisotropy around this atom [ 414 ] . the presence of such a anisotropy is important for the reactivity , as the halogen atom can simultaneously act as electron donor and acceptor [ 414 ] . let us now briefly discuss the bonding of divalent carbon ( 0 ) ligand , c(ph3)2 , with cf3i moiety , in 1-cph3 , see fig . qualitatively , the deformation density channel , 1 , exhibits similar features as in the case of 1-nh3 . c contribution as well as the charge transfer from the lone electron pair of carbon to the empty *(c however , these transfers correspond to significantly higher stabilization , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^1 = { { { - 14.2kcal } } \left/ { { mol } } \right . } $ $ \end{document } , as compared to 1-nh3 ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^1 = { { { - 8.2kcal } } \left/ { { mol } } \right . } this is probably due to stronger overlap of orbitals involved in the halogen bonding interaction . divalent carbon ( 0 ) ligands appeared to be strong electron donors , which has been recently extensively studied by tonner and frenking . a significant overlap leading to notable electronic stabilization in 1-cph3 also causes the electrostatic contribution to be more pronounced when compared to 1-nh3 ( by 12.4 kcal mol ) , see table 1 . accordingly , the total interaction energy , eint , becomes more stabilizing , by 5.8 kcal mol for 1-cph3 . after we discussed the character of halogen bonding in simple molecules , let us consider now the bonding of isopropyl bromide to the model system of catalyst [ c6h4(c3h2n2i)2][otf]2 , see 2-br in fig . it has been proven experimentally that [ c6h4(c3h2n2i)2][otf]2 is able to break a strong carbon - bromide bond . authors of this work suggested that the activation of c br bond is induced by the formation of halogen bond between bromine and the iodine atoms , see fig . we have found indeed , based on dft / bp86-d / tz2p calculations , the minimum on the potential energy surface for the complex 2-br , where the bromine center forms two halogen connections with iodine atoms . energy decomposition method ( ets ) indicates that isopropyl bromide is strongly bonded to [ c6h4(c3h2n2i)2 ] moiety , i.e. , the total interaction energy is eint = -19.9 kcal mol . br bond becomes significantly elongated , by 0.08 ( as compared to non - bonded isopropyl bromide ) , due to the formation of two br interestingly , in this case the orbital - interaction contribution ( eorb = -19.6 kcal mol ) appeared to be of the same importance compared to the electrostatic stabilization ( eelstat = -20.3 kcal mol ) . it is an important result in light of the common view that the charge transfer ( electronic factor ) is a rather inferior factor in the halogen bonding [ 9 , 21 ] . that two deformation density channels , 1 and 2 , build up the two i br connections . ets - nocv allows to conclude that the one contribution is stronger ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^1 = { { { - 8.5kcal } } \left/ { { mol } } \right . } $ $ \end{document } ) than the other one ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^2 = { { { - 6.5kcal } } \left/ { { mol } } \right . } figure 4a shows that , similar to the previously considered examples , both halogen bonds contain the covalent i br components as well as the charge transfer contributions originating from the lone electron pair donation to the empty *(c further mechanistic and kinetic studies leading to the heterolytic c br bond cleavage are under way.fig . 4the contours of deformation density contributions describing the halogen bonding in 2-br ( part a ) and in 2-h 2 ( part b ) . in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown the contours of deformation density contributions describing the halogen bonding in 2-br ( part a ) and in 2-h 2 ( part b ) . in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown keeping in mind the above important experimental finding that a strongly electrophilic system can activate covalent bond , we decided to check whether the same system can form a chemical bond with chemically inert hydrogen molecule , h2 . to our partial surprise we found a stable complex of dihydrogen with [ c6h4(c3h2n2i)2 ] moiety , see 2-h2 in fig . 1 . ets energy decomposition method indicated that the hydrogen unit is weakly bonded to iodine atom , eint = -1.9 kcal mol , see table 1 . interestingly , neither the electrostatic term ( eelstat = -1.6 kcal mol ) , nor the dispersion interaction ( edisp = -1.0 kcal mol ) , are the leading contributions to eint . it appears that the electronic factor ( eorb = -2.2 kcal mol ) is the most important for the bonding in 2-h2 , see table 1 . deformation density channel originating from nocv s , 1 , presented in the right side of fig . 4b , shows that the electronic stabilization originates predominantly from the electron donation from the occupied (h - h ) orbital to the i we believe that this result is promising in terms of searching for new reactivity patterns of molecular hydrogen . it should be added that it has already been found that transition metal - based dihydride systems can form stable complexes with halogen - containing molecules . finally , we will end our study by a brief discussion of the binding of chloride anion to a recently developed system that contains two iodine atoms and two n h groups as electron acceptors , see 3-cl in fig . 1 . in line with expectation , the simultaneous presence of two hydrogen ( cl hn ) and two halogen ( cl i ) interactions , leads to very high total stabilization energy , eint = -62.4 kcal mol , see table 1 . it is the result of significant electrostatic ( eelstat = -63.8 kcal mol ) and electronic it is important to highlight that nocv deformation density contributions combined with ets scheme allowed to estimate separately the electronic strength of hydrogen ( cl hn ) , 2 , and halogen ( cl i ) interactions , 1 , see fig . i bonding , in terms of the orbital interaction component , is more than three times stronger ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^1 = { { { - 22.3kcal } } \left/ { { mol } } \right . } $ $ \end{document } ) than the hydrogen bond connection ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^1 = { { { - 6.3kcal } } \left/ { { mol } } \right . } $ $ \end{document } ) . such separated information on the strength of halogen and hydrogen connections within the same molecule can help to modulate the selectivity and accordingly lead to rational design of new acceptor molecules.fig . 5the contours of deformation density contributions describing the halogen ( 1 ) and hydrogen bonding ( 1 ) in 3-cl . in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown . the contours of deformation density contributions describing the halogen ( 1 ) and hydrogen bonding ( 1 ) in 3-cl . in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown . in the present study we have characterized the halogen bonding in selected molecules , 1-nh3 , 1-cph3 , 2-br , 2-h2 and 3-cl , based on the recently proposed ets - nocv procedure . we have chosen the examples containing from one ( 1-nh3 , 1-cph3 ) up to four bonding connections ( 3-cl ) . in the ets energy decomposition scheme , the interaction energy between the fragments is divided into three well defined components : electrostatic , pauli repulsion , and the orbital interaction . however , it should be emphasized that any energy partitioning method includes some arbitrariness due to the fact that the contributions to the total interaction energy are not physical observables . nevertheless , the results of the present analysis , in agreement with previous studies [ 69 , 57 , 58 ] demonstrate the indisputable role of the electrostatic stabilization in halogen bonding . we have found based on the nocv - deformation density contours ( i ) that in each analyzed system the halogen bonding c xb ( x - halogen atom , b - lewis base ) , contains a large degree of covalent contribution ( charge transfer to x b inter - atomic region ) supported further by the electron donation from base atom b to empty *(c - x ) orbital . it should be emphasized that ets - nocv approach allowed to clearly visualize the formation of -hole at iodine atom of cf3i molecule . thus , the nocv - analysis confirms the vital importance of the presence of -hole for halogen bonding [ 69 ] ; an electron - density deficiency at outer part of halogen atom ( x ) along c x bond leads to both effects , electrostatic stabilization of the c xb interaction , but as well to a charge flow from a base to electron - deficient region . an importance of the observed electron - density displacements is further demonstrated by the ets orbital - interaction energy , eorb , dominated in each case by one contribution eorb corresponding to main charge transfer channel 1 . finally , in all of the analyzed systems the dispersion interaction appeared to be less important for halogen bonding . due to the fact that in each case we noted a large degree of covalency of halogen bond , we believe that the term non - covalent interaction , frequently used in literature , is not fully appropriate , at least for the systems studied in the present work . these results suggest further that in addition to the dominant electrostatic component , depending on the system , other terms can be relatively important . this observation supports the new set of criteria proposed recently by legon for definition of halogen bonding . we have also demonstrated that strongly electrophilic species , [ c6h4(c3h2n2i)2][otf]2 , ( 2 ) , can activate chemically inert molecules via formation of halogen bonding , e.g. , isopropyl bromide ( eint = -19.9 kcal mol ) . interestingly , it has also been noted that ( 2 ) can bind the hydrogen molecule , 2-h2 , eint = -1.8 kcal mol , which seems to be a promising result in terms of future findings of new reactivity patterns of hydrogen molecule . finally , ets - nocv approach allowed to qualitatively and quantitatively characterize separately the halogen ( cl i ) and hydrogen bonds ( cl hn ) within the same acceptor molecule ( 3-cl ) .

in the present study we have characterized the halogen bonding in selected molecules h3n icf3 ( 1-nh3 ) , ( ph3)2c icf3 ( 1-cph3 ) , c3h7br(in2h2c3)2c6h4 ( 2-br ) , h2(in2h2c3)2c6h4 ( 2-h2 ) and cl(ic6f5)2c7h10n2o5 ( 3-cl ) , containing from one halogen bond ( 1-nh3 , 1-cph3 ) up to four connections in 3-cl ( the two cl hn and two cl i ) , based on recently proposed ets - nocv analysis . it was found based on the nocv - deformation density components that the halogen bonding c x b ( x - halogen atom , b - lewis base ) , contains a large degree of covalent contribution ( the charge transfer to x b inter - atomic region ) supported further by the electron donation from base atom b to the empty *(c x ) orbital . such charge transfers can be of similar importance compared to the electrostatic stabilization . further , the covalent part of halogen bonding is due to the presence of -hole at outer part of halogen atom ( x ) . ets - nocv approach allowed to visualize formation of the -hole at iodine atom of cf3i molecule . it has also been demonstrated that strongly electrophilic halogen bond donor , [ c6h4(c3h2n2i)2][otf]2 , can activate chemically inert isopropyl bromide ( 2-br ) moiety via formation of br i bonding and bind the hydrogen molecule ( 2-h2 ) . finally , ets - nocv analysis performed for 3-cl leads to the conclusion that , in terms of the orbital - interaction component , the strength of halogen ( cl i ) bond is roughly three times more important than the hydrogen bonding ( cl hn).figureets - nocv reprezentation of -hole at iodine together with the molecular electrostatic potential picture

Introduction Computational details Computational methods Results and discussion Conclusions

recently , the halogen bonding , a xb ( x halogen atom , b lewis base , a electronegative atom ) , attracted considerable attention due to its strong , selective and directional character [ 414 ] . the pioneering experimental studies reporting on the existence of halogen bonding dates back to the mid nineteenth century [ 1519 ] . the first theoretical analysis of the resonance structures has been proposed by mulliken to explain the molecular spectra of halogen bonded complexes . in his nobel lecture hassel highlighted that the electrophilic part of halogen atom can be very crucial in molecular self - assembly phenomena . the authors noticed for the first time , based on the molecular electrostatic potential , that there exists an electron deficiency at the outer part of the halogen atom , so called -hole , what leads in turn to the electrostatic attraction with lewis bases . hence , the halogen bonding is driven mainly by the electrostatic term [ 69 ] . in the present study we will characterize the halogen bonding in selected molecules in terms of quantitative role of the electronic ( the charge transfer and covalency ) and electrostatic factors . the recently developed ets - nocv scheme will be used , that originates from a combination of the extended transition state ( ets ) energy decomposition approach with the natural orbitals for chemical valence ( nocv ) analysis [ 2329 ] . ets - nocv approach will also be used for the first time to describe the -hole at iodine atom of cf3i , based on deformation density contributions obtained from the nocv analysis of the bond between the cf3 and i fragments . in the next step , we will focus our attention on the catalytic system [ c6h4(c3h2n2i)2][otf]2 ( 2 in fig . it has been proven experimentally that this halogen bond donor is a very strong electrophilic species that is able to break covalent c br bond in benzhydryl bromide ( ph2hc br ) . accordingly , as the second objective of our study we will perform pioneering theoretical analysis of the interaction between isopropyl bromide ( the methyl groups were used instead of phenyl rings ) and the model [ c6h4(c3h2n2i)2 ] system ( the counter anion otf has been omitted in the calculations ) , see 2-br in fig . further , we will demonstrate that very high electrophilicity of 2 can lead to the formation of dihydrogen complex ( 2-h2 in fig . finally , the halogen ( cl i ) and hydrogen bonds ( cl hn ) co - existing within the same molecule ( 3-cl in fig . all the dft calculations presented here are based on the amsterdam density functional ( adf2009 ) program in which ets - nocv scheme was implemented [ 4145 ] . bonding analysis presented in this study is based on the ets - nocv approach which is a combination of the extended transition state ( ets ) method with the natural orbitals for chemical valence ( nocv ) scheme [ 2329 ] . historically , the natural orbitals for chemical valence ( nocv ) have been derived from the nalewajski - mrozek valence theory [ 5056 ] as eigenvectors of the deformation density matrix . it was shown [ 24 , 26 ] that the natural orbitals for chemical valence pairs ( -k,,k ) decompose the differential density into nocv - contributions ( k):1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta \rho ( r ) = \sum\limits_{{k = 1}}^{{{{m } \left/ { 2 } \right . } visual inspection of deformation density plots ( k ) helps to attribute symmetry and the direction of the charge flow . in addition , within ets - nocv scheme , the deformation - density based picture is enriched by the energetic estimations , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^k $ $ \end{document } , for each k . in the combined ets - nocv scheme the orbital interaction term ( eorb ) is expressed in terms of nocv s eigenvalues ( vk ) as:3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta { e_{{orb } } } = \sum\limits_k { \delta e_{{orb}}^k = } \sum\limits_{{k = 1}}^{{{{m } \left/ { 2 } \right . } we will start our discussion from the nocv s based description of halogen bonding in 1-nh3 . 2a that the leading deformation density channel , 1 , with the corresponding electronic stabilization \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^1 = { { { - 8.2kcal } } \left/ { { mol } } \right . } $ $ \end{document } , constitutes the halogen bonding between ammonia and cf3i molecule . qualitatively , 1 is based on the donation from the lone electron pair of nitrogen to the empty *(i - c ) . it is important to note that of similar importance is the covalent contribution that originates from the electron transfer from both the nitrogen and iodine atoms to the i it is noteworthy that , due to formation of halogen bond , the accumulation of electron density is also observed at the carbon atom of cf3i , which is in line with an increase in s character of carbon ; this issue has been deeply analyzed recently by grabowski using nbo method . 2the contour of deformation density contribution 1 describing formation of the halogen bonding in 1-nh 3 ( part a ) and in 1-cph 3 ( part b ) . 1 the contour of deformation density contribution 1 describing formation of the halogen bonding in 1-nh 3 ( part a ) and in 1-cph 3 ( part b ) . 1 an analysis of the data presented in table 1 for 1-nh3 leads to the conclusion that the electrostatic contribution ( eelstat = -15.2 kcal mol ) is visibly more important than the orbital - interaction factor ( covalent + charge transfer , as discussed above , eorb = -9.2 kcal mol ) . thus , our ets - nocv result supports the role of electrostatic factor disclosed in the previous studies based on the other theoretical approaches ( electrostatic potential , symmetry adapted perturbation theory - based energy partitioning ) [ 69 , 57 , 58 ] . however , it should be emphasized that both , the electrostatic and orbital - interaction components are crucial for total energetic stabilization . domination of the electrostatic component can be understood in terms of -hole concept that has been introduced by politzer and coworkers and extensively studied during recent years [ 59 ] . according to this concept halogen bonding is based on the electrostatic attraction between the lewis base and lewis acid parts ( -hole ) of the halogen atom [ 59 ] . such electron deficiency in the outer part of iodine atom in cf3i ( -hole ) is manifested by positive molecular electrostatic potential , as can be seen from fig . the contour of deformation density contribution 1 describing the formation of c i bond in cf3i molecule ( part b ) . the contour of deformation density contribution 1 describing the formation of c i bond in cf3i molecule ( part b ) . namely , whether we are able to visualize the formation of -hole using deformation density contributions originating from nocv . in addition , it is gratifying to see an outflow of electron density from the outer area of iodine atom , which clearly corresponds to the formation of -hole . it should further be noted that apart from the above components , the charge accumulation at iodine is observed due to formation of c i bond , which confirms significant charge anisotropy around this atom [ 414 ] . the presence of such a anisotropy is important for the reactivity , as the halogen atom can simultaneously act as electron donor and acceptor [ 414 ] . c contribution as well as the charge transfer from the lone electron pair of carbon to the empty *(c however , these transfers correspond to significantly higher stabilization , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^1 = { { { - 14.2kcal } } \left/ { { mol } } \right . } this is probably due to stronger overlap of orbitals involved in the halogen bonding interaction . after we discussed the character of halogen bonding in simple molecules , let us consider now the bonding of isopropyl bromide to the model system of catalyst [ c6h4(c3h2n2i)2][otf]2 , see 2-br in fig . authors of this work suggested that the activation of c br bond is induced by the formation of halogen bond between bromine and the iodine atoms , see fig . we have found indeed , based on dft / bp86-d / tz2p calculations , the minimum on the potential energy surface for the complex 2-br , where the bromine center forms two halogen connections with iodine atoms . br bond becomes significantly elongated , by 0.08 ( as compared to non - bonded isopropyl bromide ) , due to the formation of two br interestingly , in this case the orbital - interaction contribution ( eorb = -19.6 kcal mol ) appeared to be of the same importance compared to the electrostatic stabilization ( eelstat = -20.3 kcal mol ) . it is an important result in light of the common view that the charge transfer ( electronic factor ) is a rather inferior factor in the halogen bonding [ 9 , 21 ] . figure 4a shows that , similar to the previously considered examples , both halogen bonds contain the covalent i br components as well as the charge transfer contributions originating from the lone electron pair donation to the empty *(c further mechanistic and kinetic studies leading to the heterolytic c br bond cleavage are under way.fig . 4the contours of deformation density contributions describing the halogen bonding in 2-br ( part a ) and in 2-h 2 ( part b ) . in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown the contours of deformation density contributions describing the halogen bonding in 2-br ( part a ) and in 2-h 2 ( part b ) . in addition the corresponding ets - nocv - based energies ( in kcal mol ) are shown keeping in mind the above important experimental finding that a strongly electrophilic system can activate covalent bond , we decided to check whether the same system can form a chemical bond with chemically inert hydrogen molecule , h2 . ets energy decomposition method indicated that the hydrogen unit is weakly bonded to iodine atom , eint = -1.9 kcal mol , see table 1 . 4b , shows that the electronic stabilization originates predominantly from the electron donation from the occupied (h - h ) orbital to the i we believe that this result is promising in terms of searching for new reactivity patterns of molecular hydrogen . finally , we will end our study by a brief discussion of the binding of chloride anion to a recently developed system that contains two iodine atoms and two n h groups as electron acceptors , see 3-cl in fig . in line with expectation , the simultaneous presence of two hydrogen ( cl hn ) and two halogen ( cl i ) interactions , leads to very high total stabilization energy , eint = -62.4 kcal mol , see table 1 . it is the result of significant electrostatic ( eelstat = -63.8 kcal mol ) and electronic it is important to highlight that nocv deformation density contributions combined with ets scheme allowed to estimate separately the electronic strength of hydrogen ( cl hn ) , 2 , and halogen ( cl i ) interactions , 1 , see fig . i bonding , in terms of the orbital interaction component , is more than three times stronger ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta e_{{orb}}^1 = { { { - 22.3kcal } } \left/ { { mol } } \right . } such separated information on the strength of halogen and hydrogen connections within the same molecule can help to modulate the selectivity and accordingly lead to rational design of new acceptor molecules.fig . 5the contours of deformation density contributions describing the halogen ( 1 ) and hydrogen bonding ( 1 ) in 3-cl . the contours of deformation density contributions describing the halogen ( 1 ) and hydrogen bonding ( 1 ) in 3-cl . in the present study we have characterized the halogen bonding in selected molecules , 1-nh3 , 1-cph3 , 2-br , 2-h2 and 3-cl , based on the recently proposed ets - nocv procedure . we have chosen the examples containing from one ( 1-nh3 , 1-cph3 ) up to four bonding connections ( 3-cl ) . however , it should be emphasized that any energy partitioning method includes some arbitrariness due to the fact that the contributions to the total interaction energy are not physical observables . nevertheless , the results of the present analysis , in agreement with previous studies [ 69 , 57 , 58 ] demonstrate the indisputable role of the electrostatic stabilization in halogen bonding . we have found based on the nocv - deformation density contours ( i ) that in each analyzed system the halogen bonding c xb ( x - halogen atom , b - lewis base ) , contains a large degree of covalent contribution ( charge transfer to x b inter - atomic region ) supported further by the electron donation from base atom b to empty *(c - x ) orbital . it should be emphasized that ets - nocv approach allowed to clearly visualize the formation of -hole at iodine atom of cf3i molecule . thus , the nocv - analysis confirms the vital importance of the presence of -hole for halogen bonding [ 69 ] ; an electron - density deficiency at outer part of halogen atom ( x ) along c x bond leads to both effects , electrostatic stabilization of the c xb interaction , but as well to a charge flow from a base to electron - deficient region . an importance of the observed electron - density displacements is further demonstrated by the ets orbital - interaction energy , eorb , dominated in each case by one contribution eorb corresponding to main charge transfer channel 1 . finally , in all of the analyzed systems the dispersion interaction appeared to be less important for halogen bonding . due to the fact that in each case we noted a large degree of covalency of halogen bond , we believe that the term non - covalent interaction , frequently used in literature , is not fully appropriate , at least for the systems studied in the present work . these results suggest further that in addition to the dominant electrostatic component , depending on the system , other terms can be relatively important . we have also demonstrated that strongly electrophilic species , [ c6h4(c3h2n2i)2][otf]2 , ( 2 ) , can activate chemically inert molecules via formation of halogen bonding , e.g. interestingly , it has also been noted that ( 2 ) can bind the hydrogen molecule , 2-h2 , eint = -1.8 kcal mol , which seems to be a promising result in terms of future findings of new reactivity patterns of hydrogen molecule . finally , ets - nocv approach allowed to qualitatively and quantitatively characterize separately the halogen ( cl i ) and hydrogen bonds ( cl hn ) within the same acceptor molecule ( 3-cl ) .

procalcitonin ( pct ) is widely used as a marker before final blood culture ( bc ) confirmation in clinical diagnosis of bacteremia and sepsis , with diagnostic sensitivity 74.8100.0% , specificity 70.0100.0% , positive predictive value 55.0100.0% , and negative predictive value 56.3100.0% , and pct in combination with careful clinical parameters can discriminate infection caused bacteremia from inflammatory sepsis in 77% of cases . however , pct alone still has some limitations , especially lack of definitive cut - off in the indeterminate zone . for example , pct can not reliably differentiate sepsis from other noninfectious causes of systemic inflammatory response syndrome in critically ill patients and is of no use in determining new fever caused by bacteremia in the intensive care unit ( icu ) patients . meanwhile , although the prognostic value of endotoxin level for bacteremia and sepsis remains disparate , less than two - thirds of patients with gram - negative ( gn ) bacteremia are detected with endotoxemia and vice versa . endotoxemia broadly parallels the frequency and importance of gn patient sepsis and is only not detected in > 20% gn bacteraemic patients among the 58 studies overall or > 20% studies of patients with sepsis syndrome . while the association of endotoxemia with bacteremia is bacterial type dependent , endotoxemia is more commonly detected in patients with bacteremia caused by non - enterobacteriaceae than a commensal member of enterobacteriaceae . therefore , both of the markers have some advantages and disadvantages and can not be solely used for a definite diagnosis of bacteremia or sepsis . furthermore , there has been few study comparing the values of the two biomarkers for the diagnosis of bacteremia within the same patient cohort . the aim of this retrospective study was to compare the role of the two biomarkers in the diagnosis of bacteremia in icus of a chinese hospital . the study data were retrospectively collected from 420 patients with consecutive admissions to the emergency and icus of changzheng hospital in shanghai , china from january 1 , 2010 to december 31 , 2012 . the ethics committee of changzheng hospital in shanghai , china approved this study and patient consents were waived . all patients subjected to bc tests for the analysis of the endotoxin or pct levels during icu stay were enrolled in this study . the exclusion criteria included the presence of infection with human immunodeficiency virus and/or aids , neutropenia without sepsis , pregnancy , treatment with immunosuppressive therapies , or blood diseases such as hematological tumors . the relevant patient demographics including age , gender , comorbidities , infection sites , microbial isolates , and major laboratory test results were recorded at baseline . sepsis , severe sepsis , and septic shock were defined according to the internationally accepted criteria . the disease severity in each patient was assessed upon admission using two different scores : the acute physiology and chronic health evaluation ( apache ) ii score and the sequential organ failure assessments ( sofas ) score . the blood samples were collected through venous puncture using the bactec system ( becton dickinson diagnostic instrument systems , sparks , md , usa ) based on both standard aerobic and anaerobic media coupled with the 9240 automated bc system ( becton dickinson diagnostic instrument system , paramus , nj , usa ) . one episode of bacteraemia was defined as the recovery of any bacterial species in one or more bcs . patients from whom staphylococcus non - aureus was isolated in bcs were not eligible , except when at least two consecutive samples were grown for the same species harboring the same antibiotic resistance patterns . thus , all episodes of bc can be divided into four groups according to bc : bc ( no isolates ) , g ( gn bacteria ) , g ( gram - positive bacteria ) , and fungi ( fungi ) . notably , the pct and endotoxin levels of the mixed cultures with one or more isolates were not compared with the four groups described above . furthermore , g was separated into enterobacteriaceae and non - enterobacteriaceae groups according to the previously reported standards . the group of commensal enterobacteriaceae , predominantly comprise escherichia coli , klebsiella pneumoniae , serratia marcescens , proteus mirabilis , and providencia rettgeri . the group of non - enterobacteriaceae in the present analysis included acinetobacter baumannii , burkholderia cepacia , and pseudomonas aeruginosa . the pct levels were measured using an immunoluminometric assay ( lumitest pct kit , brahms diagnostica , hennigsdorf bei berlin , germany ) . the chemiluminescence was measured using a luminometer ( lumat , lb 9507 , berthold , wildbad , germany ) . the endotoxin concentration was assayed using a limulus test involving a turbidimetric time assay ( zhanjiang a & c biological ltd . , china ) at 450 nm with toxinometer bet-16 ( tianda tianfa technology co. , ltd . , tianjin , china ) at 37.8c . to measure the endotoxin levels in plasma , 1 ml whole blood in 1 ml of anticoagulants and eluent r / min for 10 min ; plasma ( 0.2 ml ) was added to 0.8 ml diluent and heated at 75c for 10 min . an aliquot ( 0.1 ml ) of stock solution processed above was added to 0.1 ml limulus amebocyte lysate ( lal ) reagent , and the kinetic turbidity of the mixture was measured using a tube reader ( zhanjiang a and c biological ltd . , the data are presented as the mean standard deviation , median and interquartile ranges ( 25 and 75 percentiles ) or numbers and percentages . the categorical variables were compared using chi - square or fisher 's exact tests , where appropriate . the differences in the parametric data between different strata were calculated using student 's t - test and analysis of variance ( anova ) with post - hoc lsd test for the two groups . to compare the nonparametric data , the mann wallis test with post - hoc mann whitney u - test was performed for multiple comparisons . spearman 's rank correlation or pearson 's tests were performed to evaluate the association between the two groups , where appropriate . the data were analyzed using the receiver operating characteristic ( roc ) curve for the plasma endotoxin and pct levels for the prediction of gn bacteremia . the study data were retrospectively collected from 420 patients with consecutive admissions to the emergency and icus of changzheng hospital in shanghai , china from january 1 , 2010 to december 31 , 2012 . the ethics committee of changzheng hospital in shanghai , china approved this study and patient consents were waived . all patients subjected to bc tests for the analysis of the endotoxin or pct levels during icu stay were enrolled in this study . the exclusion criteria included the presence of infection with human immunodeficiency virus and/or aids , neutropenia without sepsis , pregnancy , treatment with immunosuppressive therapies , or blood diseases such as hematological tumors . the relevant patient demographics including age , gender , comorbidities , infection sites , microbial isolates , and major laboratory test results were recorded at baseline . sepsis , severe sepsis , and septic shock were defined according to the internationally accepted criteria . the disease severity in each patient was assessed upon admission using two different scores : the acute physiology and chronic health evaluation ( apache ) ii score and the sequential organ failure assessments ( sofas ) score . the blood samples were collected through venous puncture using the bactec system ( becton dickinson diagnostic instrument systems , sparks , md , usa ) based on both standard aerobic and anaerobic media coupled with the 9240 automated bc system ( becton dickinson diagnostic instrument system , paramus , nj , usa ) . one episode of bacteraemia was defined as the recovery of any bacterial species in one or more bcs . patients from whom staphylococcus non - aureus was isolated in bcs were not eligible , except when at least two consecutive samples were grown for the same species harboring the same antibiotic resistance patterns . thus , all episodes of bc can be divided into four groups according to bc : bc ( no isolates ) , g ( gn bacteria ) , g ( gram - positive bacteria ) , and fungi ( fungi ) . notably , the pct and endotoxin levels of the mixed cultures with one or more isolates were not compared with the four groups described above . furthermore , g was separated into enterobacteriaceae and non - enterobacteriaceae groups according to the previously reported standards . the group of commensal enterobacteriaceae , predominantly comprise escherichia coli , klebsiella pneumoniae , serratia marcescens , proteus mirabilis , and providencia rettgeri . the group of non - enterobacteriaceae in the present analysis included acinetobacter baumannii , burkholderia cepacia , and pseudomonas aeruginosa . the pct levels were measured using an immunoluminometric assay ( lumitest pct kit , brahms diagnostica , hennigsdorf bei berlin , germany ) . the chemiluminescence was measured using a luminometer ( lumat , lb 9507 , berthold , wildbad , germany ) . the endotoxin concentration was assayed using a limulus test involving a turbidimetric time assay ( zhanjiang a & c biological ltd . , china ) at 450 nm with toxinometer bet-16 ( tianda tianfa technology co. , ltd . , tianjin , china ) at 37.8c . to measure the endotoxin levels in plasma , 1 ml whole blood in 1 ml of anticoagulants and eluent r / min for 10 min ; plasma ( 0.2 ml ) was added to 0.8 ml diluent and heated at 75c for 10 min . an aliquot ( 0.1 ml ) of stock solution processed above was added to 0.1 ml limulus amebocyte lysate ( lal ) reagent , and the kinetic turbidity of the mixture was measured using a tube reader ( zhanjiang a and c biological ltd . , data analysis was conducted using spss 18 software ( spss inc . , il , usa ) . the data are presented as the mean standard deviation , median and interquartile ranges ( 25 and 75 percentiles ) or numbers and percentages . the categorical variables were compared using chi - square or fisher 's exact tests , where appropriate . the differences in the parametric data between different strata were calculated using student 's t - test and analysis of variance ( anova ) with post - hoc lsd test for the two groups . to compare the nonparametric data , the mann wallis test with post - hoc mann whitney u - test was performed for multiple comparisons . spearman 's rank correlation or pearson 's tests were performed to evaluate the association between the two groups , where appropriate . the data were analyzed using the receiver operating characteristic ( roc ) curve for the plasma endotoxin and pct levels for the prediction of gn bacteremia . a total of 420 patients admitted to the icu were screened for the study ; 179 patients did not meet the inclusion criteria and were excluded , and 241 patients met the inclusion criteria ( male , 68.0% ) , of which 71 ( 29.5% ) patients had bacteremia . the primary reasons for infections were pneumonia and abdominal infections by predominant gn bacteria conformed from bc . the mortality rate for all patients was 24.5% , with a higher mortality rate for patients with severe sepsis ( 29.2% ) or septic shock ( 62.2% ) than sepsis ( 9.8% ) or nonsepsis ( 12.6% ) . the mean age , apache ii , sofa , and cci scores of the nonsurviving patients were significantly higher than those of the surviving patients . sex , infection sites , and the presence of enterobacteriaceae infection did not affect the mortality of the patients . a summary of the patient demographics and clinical parameters of the study population are listed in table 1 . patient characteristics based on the diagnosis at admission * pct and endotoxin levels from first - time bc . apache ii : the acute physiology and chronic health evaluation ii score ; sofas : the sequential organ failure assessments score ; cci : the charlson comorbidity index ; bc : blood culture ; g : gram - negative bacteria ; g : grampositive bacteria ; pct : procalcitonin ; sd : standard deviation . among 505 isolated samples , 92 ( 18.2% ) of the 92 isolates , a total of 69 ( 75.0% ) isolates were gn microorganisms , including 13 b. cepacia , 11 k. pneumoniae , 10 acinetobacter baumannii , six e. coli , and five enterobacter cloacae . whereas , only 13 ( 14.1% ) isolates were gram - positive microorganisms , of which , a total of 6 ( 6.5% ) isolates were fungi , with 4 isolates being candida albicans . more than one microorganism was found in 4 ( 4.3% ) episodes after the bc , with b. cepacia being present in two episodes of bc . microorganisms isolated from blood culture ( n = 92 ) k. pneumonia : klebsiella pneumonia ; s. marcescens : serratia marcescens ; e. coli : escherichia coli ; e. cloacae : enterobacter cloacae ; k. oxytoca : klebsiella oxytoca ; s. enteritidis : salmonella enteritidis ; b. cepacia : burkholderia cepacia ; a. baumannii : acinetobacter baumannii ; p. aeruginosa : pseudomonas aeruginosa ; a. junii : acinetobacter junii ; p. paucimobilis : pseudomonas paucimobilis ; b. diminuta : brevundimonas diminuta ; s. paucimobilis : sphingomonas paucimobilis ; c. meningosepticum : chryseobacterium meningosepticum ; a. xylosoxidans : achromobacter xylosoxidans ; h. influenza : haemophilus influenza ; s. capitis : staphylococcus capitis ; s. hemolyticus : staphylococcus hemolyticus ; s. huminis : staphylococcus huminis ; s. epidermidis : staphylococcus epidermidis ; s. aureus : staphylococcus aureus ; k. roseus : kocuria roseus ; e. faecalis : enterococcus faecalis ; g. bergeri : gemella bergeri ; c. albicans : candida albicans ; c. glabrata : candida glabrata ; c. haemulonii : candida haemulonii ; s. lentus : staphylococcus lentus ; mrsa : methicillin - resistant s. aureus . the pct concentration and endotoxin level significantly differed among the four groups g , g , fungi , and bc ( p < 0.000 , pct ; and p = 0.0244 , endotoxin ) . the pct level was significantly higher in the g group than in the bc ( p < 0.0001 ) and g ( p = 0.0484 ) groups ; patients with fungi isolates also had a higher level of pct than patients with bc ( p = 0.0244 ) . the plasma endotoxin level in the g group was significantly higher than in the bc group ( p = 0.0025 ) , and no significant difference was found between g group and the bc ( or g ) group [ figure 1 ] . comparison of the procalcitonin and plasma endotoxin levels in different groups according to the blood culture results . p < 0.05 : * compared with g ; compared with bc. bc : blood culture negative group ; g : gram - negative bacteria ; g : gram - positive bacteria . patients with non - enterobacteriaceae isolates showed a significantly higher plasma endotoxin level than patients with enterobacteriaceae ( p = 0.0276 ) ; the pct level did not differ significantly between the above two patient groups ( p = 0.2964 ) [ figure 2 ] . it was significantly different among all the different groups in the species level for endotoxin ( p = 0.0446 ) , not pct ( p = 0.5529 ) [ figure 1 ] . the endotoxin level of the patients with b. cepacia infection was significantly higher than that with s. marcescens ( p = 0.0236 ) , k. pneumoniae ( p = 0.0048 ) , and e. cloacae ( p = 0.0180 ) ; the difference between e. cloacae and p. aeruginosa in the endotoxin level was also nearly significantly different ( p = 0.0519 ) . the pct level in the patients with e. cloacae infection was significantly ( p = 0.0319 ) and almost ( p = 0.0893 ) higher than those with s. marcescens and a. baumannii , respectively [ figure 3 ] . comparison of the procalcitonin and plasma endotoxin levels in different groups according to bacterial species . the area under the roc curves for the pct and endotoxin levels used to predict gn isolates of bc were 0.741 ( 95% confidence interval [ ci ] : 0.6830.779 , p < 0.001 ) and 0.614 ( 95% ci : 0.5500.678 , p = 0.002 ) , respectively . a total of 420 patients admitted to the icu were screened for the study ; 179 patients did not meet the inclusion criteria and were excluded , and 241 patients met the inclusion criteria ( male , 68.0% ) , of which 71 ( 29.5% ) patients had bacteremia . the primary reasons for infections were pneumonia and abdominal infections by predominant gn bacteria conformed from bc . the mortality rate for all patients was 24.5% , with a higher mortality rate for patients with severe sepsis ( 29.2% ) or septic shock ( 62.2% ) than sepsis ( 9.8% ) or nonsepsis ( 12.6% ) . the mean age , apache ii , sofa , and cci scores of the nonsurviving patients were significantly higher than those of the surviving patients . sex , infection sites , and the presence of enterobacteriaceae infection did not affect the mortality of the patients . a summary of the patient demographics and clinical parameters of the study population are listed in table 1 . patient characteristics based on the diagnosis at admission * pct and endotoxin levels from first - time bc . apache ii : the acute physiology and chronic health evaluation ii score ; sofas : the sequential organ failure assessments score ; cci : the charlson comorbidity index ; bc : blood culture ; g : gram - negative bacteria ; g : grampositive bacteria ; pct : procalcitonin ; sd : standard deviation . among 505 isolated samples , 92 ( 18.2% ) isolates were positive for bc . of the 92 isolates , a total of 69 ( 75.0% ) isolates were gn microorganisms , including 13 b. cepacia , 11 k. pneumoniae , 10 acinetobacter baumannii , six e. coli , and five enterobacter cloacae . whereas , only 13 ( 14.1% ) isolates were gram - positive microorganisms , of which , a total of 6 ( 6.5% ) isolates were fungi , with 4 isolates being candida albicans . more than one microorganism was found in 4 ( 4.3% ) episodes after the bc , with b. cepacia being present in two episodes of bc . microorganisms isolated from blood culture ( n = 92 ) k. pneumonia : klebsiella pneumonia ; s. marcescens : serratia marcescens ; e. coli : escherichia coli ; e. cloacae : enterobacter cloacae ; k. oxytoca : klebsiella oxytoca ; s. enteritidis : salmonella enteritidis ; b. cepacia : burkholderia cepacia ; a. baumannii : acinetobacter baumannii ; p. aeruginosa : pseudomonas aeruginosa ; a. junii : acinetobacter junii ; p. paucimobilis : pseudomonas paucimobilis ; b. diminuta : brevundimonas diminuta ; s. paucimobilis : sphingomonas paucimobilis ; c. meningosepticum : chryseobacterium meningosepticum ; a. xylosoxidans : achromobacter xylosoxidans ; h. influenza : haemophilus influenza ; s. capitis : staphylococcus capitis ; s. hemolyticus : staphylococcus hemolyticus ; s. huminis : staphylococcus huminis ; s. epidermidis : staphylococcus epidermidis ; s. aureus : staphylococcus aureus ; k. roseus : kocuria roseus ; e. faecalis : enterococcus faecalis ; g. bergeri : gemella bergeri ; c. albicans : candida albicans ; c. glabrata : candida glabrata ; c. haemulonii : candida haemulonii ; s. lentus : staphylococcus lentus ; mrsa : methicillin - resistant s. aureus . the pct concentration and endotoxin level significantly differed among the four groups g , g , fungi , and bc ( p < 0.000 , pct ; and p = 0.0244 , endotoxin ) . the pct level was significantly higher in the g group than in the bc ( p < 0.0001 ) and g ( p = 0.0484 ) groups ; patients with fungi isolates also had a higher level of pct than patients with bc ( p = 0.0244 ) . the plasma endotoxin level in the g group was significantly higher than in the bc group ( p = 0.0025 ) , and no significant difference was found between g group and the bc ( or g ) group [ figure 1 ] . comparison of the procalcitonin and plasma endotoxin levels in different groups according to the blood culture results . p < 0.05 : * compared with g ; compared with bc. bc : blood culture negative group ; g : gram - negative bacteria ; g : gram - positive bacteria . patients with non - enterobacteriaceae isolates showed a significantly higher plasma endotoxin level than patients with enterobacteriaceae ( p = 0.0276 ) ; the pct level did not differ significantly between the above two patient groups ( p = 0.2964 ) [ figure 2 ] . it was significantly different among all the different groups in the species level for endotoxin ( p = 0.0446 ) , not pct ( p = 0.5529 ) [ figure 1 ] . the endotoxin level of the patients with b. cepacia infection was significantly higher than that with s. marcescens ( p = 0.0236 ) , k. pneumoniae ( p = 0.0048 ) , and e. cloacae ( p = 0.0180 ) ; the difference between e. cloacae and p. aeruginosa in the endotoxin level was also nearly significantly different ( p = 0.0519 ) . the pct level in the patients with e. cloacae infection was significantly ( p = 0.0319 ) and almost ( p = 0.0893 ) higher than those with s. marcescens and a. baumannii , respectively [ figure 3 ] . comparison of the procalcitonin and plasma endotoxin levels in different groups according to bacterial species . the area under the roc curves for the pct and endotoxin levels used to predict gn isolates of bc were 0.741 ( 95% confidence interval [ ci ] : 0.6830.779 , p < 0.001 ) and 0.614 ( 95% ci : 0.5500.678 , p = 0.002 ) , respectively . in this retrospective study , to investigate whether pct or endotoxin level in the blood is more valuable for diagnosis of bacteremia , we evaluated the association of the pct or plasma endotoxin level of different types of bacteria in the icu patients with sepsis , severe sepsis , or septic shock . we found that : ( 1 ) gn bacteria were predominant within the microorganisms found in the bcs ; ( 2 ) the level of pct is more closely associated with gn bacteremia than that of endotoxin ; ( 3 ) gn bacteremia exhibited a higher level of endotoxin than nonbacteremia ; and ( 4 ) bacteremia with non - enterobacteriaceae had a significantly higher level of endotoxin than bacteremia with enterobacteriaceae . in this study , we observed that age , comorbidities , severities at admission and status of bacteremia differ significantly between survivors and nonsurvivors , and sex difference or infection sites not affect mortality . gn bacteria were predominant within the microorganisms found in the bcs , and the major gram - positive bacteria were from coagulase - negative staphylococci . in some countries , gram - positive bacteria may have a high percentage of microorganisms found in the bcs . the difference in the predominant bacterial type may result from geographic variation , case mix , and antibiotic prescription habits . of note , in this study , b. cepacia was found to have the highest positive rate among all the microorganism species . however , b. cepacia was not found in patients with severe sepsis or septic shock in 22 icus across the mainland of china , and the reason was unknown . it was consistent with previous reports that patients with bacteremia have a high mortality rate than patients without isolates from bcs and especially high for patients with fungi isolates . there was no significant difference between the mortality rates caused by gn bacteremia with enterobacteriaceae and non - enterobacteriaceae . however , a previous study had shown that mortality was higher in a. baumannii ( one of non - enterobacteriaceae ) bacteremia , particularly compared with k. pneumoniae bacteremia ( one of enterobacteriaceae ) . we found that endotoxin level in gn bacteremia was higher than that in gram - positive bacteremia , and in the gn bacteremia , non - enterobacteriaceae had a significantly higher level of endotoxin than enterobacteriaceae . it was not surprised that lipopolysaccharide ( lps ) is the major component of gn bacteria outer membrane , and lal reacts with bacterial endotoxin or lps . gram - positive bacteria and fungi in some patients were also found to present higher endotoxin level than normal . this was also found in previous studies where a positive lal assay was observed with peptidoglycan derived from the cell walls of gram - positive organisms or ( 1 - 3)-b - d - glucans from fungi . in addition , lps from the gut or other infection sites might enter the blood without bacterial translocation . moreover , endotoxin level in some patients was found to be below detection limit possibly because that endotoxin can bind to monocytes , red cells , and platelets . the different endotoxin levels found in bacteremia caused by non - enterobacteriaceae and enterobacteriaceae were similar to the findings as in the aforementioned studies and might result from different lps structures between the two types of bacteria . according to lipid a structure , lps predominantly has hexa - acyl or nonhexa - acyl lipid a. enterobacteriaceae typically produces hexa - acyl lipid a structure , and non - enterobacteriaceae produces nonhexa - acyl lipid a structure . furthermore , different bacterial species or different genomovars within the same species b. cepacia differ in their ability to cause life - threatening pneumonia and possess different lipid a structures ; lipid a even from the same species can be penta- , hexa- , or hepta - acylated , and depending on the temperature , some enterobacteriaceae such as yersinia pestis can make tetra- , penta- , or hexa - acyl lipid a. we found that pct was more closely associated with gn bacteremia than endotoxin . in fact , pct has been widely used as a sepsis biomarker for discriminating bacterial and nonbacterial infections and to predict bacteremia with different statuses . . this might be because the following limitations : the lal is not specific to hexa - acyl lps ; lps recognition by the horseshoe crab likely reflects a defense against aquatic bacteria ; the assay widely recognizes diverse lipid a structure to enhance the detection of bacteria in biological fluids , leading to some of the problems in detection of endotoxin in patients . in addition , in this study e. coli lps was used as a standard to test endotoxin , but lps from each bacterium found by bc should be used as standard lps to test the corresponding endotoxin . in conclusion , this is the comparative study of the pct and endotoxin as a predictive indicator for bacteremia and sepsis . we found that gn was predominant within the microorganisms found in the bcs of the icu patients in our hospital , and the level of pct is more closely associated with gn bacteremia than that of endotoxin , with the plasma endotoxin level of gn bacteremia being species dependent . our findings demonstrated that diversified types of bacteria caused bacteremia in icus in different regions of china and that pct is a more valuable biomarker than endotoxin in the diagnosis of gn bacteremia . this study was partly supported by grants from the national natural science foundation of china ( no . this study was partly supported by grants from the national natural science foundation of china ( no .

background : both procalcitonin ( pct ) and plasma endotoxin levels can not be solely used for a definite diagnosis of bacteremia or sepsis , and there has been few study comparing the values of the two biomarkers for the diagnosis of bacteremia . the aim of this study was to identify bacteria causing bacteremia and evaluate the role of the two biomarkers in the diagnosis of bacteremia in intensive care unit ( icu).methods : the medical records of 420 patients in icu were retrospectively reviewed . patients ( n = 241 ) who met the inclusion criteria were subjected to blood culture ( bc ) for the analysis of the endotoxin or pct levels . the exclusion criteria included the presence of infection with human immunodeficiency virus and/or aids , neutropenia without sepsis , pregnancy , treatment with immunosuppressive therapies , or blood diseases such as hematological tumors . patients bc episodes were divided into bc negative , gram - negative ( gn ) bacteria , gram - positive bacteria , and fungi groups . the pct and plasma endotoxin levels were compared in the different groups.results:a total of 241 patients with 505 episodes of bc were analyzed . the gn bacteria group showed higher levels of pct and endotoxin than the bc negative , gram - positive bacteria , and fungi groups . gn bacteremia was more prevalent than gram - positive bacteremia . the gn bacteremia caused by non - enterobacteriaceae infection presented higher endotoxin level than that by enterobacteriaceae , but no significant difference in pct levels was observed between the two groups . the plasma endotoxin significantly differed among different groups and was bacterial species dependent.conclusions:plasma endotoxin was more related to gn than to gram - positive bacteremia , and that endotoxin level was species dependent , but pct level remained relatively more stable within the gn bacteria caused bacteremia . both gn and positive bacteria caused bacteremia in the icu patients in different regions of china . and pct is a more valuable biomarker than endotoxin in the diagnosis of bacteremia .

I M Patient cohort Data collection Definition Procalcitonin and plasma endotoxin assay Statistical analysis R Patient characteristics Blood culture results Association of endotoxin or procalcitonin level with different microorganisms Procalcitonin and endotoxin levels in prediction of bacteremia D Financial support and sponsorship Conflicts of interest

procalcitonin ( pct ) is widely used as a marker before final blood culture ( bc ) confirmation in clinical diagnosis of bacteremia and sepsis , with diagnostic sensitivity 74.8100.0% , specificity 70.0100.0% , positive predictive value 55.0100.0% , and negative predictive value 56.3100.0% , and pct in combination with careful clinical parameters can discriminate infection caused bacteremia from inflammatory sepsis in 77% of cases . for example , pct can not reliably differentiate sepsis from other noninfectious causes of systemic inflammatory response syndrome in critically ill patients and is of no use in determining new fever caused by bacteremia in the intensive care unit ( icu ) patients . meanwhile , although the prognostic value of endotoxin level for bacteremia and sepsis remains disparate , less than two - thirds of patients with gram - negative ( gn ) bacteremia are detected with endotoxemia and vice versa . while the association of endotoxemia with bacteremia is bacterial type dependent , endotoxemia is more commonly detected in patients with bacteremia caused by non - enterobacteriaceae than a commensal member of enterobacteriaceae . therefore , both of the markers have some advantages and disadvantages and can not be solely used for a definite diagnosis of bacteremia or sepsis . furthermore , there has been few study comparing the values of the two biomarkers for the diagnosis of bacteremia within the same patient cohort . the aim of this retrospective study was to compare the role of the two biomarkers in the diagnosis of bacteremia in icus of a chinese hospital . all patients subjected to bc tests for the analysis of the endotoxin or pct levels during icu stay were enrolled in this study . the exclusion criteria included the presence of infection with human immunodeficiency virus and/or aids , neutropenia without sepsis , pregnancy , treatment with immunosuppressive therapies , or blood diseases such as hematological tumors . thus , all episodes of bc can be divided into four groups according to bc : bc ( no isolates ) , g ( gn bacteria ) , g ( gram - positive bacteria ) , and fungi ( fungi ) . notably , the pct and endotoxin levels of the mixed cultures with one or more isolates were not compared with the four groups described above . the group of non - enterobacteriaceae in the present analysis included acinetobacter baumannii , burkholderia cepacia , and pseudomonas aeruginosa . the pct levels were measured using an immunoluminometric assay ( lumitest pct kit , brahms diagnostica , hennigsdorf bei berlin , germany ) . the differences in the parametric data between different strata were calculated using student 's t - test and analysis of variance ( anova ) with post - hoc lsd test for the two groups . spearman 's rank correlation or pearson 's tests were performed to evaluate the association between the two groups , where appropriate . the data were analyzed using the receiver operating characteristic ( roc ) curve for the plasma endotoxin and pct levels for the prediction of gn bacteremia . all patients subjected to bc tests for the analysis of the endotoxin or pct levels during icu stay were enrolled in this study . the exclusion criteria included the presence of infection with human immunodeficiency virus and/or aids , neutropenia without sepsis , pregnancy , treatment with immunosuppressive therapies , or blood diseases such as hematological tumors . thus , all episodes of bc can be divided into four groups according to bc : bc ( no isolates ) , g ( gn bacteria ) , g ( gram - positive bacteria ) , and fungi ( fungi ) . notably , the pct and endotoxin levels of the mixed cultures with one or more isolates were not compared with the four groups described above . the group of non - enterobacteriaceae in the present analysis included acinetobacter baumannii , burkholderia cepacia , and pseudomonas aeruginosa . the differences in the parametric data between different strata were calculated using student 's t - test and analysis of variance ( anova ) with post - hoc lsd test for the two groups . spearman 's rank correlation or pearson 's tests were performed to evaluate the association between the two groups , where appropriate . the data were analyzed using the receiver operating characteristic ( roc ) curve for the plasma endotoxin and pct levels for the prediction of gn bacteremia . a total of 420 patients admitted to the icu were screened for the study ; 179 patients did not meet the inclusion criteria and were excluded , and 241 patients met the inclusion criteria ( male , 68.0% ) , of which 71 ( 29.5% ) patients had bacteremia . sex , infection sites , and the presence of enterobacteriaceae infection did not affect the mortality of the patients . patient characteristics based on the diagnosis at admission * pct and endotoxin levels from first - time bc . apache ii : the acute physiology and chronic health evaluation ii score ; sofas : the sequential organ failure assessments score ; cci : the charlson comorbidity index ; bc : blood culture ; g : gram - negative bacteria ; g : grampositive bacteria ; pct : procalcitonin ; sd : standard deviation . more than one microorganism was found in 4 ( 4.3% ) episodes after the bc , with b. cepacia being present in two episodes of bc . microorganisms isolated from blood culture ( n = 92 ) k. pneumonia : klebsiella pneumonia ; s. marcescens : serratia marcescens ; e. coli : escherichia coli ; e. cloacae : enterobacter cloacae ; k. oxytoca : klebsiella oxytoca ; s. enteritidis : salmonella enteritidis ; b. cepacia : burkholderia cepacia ; a. baumannii : acinetobacter baumannii ; p. aeruginosa : pseudomonas aeruginosa ; a. junii : acinetobacter junii ; p. paucimobilis : pseudomonas paucimobilis ; b. diminuta : brevundimonas diminuta ; s. paucimobilis : sphingomonas paucimobilis ; c. meningosepticum : chryseobacterium meningosepticum ; a. xylosoxidans : achromobacter xylosoxidans ; h. influenza : haemophilus influenza ; s. capitis : staphylococcus capitis ; s. hemolyticus : staphylococcus hemolyticus ; s. huminis : staphylococcus huminis ; s. epidermidis : staphylococcus epidermidis ; s. aureus : staphylococcus aureus ; k. roseus : kocuria roseus ; e. faecalis : enterococcus faecalis ; g. bergeri : gemella bergeri ; c. albicans : candida albicans ; c. glabrata : candida glabrata ; c. haemulonii : candida haemulonii ; s. lentus : staphylococcus lentus ; mrsa : methicillin - resistant s. aureus . the pct concentration and endotoxin level significantly differed among the four groups g , g , fungi , and bc ( p < 0.000 , pct ; and p = 0.0244 , endotoxin ) . the pct level was significantly higher in the g group than in the bc ( p < 0.0001 ) and g ( p = 0.0484 ) groups ; patients with fungi isolates also had a higher level of pct than patients with bc ( p = 0.0244 ) . the plasma endotoxin level in the g group was significantly higher than in the bc group ( p = 0.0025 ) , and no significant difference was found between g group and the bc ( or g ) group [ figure 1 ] . comparison of the procalcitonin and plasma endotoxin levels in different groups according to the blood culture results . bc : blood culture negative group ; g : gram - negative bacteria ; g : gram - positive bacteria . patients with non - enterobacteriaceae isolates showed a significantly higher plasma endotoxin level than patients with enterobacteriaceae ( p = 0.0276 ) ; the pct level did not differ significantly between the above two patient groups ( p = 0.2964 ) [ figure 2 ] . it was significantly different among all the different groups in the species level for endotoxin ( p = 0.0446 ) , not pct ( p = 0.5529 ) [ figure 1 ] . the endotoxin level of the patients with b. cepacia infection was significantly higher than that with s. marcescens ( p = 0.0236 ) , k. pneumoniae ( p = 0.0048 ) , and e. cloacae ( p = 0.0180 ) ; the difference between e. cloacae and p. aeruginosa in the endotoxin level was also nearly significantly different ( p = 0.0519 ) . the pct level in the patients with e. cloacae infection was significantly ( p = 0.0319 ) and almost ( p = 0.0893 ) higher than those with s. marcescens and a. baumannii , respectively [ figure 3 ] . comparison of the procalcitonin and plasma endotoxin levels in different groups according to bacterial species . the area under the roc curves for the pct and endotoxin levels used to predict gn isolates of bc were 0.741 ( 95% confidence interval [ ci ] : 0.6830.779 , p < 0.001 ) and 0.614 ( 95% ci : 0.5500.678 , p = 0.002 ) , respectively . a total of 420 patients admitted to the icu were screened for the study ; 179 patients did not meet the inclusion criteria and were excluded , and 241 patients met the inclusion criteria ( male , 68.0% ) , of which 71 ( 29.5% ) patients had bacteremia . sex , infection sites , and the presence of enterobacteriaceae infection did not affect the mortality of the patients . patient characteristics based on the diagnosis at admission * pct and endotoxin levels from first - time bc . apache ii : the acute physiology and chronic health evaluation ii score ; sofas : the sequential organ failure assessments score ; cci : the charlson comorbidity index ; bc : blood culture ; g : gram - negative bacteria ; g : grampositive bacteria ; pct : procalcitonin ; sd : standard deviation . of the 92 isolates , a total of 69 ( 75.0% ) isolates were gn microorganisms , including 13 b. cepacia , 11 k. pneumoniae , 10 acinetobacter baumannii , six e. coli , and five enterobacter cloacae . microorganisms isolated from blood culture ( n = 92 ) k. pneumonia : klebsiella pneumonia ; s. marcescens : serratia marcescens ; e. coli : escherichia coli ; e. cloacae : enterobacter cloacae ; k. oxytoca : klebsiella oxytoca ; s. enteritidis : salmonella enteritidis ; b. cepacia : burkholderia cepacia ; a. baumannii : acinetobacter baumannii ; p. aeruginosa : pseudomonas aeruginosa ; a. junii : acinetobacter junii ; p. paucimobilis : pseudomonas paucimobilis ; b. diminuta : brevundimonas diminuta ; s. paucimobilis : sphingomonas paucimobilis ; c. meningosepticum : chryseobacterium meningosepticum ; a. xylosoxidans : achromobacter xylosoxidans ; h. influenza : haemophilus influenza ; s. capitis : staphylococcus capitis ; s. hemolyticus : staphylococcus hemolyticus ; s. huminis : staphylococcus huminis ; s. epidermidis : staphylococcus epidermidis ; s. aureus : staphylococcus aureus ; k. roseus : kocuria roseus ; e. faecalis : enterococcus faecalis ; g. bergeri : gemella bergeri ; c. albicans : candida albicans ; c. glabrata : candida glabrata ; c. haemulonii : candida haemulonii ; s. lentus : staphylococcus lentus ; mrsa : methicillin - resistant s. aureus . the pct concentration and endotoxin level significantly differed among the four groups g , g , fungi , and bc ( p < 0.000 , pct ; and p = 0.0244 , endotoxin ) . the pct level was significantly higher in the g group than in the bc ( p < 0.0001 ) and g ( p = 0.0484 ) groups ; patients with fungi isolates also had a higher level of pct than patients with bc ( p = 0.0244 ) . the plasma endotoxin level in the g group was significantly higher than in the bc group ( p = 0.0025 ) , and no significant difference was found between g group and the bc ( or g ) group [ figure 1 ] . comparison of the procalcitonin and plasma endotoxin levels in different groups according to the blood culture results . bc : blood culture negative group ; g : gram - negative bacteria ; g : gram - positive bacteria . patients with non - enterobacteriaceae isolates showed a significantly higher plasma endotoxin level than patients with enterobacteriaceae ( p = 0.0276 ) ; the pct level did not differ significantly between the above two patient groups ( p = 0.2964 ) [ figure 2 ] . the endotoxin level of the patients with b. cepacia infection was significantly higher than that with s. marcescens ( p = 0.0236 ) , k. pneumoniae ( p = 0.0048 ) , and e. cloacae ( p = 0.0180 ) ; the difference between e. cloacae and p. aeruginosa in the endotoxin level was also nearly significantly different ( p = 0.0519 ) . the pct level in the patients with e. cloacae infection was significantly ( p = 0.0319 ) and almost ( p = 0.0893 ) higher than those with s. marcescens and a. baumannii , respectively [ figure 3 ] . comparison of the procalcitonin and plasma endotoxin levels in different groups according to bacterial species . the area under the roc curves for the pct and endotoxin levels used to predict gn isolates of bc were 0.741 ( 95% confidence interval [ ci ] : 0.6830.779 , p < 0.001 ) and 0.614 ( 95% ci : 0.5500.678 , p = 0.002 ) , respectively . in this retrospective study , to investigate whether pct or endotoxin level in the blood is more valuable for diagnosis of bacteremia , we evaluated the association of the pct or plasma endotoxin level of different types of bacteria in the icu patients with sepsis , severe sepsis , or septic shock . we found that : ( 1 ) gn bacteria were predominant within the microorganisms found in the bcs ; ( 2 ) the level of pct is more closely associated with gn bacteremia than that of endotoxin ; ( 3 ) gn bacteremia exhibited a higher level of endotoxin than nonbacteremia ; and ( 4 ) bacteremia with non - enterobacteriaceae had a significantly higher level of endotoxin than bacteremia with enterobacteriaceae . gn bacteria were predominant within the microorganisms found in the bcs , and the major gram - positive bacteria were from coagulase - negative staphylococci . in some countries , gram - positive bacteria may have a high percentage of microorganisms found in the bcs . there was no significant difference between the mortality rates caused by gn bacteremia with enterobacteriaceae and non - enterobacteriaceae . we found that endotoxin level in gn bacteremia was higher than that in gram - positive bacteremia , and in the gn bacteremia , non - enterobacteriaceae had a significantly higher level of endotoxin than enterobacteriaceae . gram - positive bacteria and fungi in some patients were also found to present higher endotoxin level than normal . moreover , endotoxin level in some patients was found to be below detection limit possibly because that endotoxin can bind to monocytes , red cells , and platelets . the different endotoxin levels found in bacteremia caused by non - enterobacteriaceae and enterobacteriaceae were similar to the findings as in the aforementioned studies and might result from different lps structures between the two types of bacteria . according to lipid a structure , lps predominantly has hexa - acyl or nonhexa - acyl lipid a. enterobacteriaceae typically produces hexa - acyl lipid a structure , and non - enterobacteriaceae produces nonhexa - acyl lipid a structure . furthermore , different bacterial species or different genomovars within the same species b. cepacia differ in their ability to cause life - threatening pneumonia and possess different lipid a structures ; lipid a even from the same species can be penta- , hexa- , or hepta - acylated , and depending on the temperature , some enterobacteriaceae such as yersinia pestis can make tetra- , penta- , or hexa - acyl lipid a. we found that pct was more closely associated with gn bacteremia than endotoxin . in conclusion , this is the comparative study of the pct and endotoxin as a predictive indicator for bacteremia and sepsis . we found that gn was predominant within the microorganisms found in the bcs of the icu patients in our hospital , and the level of pct is more closely associated with gn bacteremia than that of endotoxin , with the plasma endotoxin level of gn bacteremia being species dependent . our findings demonstrated that diversified types of bacteria caused bacteremia in icus in different regions of china and that pct is a more valuable biomarker than endotoxin in the diagnosis of gn bacteremia . this study was partly supported by grants from the national natural science foundation of china ( no .

resin cements are widely used in the luting of composite resin or porcelain inlays , porcelain laminate veneers , resin bonded metallic prosthesis and ceramic restoratives using computer aided design and manufacturing ( cad / cam ) . different types of light - curing units ( lcus ) have been proposed for the photopolymerization of light activated restorative materials including conventional quartz tungsten halogen ( qth ) lights and new photoactivation techniques , such as intermittent light , plasma arc curing ( pac ) and , more recently , a new technology employing light - emitting diode ( led ) or laser . solid - state leds use junctions of doped semiconductors ( p - n junctions ) based on gallium nitride to emit blue light . the spectral output of blue leds falls between 450 and 490 nm , so these units are effective for curing materials with camphorquinone photo - initators . led units do not require a filter , have a long life span , and do not produce as much heat as quartz - tungsten halogen devices . to date there is no internationally accepted classification of the various types of led curing lights . most of the first - generation led lcus were unable to cure composites as well as correctly functioning qth lights . however , led technology has advanced and it has been reported that high power " second - generation led curing lights " can polymerize some resins as well as or better than some qth lights . high power leds have since made it possible to merge the technical developments in dental curing lights , allowing led - based systems to achieve a 50% reduction in cure time from that recommended by manufacturers however , this shorter exposure time may lead to an insufficient degree of conversion of the resin cement . especially at the bottom of restoration , this may lead to reduced physical properties and poor biocompatibility when ceramic restoration thickness ( > 2 mm ) is difficult to cure well from standard light source position for several ceramic crowns . the degree of light induced conversion of monomers to polymers is influenced by various parameters , such as the intensity of the light around the wavelength triggering level , the photoinitator system , the duration of irradiation , concentrations , types and mixtures of photoinitators , co - initators , stabilizers and inhibitors , as well as the types and proportions of monomers and fillers . adequate polymerization is the most important factor in maximizing the physical properties , clinical performance and biocompatibility . problems associated with inadequate polymerization include inferior physical properties , solubility in the oral environment and increased microleakage with resultant recurrent decay and pulpal irritation . on the other hand , the amount of leachable residual monomers may vary with the light source used for curing . although amongst the different lcus available in dental practice , halogen lamps are the most frequently used , recently the led technology has been successfully proposed . the adhesive properties depend on direct contact between the cement and the mineralized tissues . luting agents however , luting agents generally appear to cause little adverse long term pulpal effect , particularly if the remaining dentin thickness exceeds 1 mm . therefore , the biocompatibility of resin luting cements ( rlcs ) is important for the optimal advantage of these materials in clinical use . there is little information on biocompatibility tests , especially on the effect of reduced exposure time on cytotoxicity for luting resin cements which is directly related to clinical success of the prosthetic restorations . this study aimed at assessing the effect of reduced curing time of five rlcs polymerized by high - power led curing unit on the viability of a cell of l-929 fibroblast cells over 24 h. the cells used for the experiments were l-929 mouse fibroblasts ( l-929 an2 hkk 95030802 ; food and mouth disease institute , ankara , turkey ) in conformity with the iso 10993 - 5 standard . the cells were grown as monolayer cultures in t-25 flasks ( costar , cambridge , ma , usa ) , subcultured three times a week at 37c in an atmosphere of 5% co2 in air and 100% relative humidity and maintained at third passage . the culture medium was dulbecco 's modified eagle medium ( dmem)/ham 's f12 nutrient mixture ( 1:1 ; sigma , st louis , mo , usa ) supplemented with 10% ( v / v ) foetal bovine serum ( fbs ; biochrom , berlin , germany ) without antibiotics . adherent cells at a logarithmic growth phase were controlled under an inverted tissue culture microscope ( olympus ck40 , tokyo , japan ) and detached with a mixture of 0.025% trypsin ( sigma ) and 0.02% ethylenediaminetetraacetic acid ( edta ; sigma ) , incubated for 2 - 5 min at 37c and used for cell inoculation . five dual polymerizing luting resin cements were used in this study : relyx unicem ( ru , 3 m espe dental products , st . schaumburg , il , usa ) ; lute - it ( lt , pentron clinical technologies , l.l.c . , schaumburg , il , usa ) ; rely x arc ( ra , 3 m espe dental products , st . paul , usa ) the tested materials and their compositions are listed in figure 1 . photoactivation was performed with the led lcu ( elipar freelight 2 , 3 m espe , 939820014022 , seefeld , germany ) . forty disc - shaped samples ( 6 mm diameter x 1 mm thickness ) were prepared for each tested material : 20 disc samples exposed with led lcu for 20 s and remaining 20 disc samples exposed for 40 s. standard exposure mode , providing full light intensity for the entire exposure , was chosen with both 40 s and 20 s. the led lcu had a light guide tip diameter of 8 mm with irradiance of 1200 mw / cmwith a wavelength of 430 - 480 nm according to the manufacturer . tested materials and their composition according to manufacturers . bis - gma : bisphenol a diglycidyl methacrylate ; udma : urethane dimethacrylate ; hddma : 1,6-hexanediol dimethacrylate ; tegdma : triethyleneglycol dimethacrylate ; ebpadma : ethoxylated bisphenol a dimethacrylate the rlcs were placed into sterile circular polytetrafluoroethylene moulds . polyethylene films were added on the top surface of the luting resin cements . therefore to simulate clinical conditions , the tested materials were irradiated from the top through the ceramic discs ( 1 cm diameter x 2 mm thickness , ips empress 2 ; shade 1a , lot : d 50108 , ivoclar , vivadent , fl-9494 , schaan , liechtenstein ) by contacting the end of the light guide with its surface , through the use of led lcu for 20 s ( 50% of the recommended exposure time ) and 40 s ( 100% exposure time ) ( figure 2 ) , under aseptic conditions at laminar flow ( holten , class ii , denmark ) . the freshly prepared samples were placed immediately at the bottom of 6-well plates ( costar , cambridge ma , usa ) . schematic illustration of sample preparation the ratio of the surface area of the disc samples to the extraction volume was 0.5 cm ml which is in line with iso 10993 - 12 , in this study . the samples were placed in dmem / f12 with 10% fbs and incubated at 37c in an atmosphere of 5% co2 in air without agitation for 24 h incubation period . after the incubation , the extracts were filtered through 0.22 m cellulose acetate filters ( millipore ; sigma , st . louis , mo , usa ) and then they were used to evaluate cytotoxicity . the l-929 cell suspension with dmem / f12 with 10% fbs and 1% antibiotic was prepared at a concentration of 3 x 10 cell ml and inoculated onto 96-well cluster cell culture plates ( 100 l per well ) . the multiwell plates were incubated at 37c , 5% co2 in air for 24 h. after 24 h , the culture medium was removed from the wells and equal volumes ( 100 l ) of the extracts were added into each well . in control wells , 100 l dmemf12 with 10% fbs and 1% antibiotic was added . after the 24 h incubation period test extracts were removed . following removal of the test extracts , 100 l well dmemf12 with 10% fbs and 1% antibiotic and 12 l mtt ( tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide ) were added to each well and incubated in a dark environment for 4 h at 37c . after incubation 96 wells were checked for formazan crystals with inverted tissue culture microscope . mtt was aspirated and 100 l well of isopropanol ( merck , darmstadt , germany ) was added to each well . subsequently , the absorbance at 570 nm was measured using a uv - visible spectrophotometer ( lpb pharmacia , bromma , sweden ) . then the absorbance results were evaluated under a light microscope and calculated as a percentage of the controls . the cytotoxic effects of the rlcs polymerizing with led lcu and the exposure times on the fibroblast cell 's survival rates were evaluated by two way analysis of variances . then , two independent samples were compared by t - test . all statistical analysis was performed by using spss 11.5 statistical software package ( spss inc , chicago , il , usa ) . the cells used for the experiments were l-929 mouse fibroblasts ( l-929 an2 hkk 95030802 ; food and mouth disease institute , ankara , turkey ) in conformity with the iso 10993 - 5 standard . the cells were grown as monolayer cultures in t-25 flasks ( costar , cambridge , ma , usa ) , subcultured three times a week at 37c in an atmosphere of 5% co2 in air and 100% relative humidity and maintained at third passage . the culture medium was dulbecco 's modified eagle medium ( dmem)/ham 's f12 nutrient mixture ( 1:1 ; sigma , st louis , mo , usa ) supplemented with 10% ( v / v ) foetal bovine serum ( fbs ; biochrom , berlin , germany ) without antibiotics . adherent cells at a logarithmic growth phase were controlled under an inverted tissue culture microscope ( olympus ck40 , tokyo , japan ) and detached with a mixture of 0.025% trypsin ( sigma ) and 0.02% ethylenediaminetetraacetic acid ( edta ; sigma ) , incubated for 2 - 5 min at 37c and used for cell inoculation . five dual polymerizing luting resin cements were used in this study : relyx unicem ( ru , 3 m espe dental products , st . schaumburg , il , usa ) ; lute - it ( lt , pentron clinical technologies , l.l.c . , schaumburg , il , usa ) ; rely x arc ( ra , 3 m espe dental products , st . paul , usa ) the tested materials and their compositions are listed in figure 1 . photoactivation was performed with the led lcu ( elipar freelight 2 , 3 m espe , 939820014022 , seefeld , germany ) . forty disc - shaped samples ( 6 mm diameter x 1 mm thickness ) were prepared for each tested material : 20 disc samples exposed with led lcu for 20 s and remaining 20 disc samples exposed for 40 s. standard exposure mode , providing full light intensity for the entire exposure , was chosen with both 40 s and 20 s. the led lcu had a light guide tip diameter of 8 mm with irradiance of 1200 mw / cmwith a wavelength of 430 - 480 nm according to the manufacturer . tested materials and their composition according to manufacturers . bis - gma : bisphenol a diglycidyl methacrylate ; udma : urethane dimethacrylate ; hddma : 1,6-hexanediol dimethacrylate ; tegdma : triethyleneglycol dimethacrylate ; ebpadma : ethoxylated bisphenol a dimethacrylate the rlcs were placed into sterile circular polytetrafluoroethylene moulds . polyethylene films were added on the top surface of the luting resin cements . therefore to simulate clinical conditions , the tested materials were irradiated from the top through the ceramic discs ( 1 cm diameter x 2 mm thickness , ips empress 2 ; shade 1a , lot : d 50108 , ivoclar , vivadent , fl-9494 , schaan , liechtenstein ) by contacting the end of the light guide with its surface , through the use of led lcu for 20 s ( 50% of the recommended exposure time ) and 40 s ( 100% exposure time ) ( figure 2 ) , under aseptic conditions at laminar flow ( holten , class ii , denmark ) . the freshly prepared samples were placed immediately at the bottom of 6-well plates ( costar , cambridge ma , usa ) . schematic illustration of sample preparation the ratio of the surface area of the disc samples to the extraction volume was 0.5 cm ml which is in line with iso 10993 - 12 , in this study . the samples were placed in dmem / f12 with 10% fbs and incubated at 37c in an atmosphere of 5% co2 in air without agitation for 24 h incubation period . after the incubation , the extracts were filtered through 0.22 m cellulose acetate filters ( millipore ; sigma , st . the l-929 cell suspension with dmem / f12 with 10% fbs and 1% antibiotic was prepared at a concentration of 3 x 10 cell ml and inoculated onto 96-well cluster cell culture plates ( 100 l per well ) . the multiwell plates were incubated at 37c , 5% co2 in air for 24 h. after 24 h , the culture medium was removed from the wells and equal volumes ( 100 l ) of the extracts were added into each well . in control wells , 100 l dmemf12 with 10% fbs and 1% antibiotic was added . after the 24 h incubation period test extracts were removed . following removal of the test extracts , 100 l well dmemf12 with 10% fbs and 1% antibiotic and 12 l mtt ( tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide ) were added to each well and incubated in a dark environment for 4 h at 37c . after incubation 96 wells were checked for formazan crystals with inverted tissue culture microscope . mtt was aspirated and 100 l well of isopropanol ( merck , darmstadt , germany ) was added to each well . subsequently , the absorbance at 570 nm was measured using a uv - visible spectrophotometer ( lpb pharmacia , bromma , sweden ) . then the absorbance results were evaluated under a light microscope and calculated as a percentage of the controls . the cytotoxic effects of the rlcs polymerizing with led lcu and the exposure times on the fibroblast cell 's survival rates were evaluated by two way analysis of variances . then , two independent samples were compared by t - test . all statistical analysis was performed by using spss 11.5 statistical software package ( spss inc , chicago , il , usa ) . the results in figure 3 demonstrated that all freshly prepared rlcs polymerized with led for 20 s ( 50% exposure time ) and 40 s ( 100% exposure time ) reduced the cell numbers compared to the control ( culture without sample ) . distribution of cell survival rates ( % ) according to the light exposure times ( 100% and 50% ) for each resin luting cements . cell survival rates are expressed as a percentage of controls ( cultures without samples ) . ru= rely x unicem ; dk= duolink ; lt= lute it ; in= illusion ; ra= rely x arc the cytotoxic effect of the tested materials and exposure times on the cell survival rates were evaluated by two - way analysis of variance . there was a significant two - way interaction between two factors ( f=5.856 , p=0.003 ) . therefore , the time factor levels were compared in each rlc ( two independent samples were compared by t - test ) . it was observed that the cytotoxicity of the tested materials depends on the exposure time . when the interactions between exposure times and rlcs are evaluated , both dk and lt reduced cell survival rate significantly with respect to the control for 20 s and 40 s ( p<0.001 ) ( table 1 ) . cell survival rate ( csr ) according to the light exposure times for each resin luting cement as compared to the control ( cultures without sample ) for 20 s and 40 s cell survival rate results for 40 s exposure time were significantly higher than 20 s cell survival rates of dk and lt ( t=-9.399 , p=0.001 ; and t=-20.418 , p<0.001 , respectively ) . there is no significant difference between the exposure time results for ru , ra and in . however , in terms of cell survival rate , the 20 s ( 50% exposure time ) results for ru and in were higher than the 40 s ( 100% exposure time ) results for ra ( t=1.747 , p=0.156 ; t=0.164 , p=0.878 ; and t=-1.262 , p=0.276 , respectively ) . in the present study , l929 fibroblasts were used because they are the most common iso - approved cell type in the pulp and gingival tissues , which would be the target of chemicals released from the rlcs . it has been demonstrated that l929 mouse fibroblasts show comparable results to primary human gingival fibroblasts and therefore might represent a model for gingival toxicity in vitro . several in vitro studies assessing the cytotoxicity of dental materials have used fibroblast cell lines because they represent a common cell type in pulp and gingival tissues and also because of their reproducible growth rates , and advantages in handling and availability compared to primary cells . in this study , it is important for the materials to be tested immediately after mixing/ curing to avoid the loss of toxic substances released from the tested material at this initial stage . a recent study demonstrated the relevance of the cytotoxicity of rlcs and the importance of achieving sufficient polymerization with the lcus . elipar freelight 2 , used in the present study , is a high - intensity led lcu , and has exposure time options as follows : 5 , 10 , 15 and 20 s according to the manufacturer 's instructions . because of the high light intensity ( 1200 mw / cm ) of this lcu , these time periods correspond to the time periods ( 10 , 20 , 30 and 40 s ) of conventional lcu that has light intensity of 600 - 800 mw / cmfor halogen technology or 300 - 400 mw / cmlight intensity for led . thus , the normal exposure time for conventional units can be cut in half without compromising curing performance but it could have a direct affect on biocompatibility of the rlcs . ( 2009 ) evaluated the curing sufficiency under the condition of high irradiance and short irradiation time on methacrylate - based dental materials by using plasma arc and quartz tungsten halogen curing lights . higher irradiance and shorter irradiation time was found intrinsically associated with a higher free radical termination rate than that of lower irradiance but longer irradiation time . on the other hand , there is no information about the cytotoxicity of the rlcs in half the exposure time with polymerized high power led . light polymerizing of rlcs will polymerize resins in solid phase hence significantly diminishing the amount of free monomer and substantially reducing the potential for noxious stimuli . ( 2002 ) have addressed the application of led technology to cure restorative materials . typically , the advantages claimed for second - generation led lcus are more efficiency in cure , decreased heat from the light tip , consistent output over time without degradation and significantly longer useful life of the diodes compared with quartz tungsten halogen bulbs . furthermore , with the introduction of led lcus , it was asserted that the emission of blue led lcus is the ideal spectra for the conversion of dental materials containing monomer . in this way biocompatibility of light - cured dental materials may be affected by the quality of the lcu used . due to the advanced led technology , high power led curing light was able to polymerize dental resin in half exposure time ( 50% ) instead of 100% . rlcs cover the dentin walls and only indirectly interact with the pulp , by means of the tubular fluid , or with the oral mucosa , by means of saliva . an in vitro study has shown that cellular toxicity of rlcs was related to leachables containing free monomers and components such as degradation products or non reactive initiators , activators , and/or stabilizers . ( 2008 ) reported that degree of polymerization of rlc is influenced by several factors , namely , porcelain shade and thickness , lcu and curing time . inadequate degree of polymerizing is therefore necessary to improve the biocompatibility of rlcs used in prosthetic dentistry . the tested samples were irradiated from top through the ceramic disc in the present study , so as to simulate clinical condition . insufficient photoactivation contributes to an increase in level of unreacted monomers through a reduced degree of polymerization and cross linking . when light curing of rlcs are not fully polymerized , leachable components such as bis - gma , udma , tegdma , camphorquinone and hema , may penetrate through dentin tubules , exert potential pulpal injury and inhibit pulp tissue repair . according to goldberg ( 2008 ) , leaching of some ions may cause cell alterations , and some molecular mechanisms have been identified as key factors leading to apoptosis and/or pulp necrosis . ( 2009 ) investigated the genotoxic and cytotoxic effects of eluates derived from different types of dental cements on normal cultured human lymphocytes and reported that the released substances , such as tegdma included in resin cements ' composition are responsible for the cytogenetic effects . a previous study demonstrated that the cytotoxicity ranking of the most widely used monomers was bisgma > udma > tegdma > hema > mma . furthermore , a correlation between fluoride release and cytotoxicity has been observed by khalil and da'dara ( 1994 ) , reporting that high doses of fluoride inhibited cell division and caused death of rat bone marrow cells . in the current study , dk and lt showed higher cytotoxicity with both exposure times in comparison to the other tested materials . this is considered that udma and inorganic fluoride in structure of these materials may be responsible for their cytotoxic effect , as it has been previously shown that udma and fluoride are more toxic agents . furthermore , it is considered that synergistic effects of bis - gma and udma released by those tested materials lead to increased cytotoxicity on l-929 fibroblast cells . it was clear that all sufficient quantities of test materials polymerized by high power led for 20 s and 40 s leached a variety of components into cell culture medium and affected cell activity . after testing the cytotoxicity of polymerized test samples , statisticially significant differences were found regarding cell survival rates for the different exposure times and rlcs . when comparing the control cultures lt polymerized for 20 s ( 50% ) had resulted in the lowest survival rate of 28% . however , in polymerized for 20 s revealed the highest cell survival rate of 90.81% ( p<0.01 ) . in the 40 s exposure time , lt had led to the lowest survival rate ( 57.86% ) while ra had brought about the highest survival rate ( 90.91% ) compared to the control cultures ( p<0.01 ) . the results revealed that there might be several possible reasons for different effects of rlcs or exposure times ( 50% and 100% ) on their cytotoxicity such as the light transmission characteristic , the released energy during the polymerization of the rlcs and the amount as well as type of released toxic substances from the unpolymerized rlcs . generally , when the results of all samples were evaluated , materials polymerized for 20 s ( except for ru , in ) had reduced cell survival rates than the samples polymerized for 40 s with high power led . this leads to the assumption that 20-s exposure time is not sufficient for the polymerization of these materials . the obtained results indicate that the rlcs polymerized for either 20 s or 40 s with high power led may lead to unpolymerized components or toxic substances with harmful effects since they might penetrate through dentin tubules . the findings of the present study also suggest that it is important to be cautious when considering manufacturers ' recommendation that high power led light cure unit reduces the exposure time of rlcs in polymerization by 50% . based on the experimental condition , it was concluded that the period of photoactivation is related to the cytotoxic effects of the tested rlcs , and that an exposure time of 40 s is recommended for dk , lt and ra although 20 s is preferable for ru and in in operative procedures .

objectiveapplications of resin luting agents and high - power light - emitting diodes ( led ) light - curing units ( lcus ) have increased considerably over the last few years . however , it is not clear whether the effect of reduced exposure time on cytotoxicity of such products have adequate biocompatibility to meet clinical success . this study aimed at assessing the effect of reduced curing time of five resin luting cements ( rlcs ) polymerized by high - power led curing unit on the viability of a cell of l-929 fibroblast cells.material and methodsdisc - shaped samples were prepared in polytetrafluoroethylene moulds with cylindrical cavities . the samples were irradiated from the top through the ceramic discs and acetate strips using led lcu for 20 s ( 50% of the manufacturer 's recommended exposure time ) and 40 s ( 100% exposure time ) . after curing , the samples were transferred into a culture medium for 24 h. the eluates were obtained and pipetted onto l-929 fibroblast cultures ( 3x104 per well ) and incubated for evaluating after 24 h. measurements were performed by dimethylthiazol diphenyltetrazolium assay . statistical significance was determined by two - way anova and two independent samples were compared by t-test.resultsresults showed that eluates of most of the materials polymerized for 20 s ( except rely x unicem and illusion ) reduced to a higher extent cell viability compared to samples of the same materials polymerized for 40 s. illusion exhibited the least cytotoxicity for 20 s exposure time compared to the control ( culture without samples ) followed by rely x unicem and rely x arc ( 90.81% , 88.90% , and 83.11% , respectively ) . for rely x arc , duolink and lute - it 40 s exposure time was better ( t=-1.262 p=0,276 ; t=-9.399 p=0.001 ; and t=-20.418 p<0.001 , respectively).conclusionthe results of this study suggest that reduction of curing time significantly enhances the cytotoxicity of the studied resin cement materials , therefore compromising their clinical performance .

INTRODUCTION MATERIAL AND METHODS Cells Sample preparation Cytotoxicity testing (MTT assay) Statistical analysis RESULTS DISCUSSION CONCLUSIONS

different types of light - curing units ( lcus ) have been proposed for the photopolymerization of light activated restorative materials including conventional quartz tungsten halogen ( qth ) lights and new photoactivation techniques , such as intermittent light , plasma arc curing ( pac ) and , more recently , a new technology employing light - emitting diode ( led ) or laser . to date there is no internationally accepted classification of the various types of led curing lights . most of the first - generation led lcus were unable to cure composites as well as correctly functioning qth lights . however , led technology has advanced and it has been reported that high power " second - generation led curing lights " can polymerize some resins as well as or better than some qth lights . high power leds have since made it possible to merge the technical developments in dental curing lights , allowing led - based systems to achieve a 50% reduction in cure time from that recommended by manufacturers however , this shorter exposure time may lead to an insufficient degree of conversion of the resin cement . the degree of light induced conversion of monomers to polymers is influenced by various parameters , such as the intensity of the light around the wavelength triggering level , the photoinitator system , the duration of irradiation , concentrations , types and mixtures of photoinitators , co - initators , stabilizers and inhibitors , as well as the types and proportions of monomers and fillers . on the other hand , the amount of leachable residual monomers may vary with the light source used for curing . luting agents however , luting agents generally appear to cause little adverse long term pulpal effect , particularly if the remaining dentin thickness exceeds 1 mm . therefore , the biocompatibility of resin luting cements ( rlcs ) is important for the optimal advantage of these materials in clinical use . there is little information on biocompatibility tests , especially on the effect of reduced exposure time on cytotoxicity for luting resin cements which is directly related to clinical success of the prosthetic restorations . this study aimed at assessing the effect of reduced curing time of five rlcs polymerized by high - power led curing unit on the viability of a cell of l-929 fibroblast cells over 24 h. the cells used for the experiments were l-929 mouse fibroblasts ( l-929 an2 hkk 95030802 ; food and mouth disease institute , ankara , turkey ) in conformity with the iso 10993 - 5 standard . adherent cells at a logarithmic growth phase were controlled under an inverted tissue culture microscope ( olympus ck40 , tokyo , japan ) and detached with a mixture of 0.025% trypsin ( sigma ) and 0.02% ethylenediaminetetraacetic acid ( edta ; sigma ) , incubated for 2 - 5 min at 37c and used for cell inoculation . five dual polymerizing luting resin cements were used in this study : relyx unicem ( ru , 3 m espe dental products , st . schaumburg , il , usa ) ; lute - it ( lt , pentron clinical technologies , l.l.c . , schaumburg , il , usa ) ; rely x arc ( ra , 3 m espe dental products , st . forty disc - shaped samples ( 6 mm diameter x 1 mm thickness ) were prepared for each tested material : 20 disc samples exposed with led lcu for 20 s and remaining 20 disc samples exposed for 40 s. standard exposure mode , providing full light intensity for the entire exposure , was chosen with both 40 s and 20 s. the led lcu had a light guide tip diameter of 8 mm with irradiance of 1200 mw / cmwith a wavelength of 430 - 480 nm according to the manufacturer . polyethylene films were added on the top surface of the luting resin cements . therefore to simulate clinical conditions , the tested materials were irradiated from the top through the ceramic discs ( 1 cm diameter x 2 mm thickness , ips empress 2 ; shade 1a , lot : d 50108 , ivoclar , vivadent , fl-9494 , schaan , liechtenstein ) by contacting the end of the light guide with its surface , through the use of led lcu for 20 s ( 50% of the recommended exposure time ) and 40 s ( 100% exposure time ) ( figure 2 ) , under aseptic conditions at laminar flow ( holten , class ii , denmark ) . schematic illustration of sample preparation the ratio of the surface area of the disc samples to the extraction volume was 0.5 cm ml which is in line with iso 10993 - 12 , in this study . the samples were placed in dmem / f12 with 10% fbs and incubated at 37c in an atmosphere of 5% co2 in air without agitation for 24 h incubation period . the l-929 cell suspension with dmem / f12 with 10% fbs and 1% antibiotic was prepared at a concentration of 3 x 10 cell ml and inoculated onto 96-well cluster cell culture plates ( 100 l per well ) . the multiwell plates were incubated at 37c , 5% co2 in air for 24 h. after 24 h , the culture medium was removed from the wells and equal volumes ( 100 l ) of the extracts were added into each well . following removal of the test extracts , 100 l well dmemf12 with 10% fbs and 1% antibiotic and 12 l mtt ( tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide ) were added to each well and incubated in a dark environment for 4 h at 37c . subsequently , the absorbance at 570 nm was measured using a uv - visible spectrophotometer ( lpb pharmacia , bromma , sweden ) . the cytotoxic effects of the rlcs polymerizing with led lcu and the exposure times on the fibroblast cell 's survival rates were evaluated by two way analysis of variances . then , two independent samples were compared by t - test . the culture medium was dulbecco 's modified eagle medium ( dmem)/ham 's f12 nutrient mixture ( 1:1 ; sigma , st louis , mo , usa ) supplemented with 10% ( v / v ) foetal bovine serum ( fbs ; biochrom , berlin , germany ) without antibiotics . adherent cells at a logarithmic growth phase were controlled under an inverted tissue culture microscope ( olympus ck40 , tokyo , japan ) and detached with a mixture of 0.025% trypsin ( sigma ) and 0.02% ethylenediaminetetraacetic acid ( edta ; sigma ) , incubated for 2 - 5 min at 37c and used for cell inoculation . schaumburg , il , usa ) ; lute - it ( lt , pentron clinical technologies , l.l.c . , schaumburg , il , usa ) ; rely x arc ( ra , 3 m espe dental products , st . forty disc - shaped samples ( 6 mm diameter x 1 mm thickness ) were prepared for each tested material : 20 disc samples exposed with led lcu for 20 s and remaining 20 disc samples exposed for 40 s. standard exposure mode , providing full light intensity for the entire exposure , was chosen with both 40 s and 20 s. the led lcu had a light guide tip diameter of 8 mm with irradiance of 1200 mw / cmwith a wavelength of 430 - 480 nm according to the manufacturer . polyethylene films were added on the top surface of the luting resin cements . therefore to simulate clinical conditions , the tested materials were irradiated from the top through the ceramic discs ( 1 cm diameter x 2 mm thickness , ips empress 2 ; shade 1a , lot : d 50108 , ivoclar , vivadent , fl-9494 , schaan , liechtenstein ) by contacting the end of the light guide with its surface , through the use of led lcu for 20 s ( 50% of the recommended exposure time ) and 40 s ( 100% exposure time ) ( figure 2 ) , under aseptic conditions at laminar flow ( holten , class ii , denmark ) . the freshly prepared samples were placed immediately at the bottom of 6-well plates ( costar , cambridge ma , usa ) . schematic illustration of sample preparation the ratio of the surface area of the disc samples to the extraction volume was 0.5 cm ml which is in line with iso 10993 - 12 , in this study . the samples were placed in dmem / f12 with 10% fbs and incubated at 37c in an atmosphere of 5% co2 in air without agitation for 24 h incubation period . after the incubation , the extracts were filtered through 0.22 m cellulose acetate filters ( millipore ; sigma , st . the l-929 cell suspension with dmem / f12 with 10% fbs and 1% antibiotic was prepared at a concentration of 3 x 10 cell ml and inoculated onto 96-well cluster cell culture plates ( 100 l per well ) . the multiwell plates were incubated at 37c , 5% co2 in air for 24 h. after 24 h , the culture medium was removed from the wells and equal volumes ( 100 l ) of the extracts were added into each well . following removal of the test extracts , 100 l well dmemf12 with 10% fbs and 1% antibiotic and 12 l mtt ( tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide ) were added to each well and incubated in a dark environment for 4 h at 37c . the cytotoxic effects of the rlcs polymerizing with led lcu and the exposure times on the fibroblast cell 's survival rates were evaluated by two way analysis of variances . then , two independent samples were compared by t - test . the results in figure 3 demonstrated that all freshly prepared rlcs polymerized with led for 20 s ( 50% exposure time ) and 40 s ( 100% exposure time ) reduced the cell numbers compared to the control ( culture without sample ) . distribution of cell survival rates ( % ) according to the light exposure times ( 100% and 50% ) for each resin luting cements . cell survival rates are expressed as a percentage of controls ( cultures without samples ) . ru= rely x unicem ; dk= duolink ; lt= lute it ; in= illusion ; ra= rely x arc the cytotoxic effect of the tested materials and exposure times on the cell survival rates were evaluated by two - way analysis of variance . there was a significant two - way interaction between two factors ( f=5.856 , p=0.003 ) . therefore , the time factor levels were compared in each rlc ( two independent samples were compared by t - test ) . it was observed that the cytotoxicity of the tested materials depends on the exposure time . when the interactions between exposure times and rlcs are evaluated , both dk and lt reduced cell survival rate significantly with respect to the control for 20 s and 40 s ( p<0.001 ) ( table 1 ) . cell survival rate ( csr ) according to the light exposure times for each resin luting cement as compared to the control ( cultures without sample ) for 20 s and 40 s cell survival rate results for 40 s exposure time were significantly higher than 20 s cell survival rates of dk and lt ( t=-9.399 , p=0.001 ; and t=-20.418 , p<0.001 , respectively ) . however , in terms of cell survival rate , the 20 s ( 50% exposure time ) results for ru and in were higher than the 40 s ( 100% exposure time ) results for ra ( t=1.747 , p=0.156 ; t=0.164 , p=0.878 ; and t=-1.262 , p=0.276 , respectively ) . in the present study , l929 fibroblasts were used because they are the most common iso - approved cell type in the pulp and gingival tissues , which would be the target of chemicals released from the rlcs . several in vitro studies assessing the cytotoxicity of dental materials have used fibroblast cell lines because they represent a common cell type in pulp and gingival tissues and also because of their reproducible growth rates , and advantages in handling and availability compared to primary cells . in this study , it is important for the materials to be tested immediately after mixing/ curing to avoid the loss of toxic substances released from the tested material at this initial stage . a recent study demonstrated the relevance of the cytotoxicity of rlcs and the importance of achieving sufficient polymerization with the lcus . elipar freelight 2 , used in the present study , is a high - intensity led lcu , and has exposure time options as follows : 5 , 10 , 15 and 20 s according to the manufacturer 's instructions . because of the high light intensity ( 1200 mw / cm ) of this lcu , these time periods correspond to the time periods ( 10 , 20 , 30 and 40 s ) of conventional lcu that has light intensity of 600 - 800 mw / cmfor halogen technology or 300 - 400 mw / cmlight intensity for led . thus , the normal exposure time for conventional units can be cut in half without compromising curing performance but it could have a direct affect on biocompatibility of the rlcs . higher irradiance and shorter irradiation time was found intrinsically associated with a higher free radical termination rate than that of lower irradiance but longer irradiation time . on the other hand , there is no information about the cytotoxicity of the rlcs in half the exposure time with polymerized high power led . ( 2002 ) have addressed the application of led technology to cure restorative materials . typically , the advantages claimed for second - generation led lcus are more efficiency in cure , decreased heat from the light tip , consistent output over time without degradation and significantly longer useful life of the diodes compared with quartz tungsten halogen bulbs . furthermore , with the introduction of led lcus , it was asserted that the emission of blue led lcus is the ideal spectra for the conversion of dental materials containing monomer . in this way biocompatibility of light - cured dental materials may be affected by the quality of the lcu used . due to the advanced led technology , high power led curing light was able to polymerize dental resin in half exposure time ( 50% ) instead of 100% . ( 2008 ) reported that degree of polymerization of rlc is influenced by several factors , namely , porcelain shade and thickness , lcu and curing time . the tested samples were irradiated from top through the ceramic disc in the present study , so as to simulate clinical condition . according to goldberg ( 2008 ) , leaching of some ions may cause cell alterations , and some molecular mechanisms have been identified as key factors leading to apoptosis and/or pulp necrosis . a previous study demonstrated that the cytotoxicity ranking of the most widely used monomers was bisgma > udma > tegdma > hema > mma . furthermore , it is considered that synergistic effects of bis - gma and udma released by those tested materials lead to increased cytotoxicity on l-929 fibroblast cells . it was clear that all sufficient quantities of test materials polymerized by high power led for 20 s and 40 s leached a variety of components into cell culture medium and affected cell activity . after testing the cytotoxicity of polymerized test samples , statisticially significant differences were found regarding cell survival rates for the different exposure times and rlcs . when comparing the control cultures lt polymerized for 20 s ( 50% ) had resulted in the lowest survival rate of 28% . however , in polymerized for 20 s revealed the highest cell survival rate of 90.81% ( p<0.01 ) . in the 40 s exposure time , lt had led to the lowest survival rate ( 57.86% ) while ra had brought about the highest survival rate ( 90.91% ) compared to the control cultures ( p<0.01 ) . the results revealed that there might be several possible reasons for different effects of rlcs or exposure times ( 50% and 100% ) on their cytotoxicity such as the light transmission characteristic , the released energy during the polymerization of the rlcs and the amount as well as type of released toxic substances from the unpolymerized rlcs . generally , when the results of all samples were evaluated , materials polymerized for 20 s ( except for ru , in ) had reduced cell survival rates than the samples polymerized for 40 s with high power led . this leads to the assumption that 20-s exposure time is not sufficient for the polymerization of these materials . the obtained results indicate that the rlcs polymerized for either 20 s or 40 s with high power led may lead to unpolymerized components or toxic substances with harmful effects since they might penetrate through dentin tubules . the findings of the present study also suggest that it is important to be cautious when considering manufacturers ' recommendation that high power led light cure unit reduces the exposure time of rlcs in polymerization by 50% . based on the experimental condition , it was concluded that the period of photoactivation is related to the cytotoxic effects of the tested rlcs , and that an exposure time of 40 s is recommended for dk , lt and ra although 20 s is preferable for ru and in in operative procedures .

microsomal prostaglandin e2 synthase-1 ( mpges-1 ) is a key enzyme in the prostaglandin ( pg)e2 biosynthetic pathway within the arachidonic acid cascade . in this cascade , phospholipase a2 ( pla2 ) releases arachidonic acid from membrane phospholipids as a first step . then , cyclooxygenase ( cox)-1 and cox-2 catalyze the formation of the instable pgh2 . in a third step , prostaglandin e2 synthases ( pges ) catalyze the conversion of pgh2 to pge2 ( figure 1).(1 ) three isoforms of pges have been described : the two membrane - bound forms mpges-1 and mpges-2 , as well as the cytosolic pges ( cpges ) . cpges uses pgh2 produced by the constitutively expressed cox-1 , mpges-2 can use pgh2 produced by both cox isoforms , cox-1 , or the inducible cox-2 . the expression of both cox-2 and mpges-1 is increased in response to pro - inflammatory stimuli . studies indicate key roles of mpges-1 in a number of disease conditions such as inflammation , arthritis , fever , pain , anorexia , atherosclerosis , stroke , and cancer.(2 ) prostaglandin biosynthetic pathway.(1 ) pla2 , phospholipase a2 ; cox , cyclooxygenase ; pg , prostaglandin ; pgds , prostaglandin d2 synthase ; pges , prostaglandin e2 synthase ; pgfs , prostaglandin f2 synthase ; pgis , prostaglandin i2 synthase ; txs , thromboxane a2 synthase ; txa2 , thromboxane a2 . specific inhibition of mpges-1 is expected to interfere with inflammation - induced pge2 formation whereas physiological pge2 as well as other cox - derived prostanoids are not suppressed . the idea is that mpges-1 inhibitors may not lead to side effects commonly associated with nonsteroidal anti - inflammatory drugs ( nsaids ) and coxibs . thus , there is an increasing interest in this novel therapeutic strategy as an alternative to presently available anti - inflammatory drugs . however , to date , no pharmacological evidence for this theory in humans has been reported . although a few inhibitors are currently in clinical trials , no mpges-1 inhibitor is available on the market . several inhibitors of mpges-1 have been identified in vitro , including pg analogues and fatty acids . the highly potent indole compound 1 showed an ic50 value of 3 nm,(7 ) whereas an ic50 of 0.7 nm was determined for the phenanthrene imidazole compound 2.(4 ) compound 3 , also known as mk-886 ( ic50 = 2.4 m(10 ) ) , which was one of the first mpges-1 inhibitors , is commonly used as reference inhibitor in mpges-1 assays ( chart 1 ) . san juan and cho(11 ) as well as abdulhameed et al.(8 ) described theories on mpges-1 ligand binding in their 3d - quantitative structure activity relationship ( qsar ) studies on mpges-1 inhibitors . structures that were very similar to our training set compounds 4 and 5 were used in these studies . the overall binding site architecture was described similarly in both publications ; amino acid numbering was not consistent among these two studies . according to their results , the interaction site of mpges-1 consists of a so - called cationic site and an anionic site . in the cationic site of the receptor , there is a large hydrophobic region which may be important for the selectivity of ligands for mpges-1 . ser , thr , and/or ala residues might form hydrogen bonds with suitable substituents of the ligand . in the anionic site of the receptor , a basic arg , which was reported to have catalytic function,(12 ) is expected to interact with the ligand , ideally an acidic group . the aim of our study was to find novel inhibitors of mpges-1 using pharmacophore modeling and virtual screening . although jegerschld et al.(13 ) described the x - ray crystal structure of mpges-1 , a ligand - based modeling approach was applied . as already pointed out by rrsch and co - workers in a recent virtual screening report on nonacidic mpges-1 inhibitors,(14 ) the published x - ray structure represents a closed conformation of the binding site , which makes a structure - based virtual screening approach rather difficult . in contrast to the work of rrsch et al . , our study presents a ligand - based pharmacophore modeling and virtual screening strategy leading to novel acidic mpges-1 inhibitors . a workflow overview of this study including pharmacophore modeling , selection of compounds , and biological testing is provided in scheme 1 . a ligand - based pharmacophore model for acidic mpges-1 inhibitors model generation was based on the structural information of six acidic indole derivatives described in literature as inhibitors of mpges-1 . although these compounds were all members of the same chemical class , they were selected for model building due to their potent mpges-1 inhibition ( compounds 4 and 5 ) . literature data on other acidic inhibitors did not report such highly active compounds.(15 ) therefore , this was the most promising starting point to generate a high quality pharmacophore model . although the training compounds were so similar , novel active inhibitors were expected from pharmacophore - based searches due to the well - known scaffold hopping potential of pharmacophore models.(16 ) the training set structures were divided into three groups : highly active compounds with ic50 values in the low nanomolar range ( 4 and 5 ) were given priority one . the algorithm calculated numerous pharmacophore models based on the 3d alignment of these two structures . compounds 6 and 7 were given priority two ; hence , pharmacophore models that did not recognize them were discarded , which resulted in a smaller model collection . two structures showing activity in a micromolar range ( 8 and 9 ; priority three ) ( table 1 ) were used to identify the most valuable pharmacophore model . in this last step , the algorithm deleted models that recognized these structures with a high fit value . the best pharmacophore model consisted of six features : four hydrophobic ( h ) features , one aromatic ring ( ra ) , and one negatively ionizable ( ni ) feature . this model correctly recognized compounds 47 , and discarded 8 and 9 . to include additional information on compound size and shape , a steric constriction was added : compound 4 was fitted into the model , converted into a shape query , and merged with the chemical features of the initial pharmacophore ( figure 2 ) . ( a ) pharmacophore model for acidic mpges-1 inhibitors consisting of one aromatic ring ( ra , brown ) , one negatively ionizable group ( ni , dark blue ) , four hydrophobic features ( h36 , cyan ) , and a shape of the most potent inhibitor from the training set ( compound 4 ) . ( b ) compound 4 mapped to the pharmacophore model . to validate the ability of the pharmacophore model to differentiate between biologically active and inactive molecules , the model was screened against an mpges-1 inhibitor test set . this test set contained 10 mpges-1 inhibitors that were not part of the training set and have been obtained from different literature sources ( chart 2 ) . to this test set , 72 compounds that have been biologically tested but did not inhibit mpges-1 were added ( supporting information chart s1 ) . the pharmacophore model for mpges-1 inhibitors recognized 2 out of 10 active test set structures ( 20% ) , both indole derivatives ( table 2 ) . the fit value is a quantitative metric , that indicates how well the chemical functions of the ligand geometrically map the features of the pharmacophore model . the maximum fit value for the reported models was 6 ( each feature had a weight of 1 ) . the enrichment factor for this validation result was 8.2 , which indicated an excellent discriminatory power and usability for virtual screening . however , the retrieval of only two active molecules showed the models restriction to certain chemical scaffolds . thus , a second model was developed with the aim of broader focus on novel scaffolds . therefore , the test set compounds from chart 2 were added to the training compounds and fitted into the initial model , allowing one feature not to be mapped . according to the intention of this study to find new acidic inhibitors of mpges-1 , the shape was not modified either because the hit list should not comprise structures of very high molecular weight . thereby , all 14 compounds from table 1 and chart 2 with ic50 values < 5 m ( the two least active compounds 9 and 10 were not found ) were retrieved by the model , which means that they matched the ni feature , the shape , and four out of the five h / ra features . an analysis of mapping features did not give a clear preference of some h features or the ra feature over others . accordingly , it could not be figured out which of the remaining features ( the h ones or the ra ) are most important for ligand activity . thus , a partial query model was selected , allowing for missing any one of the h or the ra features . the partial query model retrieved 12 out of 72 inactive compounds ( s16 , s20 , s30 , s36 , s40 , s45 , s48 , s49 , s51 , s53 , s59 , s61 ) . this indicated that the partial query model with a good retrieval of active compounds ( actives hit rate 87.5% ) and sufficient selectivity against inactive ones ( inactives hit rate : 16.7% ) has a high probability to identify structurally diverse scaffolds . because our intention was not only to identify novel analogues of known mpges-1 inhibitors but also to discover new classes of chemical scaffolds , we decided to use both the selective original model and the promiscuous partial query model for virtual screening . the pharmacophore models were experimentally validated using substances provided by the national cancer institute ( nci ) , usa , and specs , the netherlands . first , the nci database ( version 2003 , comprising 247041 compounds ) was screened with the restrictive model , which led to a hit list of 81 compounds mapping all six pharmacophore model features . because of the low number of vh , the specs database ( version 09/2010 , comprising 200015 compounds ) was screened , leading to 70 vh . these 151 vh were clustered by structural diversity and inspected for reactive and nondrug - like groups . in total , 20 chemically diverse compounds were selected for biological testing ( compounds s73s82 from nci , supporting information chart s2 , compounds 2025 and s83s86 from specs , supporting information chart s3 ) . 1,337 vh were retrieved from screening of the nci database with the partial query model . they were submitted to the same selection process as described above , and 14 chemically diverse structures were selected for biological testing ( compounds s87s97 and 2628 , supporting information chart s4 ) . five out of the 34 acquired compounds ( s76 , s81 , s89 , s90 , and s97 ) could not be tested due to solubility problems in dimethyl sulfoxide ( dmso ) , ethanol , and water . the other 29 compounds were investigated in a cell - free mpges-1 assay which is based on the mpges-1-mediated enzymatic conversion of pgh2 as substrate to pge2 ; the latter was analyzed by high performance liquid chromatography ( hplc ) as described below . in a first screening round , all compounds ( solubilized in dmso ) were tested at a concentration of 10 m . the mpges-1 inhibitor 3(10 ) was used as reference control , and dmso ( 0.3% , v / v ) was used as vehicle control . as shown in figure 3a , nine compounds ( i.e. , 2028 , chart 3 ) showed significant inhibition of mpges-1 activity . the purity of these nine active compounds was determined using hplc coupled to mass spectrometry ( hplc - ms ) . inhibition of mpges-1 in cell - free assay , given as mean se residual activity in % of uninhibited control ( 100% , vehicle ) . ( a ) inhibition by test compounds ( 10 m ) , n = 310 . ( b ) concentration - dependent inhibition of mpges-1 by compounds 20 to 28 , n = 37 . a more detailed analysis of the nine active compounds in concentration response studies revealed a potent , concentration - dependent inhibition of mpges-1 activity by 26 and 28 with ic50 values of 0.4 and 0.5 m , respectively . compounds 2025 and 27 were less active in this test system with ic50 values in the range of 2.37.9 m , respectively ( figure 3b , table 3 ) . compounds 2025 were found by the restrictive model and 2628 by the partial query model , respectively . both pharmacophore models for acidic inhibitors of mpges-1 were therefore successfully experimentally validated with a total hit rate of 31% active compounds in the virtual screening hit lists . the structures of the nine identified mpges-1 inhibitors are chemically diverse , which proves that the pharmacophore models are applicable to scaffold hopping . these results confirm the predictive power of the models , which will be used in further virtual screening experiments . three identified active compounds omitted one feature of the restrictive pharmacophore model for acidic mpges-1 inhibitors , which suggests that they do not cover the whole mpges-1 binding site . thus , compounds 26 , 27 , and 28 omitted the h feature h6 , the ra , and the h feature h4 , respectively ( figure 4 ) . neglecting such diverse features does not indicate which one of these might be of less importance for the bioactivity . chen et al.(22 ) suggested in their study on 3d - qsar pharmacophore mapping of 35 nonacidic mpges-1 inhibitors that a hydrogen bond donor and three h features are crucial for ligand activity . some of the generated qsar models also included ra features , occasionally replacing an h feature . in our model , a ra feature overlapped with an h feature , thereby supporting the results from chen et al.(22 ) in the absence of a cocrystal structure with an inhibitor , the actual impact of hydrophobic or aromatic functionality is hard to predict at this step . fitting of the most active mpges-1 inhibitors 26 ( a ) and 28 ( b ) into the mpges-1 partial query pharmacophore model . as a comparison of the ligand - based pharmacophore models with information from the x - ray crystal structure , induced fit docking experiments were performed with compounds from literature as well as with the most active , newly identified inhibitor 26 . docking of compound 4 and the analysis of predicted protein ligand interactions confirmed the locations of ni and h chemical features contained in the pharmacophore models . for the ra feature , no corresponding protein docking of compound 26 resulted in a binding mode that is shown in figure 6 . more detailed information on the molecular docking experiments performed in this study is given in the supporting information . predicted binding mode of compound 4 to mpges-1 . ( b ) protein ligand interactions corresponding to the ligand - based pharmacophore features . chemical interactions are color - coded : hydrophobic , yellow ; negative charge , red . arg110 and arg126 from the basic subpocket are shown in ball - and - stick style . ( b ) protein ligand interactions corresponding to the ligand - based pharmacophore features . chemical interactions are color - coded : hydrophobic , yellow ; negative charge , red . previous studies with other mpges-1 inhibitors showed that some of them also interfere with the activity of 5-lipoxygenase ( 5-lo ) . such dual mpges-1/5-lo inhibitors are , for instance , licofelone and pirinixic acid derivatives.(17 ) thus , the newly identified mpges-1 inhibitors were further investigated concerning their potential to interfere with 5-lo activity . 5-lo catalyzes the initial steps in the biosynthesis of leukotrienes ( lts ) , which are further central mediators in inflammatory reactions , as reviewed by peters - golden and henderson.(25 ) inhibition of the activity of human recombinant 5-lo in a cell - free assay by compounds 2028 was determined . compound 20 was inactive in this test system , but the remaining eight compounds suppressed 5-lo activity in a concentration - dependent manner . compound 21 even showed to be highly active with an ic50 = 0.8 m , comparable to the control 5-lo inhibitor s98 ( bwa4c , supporting information chart s5 ) . to confirm the bioactivity of the compounds on 5-lo , human polymorphonuclear leukocytes ( pmnl ) expressing 5-lo were used to investigate 5-lo inhibition in a cell - based test system . therein , compounds 2023 and 2528 ( at a concentration of 10 m , respectively ) proved to inhibit 5-lo with residual activity below 50% of control , whereas compound 24 was hardly active ( table 3 ) . further experiments at different concentrations of the inhibitors were carried out in order to determine the ic50 values . 5-lo product synthesis was reduced concentration - dependently by all nine inhibitors ( figure 7 ) . of interest , compound 25 was the most potent inhibitor in this test system with an ic50 = 0.85 m . an overview of the pharmacological results determined within this study is provided in table 3 . concentration - dependent inhibition of 5-lo product formation in human pmnl by compounds 2028 , given as mean se residual activity in % of uninhibited control ( 100% , vehicle ) ; n = 4 . finally , the ability of the most potent novel mpges-1 inhibitors ( i.e. , compounds 26 and 28 ) to induce cytotoxicity was determined . neither compound 26 nor compound 28 ( 10 m , each ) reduced the viability of human lung epithelial carcinoma a549 cells or leukemic jurkat a3 t lymphocytes ( supporting information figure s1 ) . within this study , pharmacophore modeling and virtual screening led to the identification of novel dual inhibitors of mpges-1 and 5-lo activity . therefore , our novel pharmacophore models are valuable tools for selecting test compounds from various databases . the nine identified active compounds 2028 were not only strong inhibitors of mpges-1 in cell - free assays but were also able to inhibit 5-lo in cell - free assays and/or in intact cells . on the one hand , selective inhibitors are desired to learn more about the target and its binding site . on the other hand , simultaneous inhibition of several physiologically related targets supports the multitarget idea.(26 ) the interference with several anti - inflammatory targets from the arachidonic acid cascade should provide benefits in pharmacotherapy in terms of synergistic therapeutic effects as well as reduction of the incidence of typical nsaid - related side effects . future studies will address the investigation of the overall pharmacological profile of compounds 2028 in more detail and will aim to assess their anti - inflammatory activity in vivo . pharmacophore modeling and virtual screening experiments were performed using catalyst 4.11 software ( accelrys inc . , structural clustering of vh compounds was calculated using pipelinepilot version 7.5 ( accelrys inc . , san diego , ca , usa ) . all compounds from the training and test sets as well as the nci and the specs compound collection underwent 3d structure generation , minimization , and conformational analysis before the pharmacophore model development and virtual screening . the ligands from the training and the active compounds from the test set were built using the view compound workbench module of catalyst 4.11 . compounds were optimized in 3d geometry and energetically minimized using the charmm force field implemented in catalyst . a maximum of 250 conformers per ligand with a maximal energy threshold of 20 kcal / mol above the calculated energy minimum was computed using catalyst s best conformation generation mode . the nci database was downloaded from the nci download page ( dtp.nci.nih.gov/docs/3d_database/structural_information/structural_data.html ) and converted into a 3d multiconformational database using the catdb module of catalyst 4.11 . for each molecule , the conformational model for the database entries was calculated using the catdb module and fast mode , with up to 100 conformers per molecule . in addition , 72 structurally unique compounds were found in literature that showed no inhibitory activity for mpges-1 in biological assays . energetically minimized 3d structures and conformers for the 10 active and 72 inactive molecules of the test set were generated as mentioned above . for model building and refinement , the hiphop refine algorithm of catalyst 4.11 was employed . this algorithm considers a maximum of five different chemical feature types . on the basis of an analysis of the chemical features present in the training set structures , hydrogen bond acceptor , hydrogen bond donor , h , ra , and ni features a high principal value of 2 gives the molecule top priority and labels it as a reference molecule of which all chemical features are considered in building the pharmacophore space during model generation . the principal value of 1 labels a molecule as moderately active . finally , a principal value of 0 indicates low or absent activity of this molecule . those compounds are not considered for common feature pharmacophore model building . a maxomitfeat value of 0 indicates that all features in the generated model must map the compound . maxomitfeat 1 allows that all but one of the features in the generated pharmacophore must map the compound . a maxomitfeat value of 2 indicates that no features from the model need to map the compound . for the calculation of the mpges-1 model , compounds 4 and 5 had a principal value of 2 and a maxomitfeat value of 0 . compounds 6 and 7 were assigned principal and maxomitfeat values of 1 , while compounds 8 and 9 were given a principal value of 0 and a maxomitfeat value of 2 . the shape of compound 4 was generated by mapping the molecule into the initial pharmacophore model using the compare / fit tool of catalyst 4.11 with 0 allowed omitted features . the best fitting conformation , which showed the maximum possible fit value , was transformed into a shape query and merged with the chemical features of the initial model . virtual screening of the test set was performed using the fast ( rigid ) search algorithm of catalyst 4.11 . mode , which allows limited conformational flexibility of the ligand while fitting to the pharmacophore model . the nci and the specs databases were screened using the same settings as for the test set compound screening . the script converted the structures into scitegic s ecfps and determined the similarity between these fingerprints applying the tanimoto coefficient . from each cluster , compounds were mapped to the model for visual inspection . test substances used for the biological investigation were either purchased from specs , the netherlands , or kindly provided by the drug synthesis and chemistry branch , developmental therapeutics program , division of cancer treatment and diagnosis , nci , usa . the purity of compounds 2028 , which inhibited mpges-1 formation with an ic50 below 10 m , was determined by hplc - ms to be 95% . g / l ) medium , rpmi medium , penicillin , streptomycin , trypsin / ethylenediaminetetraacetate ( edta ) solution , n-2-hydroxyethylpiperazine - n-2-ethanesulfonic acid ( hepes ) , and lsm 1077 lymphocyte separation medium were obtained from paa ( pasching , austria ) . il-1 was obtained from reprotech ( hamburg , germany ) . fetal calf serum ( fcs ) , phenylmethanesulphonyl fluoride ( pmsf ) , leupeptin , soybean trypsin inhibitor ( sti ) , glutathione ( reduced ) , compound s98 , pgb1 , lysozyme , ca - ionophore a23187 , arachidonic acid , thiazolyl blue tetrazolium bromide ( mtt ) , and cycloheximide were obtained from sigma - aldrich ( deisenhofen , germany ) . compound 3 and 11-pge2 were obtained from cayman chemical ( ann arbor , mi ) . pgh2 , adenosine triphosphate ( atp ) , isopropyl--d-1-thiogalactopyranoside ( iptg ) , dextrane , and staurosporine were obtained from larodan ( malmoe , sweden ) , roche diagnostics ( mannheim , germany ) , applichem ( darmstadt , germany ) , fluka ( neu - ulm , germany ) , and calbiochem ( san diego , ca ) , respectively . a549 and jurkat a3 cells were provided by the karolinska institute ( stockholm , sweden ) and dr . leukocyte concentrates from human healthy volunteers were provided by the institute for clinical transfusion medicine ( university hospital tuebingen , germany ) . cells were cultured in the respective media at 37 c in a 6% co2 incubator . jurkat a3 cells were grown in rpmi medium containing heat - inactivated fcs ( 10% , v / v ) , hepes ( 10 mm ) , penicillin ( 100 u / ml ) , and streptomycin ( 100 g / ml ) and reseeded with a density of 2 10 cells / ml medium after three days . a549 cells were grown in dmem high glucose ( 4.5 g / ml ) medium supplemented with heat - inactivated fcs ( 10% , v / v ) , penicillin ( 100 u / ml ) , and streptomycin ( 100 g / ml ) . after three days , confluent cells were detached using 1 trypsin / edta and reseeded with a density of 10 cells / ml medium . preparation of a549 cells and determination of mpges-1 activity was performed as described previously.(24 ) in brief , a549 cells were treated with 1 ng / ml interleukin-1 for 48 h at 37 c and 5% co2 . after sonification , the homogenate was subjected to differential centrifugation at 10000 g for 10 min and 174000 g for 1 h at 4 c . the pellet ( microsomal fraction ) was resuspended in 1 ml homogenization buffer ( 0.1 m potassium phosphate buffer ph 7.4 , 1 mm pmsf , 60 g / ml sti , 1 g / ml leupeptin , 2.5 mm glutathione , and 250 mm sucrose ) , and the total protein concentration was determined . microsomal membranes were diluted in potassium phosphate buffer ( 0.1 m , ph 7.4 ) containing 2.5 mm glutathione . test compounds or vehicle were added , and after 15 min at 4 c , the reaction ( 100 l total volume ) was initiated by addition of pgh2 ( 20 m , final concentration , unless stated otherwise ) . after 1 min at 4 c , the reaction was terminated using stop solution ( 100 l ; 40 mm fecl2 , 80 mm citric acid , and 10 m of 11-pge2 as internal standard ) . pge2 was separated by solid phase extraction and analyzed by rp - hplc as described.(24 ) docking was performed using the induced fit docking module available within maestro ( www.schrdinger.com ) . the x - ray crystal structure of mpges-1 , which does not include a substrate or inhibitor of the enzyme and is crystallized in a closed conformation , was derived from the pdb ( code 3dww)(13 ) and preprocessed using the protein preparation wizard of maestro software suite . details on ligand and protein preparation , setting for induced fit docking , and docking validation are available as supporting information . e. coli mv1190 was transformed with pt35-lo plasmid expressing human recombinant 5-lo and the protein was expressed at 27 c as described.(27 ) cells were lysed in 50 mm triethanolamine / hcl ph 8.0 , 5 mm edta , sti ( 60 g / ml ) , 1 mm pmsf , and lysozyme ( 500 g / ml ) , homogenized by sonication ( 3 15 s ) , and centrifuged at 40000 g for 20 min at 4 c . the 40000 g supernatant ( s40 ) was applied to an atp - agarose column to partially purify 5-lo as described previously.(27 ) semipurified 5-lo was immediately used for activity assays . aliquots of semipurified 5-lo ( 0.5 g ) were diluted with ice - cold pbs containing 1 mm edta , and 1 mm atp was added ; final volume was 1 ml . samples were preincubated with the test compounds as indicated . after 10 min at 4 c , samples were prewarmed for 30 s at 37 c , and 2 mm cacl2 plus 20 m aa was added to start 5-lo product formation . the reaction was stopped after 10 min at 37 c by addition of 1 ml of ice - cold methanol , and the formed metabolites were analyzed by rp - hplc as described.(28 ) 5-lo products include the all - trans isomers of ltb4 and 5(s)-hydro(pero)xy-6-trans-8,11,14-cis - eicosatetraenoic acid . pmnl were freshly isolated from leukocyte concentrates obtained at the blood center of the university hospital tuebingen ( germany ) as described.(29 ) in brief , venous blood was taken from healthy adult donors that did not take any medication for at least 7 days , and leukocyte concentrates were prepared by centrifugation ( 4000 g , 20 min , 20 c ) . pmnl were immediately isolated from the pellet after centrifugation on nycoprep cushions , and hypotonic lysis of erythrocytes was performed . pmnl were finally resuspended in pbs ph 7.4 ( pbs ) containing 1 mg / ml glucose and 1 mm cacl2 ( pgc buffer ) ( purity > 9697% ) . freshly isolated pmnl ( 10/ml pgc buffer ) were preincubated with the test compounds for 15 min at 37 c , and 5-lo product formation was started by addition of 2.5 m ionophore a23187 plus 20 m aa . after 10 min at 37 c , the reaction was stopped with 1 ml of methanol and 30 l of 1 n hcl , and then 200 ng pgb1 and 500 l pbs were added . formed 5-lo metabolites were extracted and analyzed by hplc as described.(28 ) 5-lo products include ltb4 and its all - trans isomers , and 5(s)-hydro(pero)xy-6-trans-8,11,14-cis - eicosatetraenoic acid . cysteinyl - lts c4 , d4 , and e4 were not detected , and oxidation products of ltb4 were not determined . cell viability was assessed by trypan blue staining and light microscopy as well as by mtt assay . for mtt assay , jurkat a3 ( 3 10 cells / ml ) or a549 cells ( 4 10 ) were plated in a 75 cm cell culture flask and incubated at 37 c and 5% co2 for 72 h. then , test compounds were added and the incubation was continued for 24 h before cell viability was determined as described.(30 ) the cytotoxic compounds cycloheximide ( chx ) and staurosporine ( stauro ) were used as controls .

microsomal prostaglandin e2 synthase-1 ( mpges-1 ) catalyzes prostaglandin e2 formation and is considered as a potential anti - inflammatory pharmacological target . to identify novel chemical scaffolds active on this enzyme , two pharmacophore models for acidic mpges-1 inhibitors were developed and theoretically validated using information on mpges-1 inhibitors from literature . the models were used to screen chemical databases supplied from the national cancer institute ( nci ) and the specs . out of 29 compounds selected for biological evaluation , nine chemically diverse compounds caused concentration - dependent inhibition of mpges-1 activity in a cell - free assay with ic50 values between 0.4 and 7.9 m , respectively . further pharmacological characterization revealed that also 5-lipoxygenase ( 5-lo ) was inhibited by most of these active compounds in cell - free and cell - based assays with ic50 values in the low micromolar range . together , nine novel chemical scaffolds inhibiting mpges-1 are presented that may possess anti - inflammatory properties based on the interference with eicosanoid biosynthesis .

Introduction Results and Discussion Conclusions Experimental Section

microsomal prostaglandin e2 synthase-1 ( mpges-1 ) is a key enzyme in the prostaglandin ( pg)e2 biosynthetic pathway within the arachidonic acid cascade . in this cascade , phospholipase a2 ( pla2 ) releases arachidonic acid from membrane phospholipids as a first step . in a third step , prostaglandin e2 synthases ( pges ) catalyze the conversion of pgh2 to pge2 ( figure 1). the expression of both cox-2 and mpges-1 is increased in response to pro - inflammatory stimuli . studies indicate key roles of mpges-1 in a number of disease conditions such as inflammation , arthritis , fever , pain , anorexia , atherosclerosis , stroke , and cancer. (1 ) pla2 , phospholipase a2 ; cox , cyclooxygenase ; pg , prostaglandin ; pgds , prostaglandin d2 synthase ; pges , prostaglandin e2 synthase ; pgfs , prostaglandin f2 synthase ; pgis , prostaglandin i2 synthase ; txs , thromboxane a2 synthase ; txa2 , thromboxane a2 . specific inhibition of mpges-1 is expected to interfere with inflammation - induced pge2 formation whereas physiological pge2 as well as other cox - derived prostanoids are not suppressed . the idea is that mpges-1 inhibitors may not lead to side effects commonly associated with nonsteroidal anti - inflammatory drugs ( nsaids ) and coxibs . thus , there is an increasing interest in this novel therapeutic strategy as an alternative to presently available anti - inflammatory drugs . although a few inhibitors are currently in clinical trials , no mpges-1 inhibitor is available on the market . several inhibitors of mpges-1 have been identified in vitro , including pg analogues and fatty acids . (4 ) compound 3 , also known as mk-886 ( ic50 = 2.4 m(10 ) ) , which was one of the first mpges-1 inhibitors , is commonly used as reference inhibitor in mpges-1 assays ( chart 1 ) . (8 ) described theories on mpges-1 ligand binding in their 3d - quantitative structure activity relationship ( qsar ) studies on mpges-1 inhibitors . structures that were very similar to our training set compounds 4 and 5 were used in these studies . in the cationic site of the receptor , there is a large hydrophobic region which may be important for the selectivity of ligands for mpges-1 . in the anionic site of the receptor , a basic arg , which was reported to have catalytic function,(12 ) is expected to interact with the ligand , ideally an acidic group . the aim of our study was to find novel inhibitors of mpges-1 using pharmacophore modeling and virtual screening . (13 ) described the x - ray crystal structure of mpges-1 , a ligand - based modeling approach was applied . as already pointed out by rrsch and co - workers in a recent virtual screening report on nonacidic mpges-1 inhibitors,(14 ) the published x - ray structure represents a closed conformation of the binding site , which makes a structure - based virtual screening approach rather difficult . , our study presents a ligand - based pharmacophore modeling and virtual screening strategy leading to novel acidic mpges-1 inhibitors . a ligand - based pharmacophore model for acidic mpges-1 inhibitors model generation was based on the structural information of six acidic indole derivatives described in literature as inhibitors of mpges-1 . although these compounds were all members of the same chemical class , they were selected for model building due to their potent mpges-1 inhibition ( compounds 4 and 5 ) . literature data on other acidic inhibitors did not report such highly active compounds. although the training compounds were so similar , novel active inhibitors were expected from pharmacophore - based searches due to the well - known scaffold hopping potential of pharmacophore models. (16 ) the training set structures were divided into three groups : highly active compounds with ic50 values in the low nanomolar range ( 4 and 5 ) were given priority one . the algorithm calculated numerous pharmacophore models based on the 3d alignment of these two structures . compounds 6 and 7 were given priority two ; hence , pharmacophore models that did not recognize them were discarded , which resulted in a smaller model collection . two structures showing activity in a micromolar range ( 8 and 9 ; priority three ) ( table 1 ) were used to identify the most valuable pharmacophore model . to include additional information on compound size and shape , a steric constriction was added : compound 4 was fitted into the model , converted into a shape query , and merged with the chemical features of the initial pharmacophore ( figure 2 ) . ( a ) pharmacophore model for acidic mpges-1 inhibitors consisting of one aromatic ring ( ra , brown ) , one negatively ionizable group ( ni , dark blue ) , four hydrophobic features ( h36 , cyan ) , and a shape of the most potent inhibitor from the training set ( compound 4 ) . this test set contained 10 mpges-1 inhibitors that were not part of the training set and have been obtained from different literature sources ( chart 2 ) . the pharmacophore model for mpges-1 inhibitors recognized 2 out of 10 active test set structures ( 20% ) , both indole derivatives ( table 2 ) . however , the retrieval of only two active molecules showed the models restriction to certain chemical scaffolds . according to the intention of this study to find new acidic inhibitors of mpges-1 , the shape was not modified either because the hit list should not comprise structures of very high molecular weight . thereby , all 14 compounds from table 1 and chart 2 with ic50 values < 5 m ( the two least active compounds 9 and 10 were not found ) were retrieved by the model , which means that they matched the ni feature , the shape , and four out of the five h / ra features . the partial query model retrieved 12 out of 72 inactive compounds ( s16 , s20 , s30 , s36 , s40 , s45 , s48 , s49 , s51 , s53 , s59 , s61 ) . this indicated that the partial query model with a good retrieval of active compounds ( actives hit rate 87.5% ) and sufficient selectivity against inactive ones ( inactives hit rate : 16.7% ) has a high probability to identify structurally diverse scaffolds . because our intention was not only to identify novel analogues of known mpges-1 inhibitors but also to discover new classes of chemical scaffolds , we decided to use both the selective original model and the promiscuous partial query model for virtual screening . the pharmacophore models were experimentally validated using substances provided by the national cancer institute ( nci ) , usa , and specs , the netherlands . first , the nci database ( version 2003 , comprising 247041 compounds ) was screened with the restrictive model , which led to a hit list of 81 compounds mapping all six pharmacophore model features . because of the low number of vh , the specs database ( version 09/2010 , comprising 200015 compounds ) was screened , leading to 70 vh . in total , 20 chemically diverse compounds were selected for biological testing ( compounds s73s82 from nci , supporting information chart s2 , compounds 2025 and s83s86 from specs , supporting information chart s3 ) . they were submitted to the same selection process as described above , and 14 chemically diverse structures were selected for biological testing ( compounds s87s97 and 2628 , supporting information chart s4 ) . five out of the 34 acquired compounds ( s76 , s81 , s89 , s90 , and s97 ) could not be tested due to solubility problems in dimethyl sulfoxide ( dmso ) , ethanol , and water . the other 29 compounds were investigated in a cell - free mpges-1 assay which is based on the mpges-1-mediated enzymatic conversion of pgh2 as substrate to pge2 ; the latter was analyzed by high performance liquid chromatography ( hplc ) as described below . in a first screening round , all compounds ( solubilized in dmso ) were tested at a concentration of 10 m . the mpges-1 inhibitor 3(10 ) was used as reference control , and dmso ( 0.3% , v / v ) was used as vehicle control . as shown in figure 3a , nine compounds ( i.e. , 2028 , chart 3 ) showed significant inhibition of mpges-1 activity . the purity of these nine active compounds was determined using hplc coupled to mass spectrometry ( hplc - ms ) . inhibition of mpges-1 in cell - free assay , given as mean se residual activity in % of uninhibited control ( 100% , vehicle ) . ( b ) concentration - dependent inhibition of mpges-1 by compounds 20 to 28 , n = 37 . a more detailed analysis of the nine active compounds in concentration response studies revealed a potent , concentration - dependent inhibition of mpges-1 activity by 26 and 28 with ic50 values of 0.4 and 0.5 m , respectively . compounds 2025 and 27 were less active in this test system with ic50 values in the range of 2.37.9 m , respectively ( figure 3b , table 3 ) . compounds 2025 were found by the restrictive model and 2628 by the partial query model , respectively . both pharmacophore models for acidic inhibitors of mpges-1 were therefore successfully experimentally validated with a total hit rate of 31% active compounds in the virtual screening hit lists . the structures of the nine identified mpges-1 inhibitors are chemically diverse , which proves that the pharmacophore models are applicable to scaffold hopping . three identified active compounds omitted one feature of the restrictive pharmacophore model for acidic mpges-1 inhibitors , which suggests that they do not cover the whole mpges-1 binding site . thus , compounds 26 , 27 , and 28 omitted the h feature h6 , the ra , and the h feature h4 , respectively ( figure 4 ) . neglecting such diverse features does not indicate which one of these might be of less importance for the bioactivity . (22 ) suggested in their study on 3d - qsar pharmacophore mapping of 35 nonacidic mpges-1 inhibitors that a hydrogen bond donor and three h features are crucial for ligand activity . fitting of the most active mpges-1 inhibitors 26 ( a ) and 28 ( b ) into the mpges-1 partial query pharmacophore model . as a comparison of the ligand - based pharmacophore models with information from the x - ray crystal structure , induced fit docking experiments were performed with compounds from literature as well as with the most active , newly identified inhibitor 26 . docking of compound 4 and the analysis of predicted protein ligand interactions confirmed the locations of ni and h chemical features contained in the pharmacophore models . for the ra feature , no corresponding protein docking of compound 26 resulted in a binding mode that is shown in figure 6 . more detailed information on the molecular docking experiments performed in this study is given in the supporting information . ( b ) protein ligand interactions corresponding to the ligand - based pharmacophore features . arg110 and arg126 from the basic subpocket are shown in ball - and - stick style . ( b ) protein ligand interactions corresponding to the ligand - based pharmacophore features . previous studies with other mpges-1 inhibitors showed that some of them also interfere with the activity of 5-lipoxygenase ( 5-lo ) . (17 ) thus , the newly identified mpges-1 inhibitors were further investigated concerning their potential to interfere with 5-lo activity . 5-lo catalyzes the initial steps in the biosynthesis of leukotrienes ( lts ) , which are further central mediators in inflammatory reactions , as reviewed by peters - golden and henderson. (25 ) inhibition of the activity of human recombinant 5-lo in a cell - free assay by compounds 2028 was determined . compound 20 was inactive in this test system , but the remaining eight compounds suppressed 5-lo activity in a concentration - dependent manner . compound 21 even showed to be highly active with an ic50 = 0.8 m , comparable to the control 5-lo inhibitor s98 ( bwa4c , supporting information chart s5 ) . to confirm the bioactivity of the compounds on 5-lo , human polymorphonuclear leukocytes ( pmnl ) expressing 5-lo were used to investigate 5-lo inhibition in a cell - based test system . therein , compounds 2023 and 2528 ( at a concentration of 10 m , respectively ) proved to inhibit 5-lo with residual activity below 50% of control , whereas compound 24 was hardly active ( table 3 ) . further experiments at different concentrations of the inhibitors were carried out in order to determine the ic50 values . 5-lo product synthesis was reduced concentration - dependently by all nine inhibitors ( figure 7 ) . concentration - dependent inhibition of 5-lo product formation in human pmnl by compounds 2028 , given as mean se residual activity in % of uninhibited control ( 100% , vehicle ) ; n = 4 . finally , the ability of the most potent novel mpges-1 inhibitors ( i.e. neither compound 26 nor compound 28 ( 10 m , each ) reduced the viability of human lung epithelial carcinoma a549 cells or leukemic jurkat a3 t lymphocytes ( supporting information figure s1 ) . within this study , pharmacophore modeling and virtual screening led to the identification of novel dual inhibitors of mpges-1 and 5-lo activity . therefore , our novel pharmacophore models are valuable tools for selecting test compounds from various databases . the nine identified active compounds 2028 were not only strong inhibitors of mpges-1 in cell - free assays but were also able to inhibit 5-lo in cell - free assays and/or in intact cells . on the other hand , simultaneous inhibition of several physiologically related targets supports the multitarget idea. (26 ) the interference with several anti - inflammatory targets from the arachidonic acid cascade should provide benefits in pharmacotherapy in terms of synergistic therapeutic effects as well as reduction of the incidence of typical nsaid - related side effects . future studies will address the investigation of the overall pharmacological profile of compounds 2028 in more detail and will aim to assess their anti - inflammatory activity in vivo . all compounds from the training and test sets as well as the nci and the specs compound collection underwent 3d structure generation , minimization , and conformational analysis before the pharmacophore model development and virtual screening . the ligands from the training and the active compounds from the test set were built using the view compound workbench module of catalyst 4.11 . the nci database was downloaded from the nci download page ( dtp.nci.nih.gov/docs/3d_database/structural_information/structural_data.html ) and converted into a 3d multiconformational database using the catdb module of catalyst 4.11 . on the basis of an analysis of the chemical features present in the training set structures , hydrogen bond acceptor , hydrogen bond donor , h , ra , and ni features a high principal value of 2 gives the molecule top priority and labels it as a reference molecule of which all chemical features are considered in building the pharmacophore space during model generation . maxomitfeat 1 allows that all but one of the features in the generated pharmacophore must map the compound . a maxomitfeat value of 2 indicates that no features from the model need to map the compound . for the calculation of the mpges-1 model , compounds 4 and 5 had a principal value of 2 and a maxomitfeat value of 0 . the nci and the specs databases were screened using the same settings as for the test set compound screening . the purity of compounds 2028 , which inhibited mpges-1 formation with an ic50 below 10 m , was determined by hplc - ms to be 95% . pgh2 , adenosine triphosphate ( atp ) , isopropyl--d-1-thiogalactopyranoside ( iptg ) , dextrane , and staurosporine were obtained from larodan ( malmoe , sweden ) , roche diagnostics ( mannheim , germany ) , applichem ( darmstadt , germany ) , fluka ( neu - ulm , germany ) , and calbiochem ( san diego , ca ) , respectively . a549 and jurkat a3 cells were provided by the karolinska institute ( stockholm , sweden ) and dr . cells were cultured in the respective media at 37 c in a 6% co2 incubator . jurkat a3 cells were grown in rpmi medium containing heat - inactivated fcs ( 10% , v / v ) , hepes ( 10 mm ) , penicillin ( 100 u / ml ) , and streptomycin ( 100 g / ml ) and reseeded with a density of 2 10 cells / ml medium after three days . preparation of a549 cells and determination of mpges-1 activity was performed as described previously. the pellet ( microsomal fraction ) was resuspended in 1 ml homogenization buffer ( 0.1 m potassium phosphate buffer ph 7.4 , 1 mm pmsf , 60 g / ml sti , 1 g / ml leupeptin , 2.5 mm glutathione , and 250 mm sucrose ) , and the total protein concentration was determined . microsomal membranes were diluted in potassium phosphate buffer ( 0.1 m , ph 7.4 ) containing 2.5 mm glutathione . test compounds or vehicle were added , and after 15 min at 4 c , the reaction ( 100 l total volume ) was initiated by addition of pgh2 ( 20 m , final concentration , unless stated otherwise ) . the x - ray crystal structure of mpges-1 , which does not include a substrate or inhibitor of the enzyme and is crystallized in a closed conformation , was derived from the pdb ( code 3dww)(13 ) and preprocessed using the protein preparation wizard of maestro software suite . e. coli mv1190 was transformed with pt35-lo plasmid expressing human recombinant 5-lo and the protein was expressed at 27 c as described. the reaction was stopped after 10 min at 37 c by addition of 1 ml of ice - cold methanol , and the formed metabolites were analyzed by rp - hplc as described. pmnl were immediately isolated from the pellet after centrifugation on nycoprep cushions , and hypotonic lysis of erythrocytes was performed . for mtt assay , jurkat a3 ( 3 10 cells / ml ) or a549 cells ( 4 10 ) were plated in a 75 cm cell culture flask and incubated at 37 c and 5% co2 for 72 h. then , test compounds were added and the incubation was continued for 24 h before cell viability was determined as described. (30 ) the cytotoxic compounds cycloheximide ( chx ) and staurosporine ( stauro ) were used as controls .

the composite international diagnostic interview ( cidi ) is a comprehensive and fully standardized diagnostic interview , primarily for use in epidemiological surveys and mental health research . this instrument has not only been used worldwide in numerous epidemiological studies , but it was also psychometrically explored and reviewed in a considerable number of studies . although standardized diagnostic interviews such as cidi have been shown to be quite reliable in various cultural settings and populations , very few studies have examined the clinical validity of these diagnostic interviews in chinese population . therefore , there are still considerable concerns about the cross - cultural validity of diagnoses generated from these instruments when compared with careful and well - documented diagnoses assigned by clinical psychiatrists . acceptable , but less promising estimates have also been obtained for some diagnoses in a few validity studies with quite variable designs in clinical samples and considerably less frequently in general population samples . consistently poor validity estimates were obtained for several types of mental disorders , whereas anxiety and depressive disorders were demonstrated across studies to have good validity estimates , with kappa values of 0.5 or above . given the widespread use of the cidi in chinese epidemiological studies , there is an urgent need to determine the degree of concordance and the types of discrepancies between cidi and clinical diagnoses made by mental health professionals . this paper presents the data from a systematic comparison of the translated cidi version , known as the cidi-3.0 , in an unselected clinical sample of patients with mental disorders , by comparing cidi / diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) diagnoses with those assigned by the treating clinical psychiatrists . the aim of this study was to investigate the validity of the cidi in relation to the structured clinical interview for dsm - iv axis i disorders ( scid - i ) , a gold standard the protocol was approved by the institutional reviewed board of institution of mental health , peking university . translation of the composite international diagnostic interview there are several version of cidi in china . the standard procedure of back - translation was followed for the cross - cultural adaptation of an original english psychometric instrument . a team of experts completed the initial translation and a separate team then carried out an independent back - translation to confirm preservation of the meaning of the original english version . an expert panel of four academic psychiatrists evaluated its content validity , tested it with chinese patients , and revised it to ensure its questions easily understandable and practically answerable by lay people . in the pilot study of the validity of cidi , the sensitivity ranged from 65.6% ( mood disorders ) to 87.0% ( substance use disorder ) and the specificity ranged from 83.3% ( anxiety disorders ) to 98.3% ( substance use disorders ) . ( = 0.05 , = 0.15 ) . to achieve adequate representation of the major mental disorders and sufficient nonpatient controls , the study was designed to include at least 71 cases with major depressive disorder , 30 with mania , 40 with anxiety disorder , 20 with an alcohol dependence disorder , 20 with eating disorder , and 60 healthy community - residing adults . all patients were consecutively recruited from two participating psychiatric institutions between september 2006 and february 2008 . exclusion criteria were age under 16 and having difficulties in completing cidi interview because of functional and cognitive disabilities including hearing and reading impairments , dementia , mental retardation , serious medical illnesses , marked and persisting hallucinations , thought disorders and disorganized speech . a total of 194 patients , 46 were excluded , either because they satisfied the exclusion criteria or because they did not complete the interview , leaving 148 patients completed the study . the healthy controls were selected from communities nearby the two participating psychiatric institutions , and they voluntarily participated in the study . the two psychiatrists were fully licensed psychiatrists with at least 3 years of clinical experiences in psychiatry and had received an intensive training in the use of scid - i . the six lay interviewers received 2-week training in the use of cidi that was provide by the beijing cidi training and resource center and completed at least 20 interviews under the supervision of experienced cidi trainers . design and the clinical assessment procedure clinicians diagnoses the two psychiatrists interviewed all subjects with scid - i according to the definitions and criteria of dsm - iv , blinded to the results of cidi interviews . both were informed about aim and procedures of the study and the necessity of a complete and detailed documentation of the psychopathology , course of illness and final diagnostic decision for each participating patient ; they were also encouraged to consult the dsm - iv manual and use all information available ( e.g. , history of psychiatric illnesses and treatment ) whenever necessary . all probable and definite lifetime diagnoses recorded in scid - i interview charts were used as the gold standard in the final analyses . discrepancy and case - by - case review after analyzing the diagnostic concordance , one psychiatrist reviewed interview records of all discrepant cases , in an attempt to identify why either the cidi did not pick up significant psychopathology or why the psychiatrist did not confirm the cidi diagnoses . the validity of cidi against scid - i was assessed using the sensitivity ( sn ) , specificity ( sp ) , positive predictive value ( ppv ) , and negative predictive value ( npv ) . the raw data were also cross tabulated . individual - level cidi - scid diagnostic concordance was evaluated using two different descriptive measures : the area under the receiver operator characteristic curve ( auc ) and cohen 's k. although k is the most widely used measure of concordance in validity studies of mental disorders , it has been criticized for its dependency on prevalence . various populations are different in prevalence when the populations do not differ in sn or sp . as sn and sp are considered to be fundamental parameters , this means that the comparisons across different populations can not be used to evaluate the cross - population performance of a test . the odds ratio ( or ) meets this requirement , as or = ( sn sp)/([1 sn ] [ 1 sp ] ) . however , the upper end of the or is unbounded , making it difficult to evaluate the extent to which cidi diagnoses are consistent with clinical diagnoses . yules q has been proposed as an alternative measure to resolve this problem , as q is a bounded transformation of or ( q = [ or 1]/[or + 1 ] ) that ranges between 1 and + 1 . q can be interpreted as the difference in the probabilities of a clinical case and a clinical noncase that differ in their classification on the cidi . ( clinical cases and noncases that have the same cidi classification ) are excluded , which means that q does not tell about actual prediction accuracy . the auc is a measure that resolves this problem , as auc can be interpreted as the probability that a randomly selected clinical case will score higher on the cidi than a randomly selected noncase . the protocol was approved by the institutional reviewed board of institution of mental health , peking university . translation of the composite international diagnostic interview there are several version of cidi in china . the standard procedure of back - translation was followed for the cross - cultural adaptation of an original english psychometric instrument . a team of experts completed the initial translation and a separate team then carried out an independent back - translation to confirm preservation of the meaning of the original english version . an expert panel of four academic psychiatrists evaluated its content validity , tested it with chinese patients , and revised it to ensure its questions easily understandable and practically answerable by lay people . in the pilot study of the validity of cidi , the sensitivity ranged from 65.6% ( mood disorders ) to 87.0% ( substance use disorder ) and the specificity ranged from 83.3% ( anxiety disorders ) to 98.3% ( substance use disorders ) . ( = 0.05 , = 0.15 ) . to achieve adequate representation of the major mental disorders and sufficient nonpatient controls , the study was designed to include at least 71 cases with major depressive disorder , 30 with mania , 40 with anxiety disorder , 20 with an alcohol dependence disorder , 20 with eating disorder , and 60 healthy community - residing adults . all patients were consecutively recruited from two participating psychiatric institutions between september 2006 and february 2008 . exclusion criteria were age under 16 and having difficulties in completing cidi interview because of functional and cognitive disabilities including hearing and reading impairments , dementia , mental retardation , serious medical illnesses , marked and persisting hallucinations , thought disorders and disorganized speech . a total of 194 patients , 46 were excluded , either because they satisfied the exclusion criteria or because they did not complete the interview , leaving 148 patients completed the study . the healthy controls were selected from communities nearby the two participating psychiatric institutions , and they voluntarily participated in the study . the two psychiatrists were fully licensed psychiatrists with at least 3 years of clinical experiences in psychiatry and had received an intensive training in the use of scid - i . the six lay interviewers received 2-week training in the use of cidi that was provide by the beijing cidi training and resource center and completed at least 20 interviews under the supervision of experienced cidi trainers . design and the clinical assessment procedure clinicians diagnoses the two psychiatrists interviewed all subjects with scid - i according to the definitions and criteria of dsm - iv , blinded to the results of cidi interviews . both were informed about aim and procedures of the study and the necessity of a complete and detailed documentation of the psychopathology , course of illness and final diagnostic decision for each participating patient ; they were also encouraged to consult the dsm - iv manual and use all information available ( e.g. , history of psychiatric illnesses and treatment ) whenever necessary . all probable and definite lifetime diagnoses recorded in scid - i interview charts were used as the gold standard in the final analyses . discrepancy and case - by - case review after analyzing the diagnostic concordance , one psychiatrist reviewed interview records of all discrepant cases , in an attempt to identify why either the cidi did not pick up significant psychopathology or why the psychiatrist did not confirm the cidi diagnoses . the validity of cidi against scid - i was assessed using the sensitivity ( sn ) , specificity ( sp ) , positive predictive value ( ppv ) , and negative predictive value ( npv ) . individual - level cidi - scid diagnostic concordance was evaluated using two different descriptive measures : the area under the receiver operator characteristic curve ( auc ) and cohen 's k. although k is the most widely used measure of concordance in validity studies of mental disorders , it has been criticized for its dependency on prevalence . various populations are different in prevalence when the populations do not differ in sn or sp . as sn and sp are considered to be fundamental parameters , this means that the comparisons across different populations can not be used to evaluate the cross - population performance of a test . the odds ratio ( or ) meets this requirement , as or = ( sn sp)/([1 sn ] [ 1 sp ] ) . however , the upper end of the or is unbounded , making it difficult to evaluate the extent to which cidi diagnoses are consistent with clinical diagnoses . yules q has been proposed as an alternative measure to resolve this problem , as q is a bounded transformation of or ( q = [ or 1]/[or + 1 ] ) that ranges between 1 and + 1 . q can be interpreted as the difference in the probabilities of a clinical case and a clinical noncase that differ in their classification on the cidi . ( clinical cases and noncases that have the same cidi classification ) are excluded , which means that q does not tell about actual prediction accuracy . the auc is a measure that resolves this problem , as auc can be interpreted as the probability that a randomly selected clinical case will score higher on the cidi than a randomly selected noncase . the mean age of all patient participants was 33.5 14.2 years , 61.5% were females , and the mean educational year of the respondents was 10.1 years . the healthy controls and patients differed not significantly in terms of education , age group , and gender . within the subjects with mental disorders , women were somewhat overrepresented in the anxiety depression and eating groups and underrepresented in the substance use disorder group . table 2 shows the frequency of dsm - iv lifetime diagnoses identified in the scid - i and cidi interviews . in total , scid - i interviews recognized 46 cases with major depressive disorder , 20 with bipolar disorders , 59 with anxiety disorders , 20 with eating disorders , 33 with psychotic disorders , 20 with alcohol or drug disorders . demographic characteristics of patients with mental disorders and healthy people , n ( % ) frequency of dsm - iv lifetime diagnoses assigned by the clinician using scid - p and by the cidi 3.0 dsm - iv algorithms * 33 subjects was diagnosis as psychosis by clinicians ; other anxiety disorders : specific phobia disorder , social phobia , agoraphobia , panic disorder , ptsd . dsm - iv : diagnostic and statistical manual of mental disorders , fourth edition ; scid : structured clinical interview for dsm - iv axis i disorders ; cidi-3.0 : composite international diagnostic interview-3.0 ; ocd : obsessive and compulsive disorder . table 3 provides crosstab number of cidi - scid diagnose , auc , or , q , sns , sps , ppvs , and npvs for each diagnosis generated by the cidi . the separate disorder - specific cidi - scid comparisons of lifetime prevalence were made for major depression , bipolar disorder , general anxiety disorder , obsessive and compulsive disorder ( ocd ) , other anxiety disorders ( including panic disorder , phobias , posttraumatic stress disorder [ ptsd ] ) , alcohol abuse with or without dependence , anorexia nervosa , bulimia nervosa , binge eating disorder , and psychosis . the individual - level cidi - scid lifetime concordance for dsm - iv diagnosis was substantial to excellent for three disorders : substance use disorders ( auc = 0.926 ) , anxiety disorders ( auc = 0.807 ) , and mood disorders ( auc = 0.806 ) . in detail , the concordance was moderate ( auc : 0.70.8 ) for general anxiety disorder , ocd , other anxiety disorder , and psychosis . the concordance was almost perfect ( auc 0.9 ) for alcohol and substance use disorders and major depression , but fairly inadequate ( auc in the range : 0.60.7 ) for the remaining disorders ( bulimia nervosa ) . the concordance between the cidi and the scid for two individual disorders , bipolar disorders ( auc = 0.55 ) and anorexia nervosa ( auc = 0.50 ) was poor with very high sps and very poor sns . the majority of scid cases were detected by the cidi ( sn ) for major depression ( 69.6% ) , general anxiety disorders ( 58.6% ) , ocd ( 82.8% ) , substance dependence ( 87.0% ) , and psychosis screen ( 63.6% ) . the vast majority of cidi cases , in comparison , were confirmed by the scid ( ppv ) , including 83.3% ( 85.0100.0% ) with anxiety disorder , 96.7100% with mood disorder , and 98.3% with substance disorder . the sn values were particularly low for the diagnoses of bipolar disorder and eating disorder ( 0.040.0% ) . consistency of lifetime dsm - iv cidi and scid diagnoses with cross tabulation of findings ( n = 148 ) * other anxiety disorders : specific phobia disorder , social phobia , agoraphobia , panic disorder , ptsd . tn : true negatives ; fn : false - negatives ; fp : false - positives ; tp : true - positives ; or : odds ratio ; auc : area under the roc curve ; roc : receiver operating characteristic ; sn : sensitivity ; sp : specificity ; ppv : positive predictive value ; npv : negative predictive value . q : yules q ; se : standard error ; ptsd : posttraumatic stress disorder ; ocd : obsessive and compulsive disorder ; dsm - iv : diagnostic and statistical manual of mental disorders ; fourth edition ; scid : structured clinical interview for dsm - iv axis i disorders ; cidi : composite international diagnostic interview . the mean age of all patient participants was 33.5 14.2 years , 61.5% were females , and the mean educational year of the respondents was 10.1 years . the healthy controls and patients differed not significantly in terms of education , age group , and gender . within the subjects with mental disorders , women were somewhat overrepresented in the anxiety depression and eating groups and underrepresented in the substance use disorder group . table 2 shows the frequency of dsm - iv lifetime diagnoses identified in the scid - i and cidi interviews . in total , scid - i interviews recognized 46 cases with major depressive disorder , 20 with bipolar disorders , 59 with anxiety disorders , 20 with eating disorders , 33 with psychotic disorders , 20 with alcohol or drug disorders . demographic characteristics of patients with mental disorders and healthy people , n ( % ) frequency of dsm - iv lifetime diagnoses assigned by the clinician using scid - p and by the cidi 3.0 dsm - iv algorithms * 33 subjects was diagnosis as psychosis by clinicians ; other anxiety disorders : specific phobia disorder , social phobia , agoraphobia , panic disorder , ptsd . dsm - iv : diagnostic and statistical manual of mental disorders , fourth edition ; scid : structured clinical interview for dsm - iv axis i disorders ; cidi-3.0 : composite international diagnostic interview-3.0 ; ocd : obsessive and compulsive disorder . table 3 provides crosstab number of cidi - scid diagnose , auc , or , q , sns , sps , ppvs , and npvs for each diagnosis generated by the cidi . the separate disorder - specific cidi - scid comparisons of lifetime prevalence were made for major depression , bipolar disorder , general anxiety disorder , obsessive and compulsive disorder ( ocd ) , other anxiety disorders ( including panic disorder , phobias , posttraumatic stress disorder [ ptsd ] ) , alcohol abuse with or without dependence , anorexia nervosa , bulimia nervosa , binge eating disorder , and psychosis . the individual - level cidi - scid lifetime concordance for dsm - iv diagnosis was substantial to excellent for three disorders : substance use disorders ( auc = 0.926 ) , anxiety disorders ( auc = 0.807 ) , and mood disorders ( auc = 0.806 ) . in detail , the concordance was moderate ( auc : 0.70.8 ) for general anxiety disorder , ocd , other anxiety disorder , and psychosis . the concordance was almost perfect ( auc 0.9 ) for alcohol and substance use disorders and major depression , but fairly inadequate ( auc in the range : 0.60.7 ) for the remaining disorders ( bulimia nervosa ) . the concordance between the cidi and the scid for two individual disorders , bipolar disorders ( auc = 0.55 ) and anorexia nervosa ( auc = 0.50 ) was poor with very high sps and very poor sns . the majority of scid cases were detected by the cidi ( sn ) for major depression ( 69.6% ) , general anxiety disorders ( 58.6% ) , ocd ( 82.8% ) , substance dependence ( 87.0% ) , and psychosis screen ( 63.6% ) . the vast majority of cidi cases , in comparison , were confirmed by the scid ( ppv ) , including 83.3% ( 85.0100.0% ) with anxiety disorder , 96.7100% with mood disorder , and 98.3% with substance disorder . the sn values were particularly low for the diagnoses of bipolar disorder and eating disorder ( 0.040.0% ) . consistency of lifetime dsm - iv cidi and scid diagnoses with cross tabulation of findings ( n = 148 ) * other anxiety disorders : specific phobia disorder , social phobia , agoraphobia , panic disorder , ptsd . tn : true negatives ; fn : false - negatives ; fp : false - positives ; tp : true - positives ; or : odds ratio ; auc : area under the roc curve ; roc : receiver operating characteristic ; sn : sensitivity ; sp : specificity ; ppv : positive predictive value ; npv : negative predictive value . q : yules q ; se : standard error ; ptsd : posttraumatic stress disorder ; ocd : obsessive and compulsive disorder ; dsm - iv : diagnostic and statistical manual of mental disorders ; fourth edition ; scid : structured clinical interview for dsm - iv axis i disorders ; cidi : composite international diagnostic interview . the present study estimated the concordance of diagnoses based on the cidi-3.0 with diagnoses based on scid - i interviews . overall , our data suggested that the cidi-3.0 succeeds in validly eliciting diagnoses used in making dsm - iv diagnoses . there are several possible factors that may have contributed to the low concordance figures for the two disorders in this study . one major reason is the difference between the information - collecting methods of the cidi and scid . the patients with a diagnosis of bipolar disorder might refuse to confirm about some of their symptom . have you ever had a period lasting 4 days or longer when you became so happy or excited that you either got into trouble , people worried about you , or a doctor said you were manic ? ) . patients with anorexia nervosa were not more likely to endorse to some questions ( ea1 . this part of the interview is about problems you might have had either with eating or with your weight . was a time in your life when you had a great deal of concern about or strongly feared being too fat or overweight ? ) . because of the cultural differences in presentation of symptoms , some chinese patients do not admit the symptom of strongly feared being too fat or overweight , the study was used a clinical sample with the advantage of complete and comprehensive longitudinal symptom and diagnostic documentation . however , it is necessary to be cautious about generalizing the findings to nonclinical general population samples , for which one might expect more close - to - threshold conditions . another possibility is that in the current study , the clinical samples are known to have a considerable degree of comorbidity . this has the advantage of allowing for more disorder - specific comparisons with small samples . however , this approach might also inflate the concordance estimations , due to the inclusions of the same subject in testing several diagnoses . similar risk existed in our choice of not using the optional dsm - iv / cidi-3.0 diagnostic hierarchy rules because it would considerably reduce the number of cidi-3.0 diagnoses assigned . second , both ppv and npv depend on prevalence . although sn and sp do not depend on prevalence , they still depend on the case mix . finally , the small sample size did not allow for more extensive subgroup comparisons by characteristics of interviewers and subjects , but we could expect , based on similar previous studies , that the cidi-3.0 is quite robust in empirical validity . further , we focused on lifetime mental disorders , therefore , the validity of cidi in diagnosing 12-month or 1-month mental disorders needed to be examined in future studies . in conclusion , the validity study has shown that cidi - scid concordance in dsm - iv diagnoses is generally good , however , for specific mental disorders , such as bipolar and eating disorders , we should be cautious in using cidi . this work was supported by grants from special research project of ministry of health ( no . 201202022 ) , the national 11 5-year plan of ministry of science and technology of china ( no . 2007bai17b01 ) , and national key technology research and development program of the ministry of science and technology of china ( no . this work was supported by grants from special research project of ministry of health ( no . 201202022 ) , the national 11 5-year plan of ministry of science and technology of china ( no . 2007bai17b01 ) , and national key technology research and development program of the ministry of science and technology of china ( no .

background : the composite international diagnostic interview-3.0 ( cidi-3.0 ) is a fully structured lay - administered diagnostic interview for the assessment of mental disorders according to icd-10 and diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) criteria . the aim of the study was to investigate the concurrent validity of the chinese cidi in diagnosing mental disorders in psychiatric settings.methods:we recruited 208 participants , of whom 148 were patients from two psychiatric hospitals and 60 healthy people from communities . these participants were administered with cidi by six trained lay interviewers and the structured clinical interview for dsm - iv axis i disorders ( scid - i , gold standard ) by two psychiatrists . agreement between cidi and scid - i was assessed with sensitivity , specificity , positive predictive value and negative predictive value . individual - level cidi - scid diagnostic concordance was evaluated using the area under the receiver operator characteristic curve and cohen 's k.results:substantial to excellent cidi to scid concordance was found for any substance use disorder ( area under the receiver operator characteristic curve [ auc ] = 0.926 ) , any anxiety disorder ( auc = 0.807 ) and any mood disorder ( auc = 0.806 ) . the concordance between the cidi and the scid for psychotic and eating disorders is moderate . however , for individual mental disorders , the cidi - scid concordance for bipolar disorders ( auc = 0.55 ) and anorexia nervosa ( auc = 0.50 ) was insufficient.conclusions:overall , the chinese version of cidi-3.0 has acceptable validity in diagnosing the substance use disorder , anxiety disorder and mood disorder among chinese adult population . however , we should be cautious when using it for bipolar disorders and anorexia nervosa .

I M Ethnics Study design Statistics R Clinical and demographic characteristics of study sample Lifetime individual-level concordance between the Composite International Diagnostic Interview-3.0 and the Structured Clinical Interview for DSM-IV Axis I Disorder D Financial support and sponsorship Conflicts of interest

the composite international diagnostic interview ( cidi ) is a comprehensive and fully standardized diagnostic interview , primarily for use in epidemiological surveys and mental health research . therefore , there are still considerable concerns about the cross - cultural validity of diagnoses generated from these instruments when compared with careful and well - documented diagnoses assigned by clinical psychiatrists . consistently poor validity estimates were obtained for several types of mental disorders , whereas anxiety and depressive disorders were demonstrated across studies to have good validity estimates , with kappa values of 0.5 or above . given the widespread use of the cidi in chinese epidemiological studies , there is an urgent need to determine the degree of concordance and the types of discrepancies between cidi and clinical diagnoses made by mental health professionals . this paper presents the data from a systematic comparison of the translated cidi version , known as the cidi-3.0 , in an unselected clinical sample of patients with mental disorders , by comparing cidi / diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) diagnoses with those assigned by the treating clinical psychiatrists . the aim of this study was to investigate the validity of the cidi in relation to the structured clinical interview for dsm - iv axis i disorders ( scid - i ) , a gold standard the protocol was approved by the institutional reviewed board of institution of mental health , peking university . translation of the composite international diagnostic interview there are several version of cidi in china . in the pilot study of the validity of cidi , the sensitivity ranged from 65.6% ( mood disorders ) to 87.0% ( substance use disorder ) and the specificity ranged from 83.3% ( anxiety disorders ) to 98.3% ( substance use disorders ) . ( = 0.05 , = 0.15 ) . to achieve adequate representation of the major mental disorders and sufficient nonpatient controls , the study was designed to include at least 71 cases with major depressive disorder , 30 with mania , 40 with anxiety disorder , 20 with an alcohol dependence disorder , 20 with eating disorder , and 60 healthy community - residing adults . all patients were consecutively recruited from two participating psychiatric institutions between september 2006 and february 2008 . the healthy controls were selected from communities nearby the two participating psychiatric institutions , and they voluntarily participated in the study . the two psychiatrists were fully licensed psychiatrists with at least 3 years of clinical experiences in psychiatry and had received an intensive training in the use of scid - i . the six lay interviewers received 2-week training in the use of cidi that was provide by the beijing cidi training and resource center and completed at least 20 interviews under the supervision of experienced cidi trainers . design and the clinical assessment procedure clinicians diagnoses the two psychiatrists interviewed all subjects with scid - i according to the definitions and criteria of dsm - iv , blinded to the results of cidi interviews . both were informed about aim and procedures of the study and the necessity of a complete and detailed documentation of the psychopathology , course of illness and final diagnostic decision for each participating patient ; they were also encouraged to consult the dsm - iv manual and use all information available ( e.g. all probable and definite lifetime diagnoses recorded in scid - i interview charts were used as the gold standard in the final analyses . discrepancy and case - by - case review after analyzing the diagnostic concordance , one psychiatrist reviewed interview records of all discrepant cases , in an attempt to identify why either the cidi did not pick up significant psychopathology or why the psychiatrist did not confirm the cidi diagnoses . the validity of cidi against scid - i was assessed using the sensitivity ( sn ) , specificity ( sp ) , positive predictive value ( ppv ) , and negative predictive value ( npv ) . individual - level cidi - scid diagnostic concordance was evaluated using two different descriptive measures : the area under the receiver operator characteristic curve ( auc ) and cohen 's k. although k is the most widely used measure of concordance in validity studies of mental disorders , it has been criticized for its dependency on prevalence . however , the upper end of the or is unbounded , making it difficult to evaluate the extent to which cidi diagnoses are consistent with clinical diagnoses . yules q has been proposed as an alternative measure to resolve this problem , as q is a bounded transformation of or ( q = [ or 1]/[or + 1 ] ) that ranges between 1 and + 1 . the auc is a measure that resolves this problem , as auc can be interpreted as the probability that a randomly selected clinical case will score higher on the cidi than a randomly selected noncase . translation of the composite international diagnostic interview there are several version of cidi in china . in the pilot study of the validity of cidi , the sensitivity ranged from 65.6% ( mood disorders ) to 87.0% ( substance use disorder ) and the specificity ranged from 83.3% ( anxiety disorders ) to 98.3% ( substance use disorders ) . to achieve adequate representation of the major mental disorders and sufficient nonpatient controls , the study was designed to include at least 71 cases with major depressive disorder , 30 with mania , 40 with anxiety disorder , 20 with an alcohol dependence disorder , 20 with eating disorder , and 60 healthy community - residing adults . all patients were consecutively recruited from two participating psychiatric institutions between september 2006 and february 2008 . a total of 194 patients , 46 were excluded , either because they satisfied the exclusion criteria or because they did not complete the interview , leaving 148 patients completed the study . the healthy controls were selected from communities nearby the two participating psychiatric institutions , and they voluntarily participated in the study . the two psychiatrists were fully licensed psychiatrists with at least 3 years of clinical experiences in psychiatry and had received an intensive training in the use of scid - i . the six lay interviewers received 2-week training in the use of cidi that was provide by the beijing cidi training and resource center and completed at least 20 interviews under the supervision of experienced cidi trainers . design and the clinical assessment procedure clinicians diagnoses the two psychiatrists interviewed all subjects with scid - i according to the definitions and criteria of dsm - iv , blinded to the results of cidi interviews . both were informed about aim and procedures of the study and the necessity of a complete and detailed documentation of the psychopathology , course of illness and final diagnostic decision for each participating patient ; they were also encouraged to consult the dsm - iv manual and use all information available ( e.g. all probable and definite lifetime diagnoses recorded in scid - i interview charts were used as the gold standard in the final analyses . discrepancy and case - by - case review after analyzing the diagnostic concordance , one psychiatrist reviewed interview records of all discrepant cases , in an attempt to identify why either the cidi did not pick up significant psychopathology or why the psychiatrist did not confirm the cidi diagnoses . the validity of cidi against scid - i was assessed using the sensitivity ( sn ) , specificity ( sp ) , positive predictive value ( ppv ) , and negative predictive value ( npv ) . individual - level cidi - scid diagnostic concordance was evaluated using two different descriptive measures : the area under the receiver operator characteristic curve ( auc ) and cohen 's k. although k is the most widely used measure of concordance in validity studies of mental disorders , it has been criticized for its dependency on prevalence . however , the upper end of the or is unbounded , making it difficult to evaluate the extent to which cidi diagnoses are consistent with clinical diagnoses . yules q has been proposed as an alternative measure to resolve this problem , as q is a bounded transformation of or ( q = [ or 1]/[or + 1 ] ) that ranges between 1 and + 1 . the auc is a measure that resolves this problem , as auc can be interpreted as the probability that a randomly selected clinical case will score higher on the cidi than a randomly selected noncase . the mean age of all patient participants was 33.5 14.2 years , 61.5% were females , and the mean educational year of the respondents was 10.1 years . within the subjects with mental disorders , women were somewhat overrepresented in the anxiety depression and eating groups and underrepresented in the substance use disorder group . table 2 shows the frequency of dsm - iv lifetime diagnoses identified in the scid - i and cidi interviews . in total , scid - i interviews recognized 46 cases with major depressive disorder , 20 with bipolar disorders , 59 with anxiety disorders , 20 with eating disorders , 33 with psychotic disorders , 20 with alcohol or drug disorders . demographic characteristics of patients with mental disorders and healthy people , n ( % ) frequency of dsm - iv lifetime diagnoses assigned by the clinician using scid - p and by the cidi 3.0 dsm - iv algorithms * 33 subjects was diagnosis as psychosis by clinicians ; other anxiety disorders : specific phobia disorder , social phobia , agoraphobia , panic disorder , ptsd . dsm - iv : diagnostic and statistical manual of mental disorders , fourth edition ; scid : structured clinical interview for dsm - iv axis i disorders ; cidi-3.0 : composite international diagnostic interview-3.0 ; ocd : obsessive and compulsive disorder . table 3 provides crosstab number of cidi - scid diagnose , auc , or , q , sns , sps , ppvs , and npvs for each diagnosis generated by the cidi . the separate disorder - specific cidi - scid comparisons of lifetime prevalence were made for major depression , bipolar disorder , general anxiety disorder , obsessive and compulsive disorder ( ocd ) , other anxiety disorders ( including panic disorder , phobias , posttraumatic stress disorder [ ptsd ] ) , alcohol abuse with or without dependence , anorexia nervosa , bulimia nervosa , binge eating disorder , and psychosis . the individual - level cidi - scid lifetime concordance for dsm - iv diagnosis was substantial to excellent for three disorders : substance use disorders ( auc = 0.926 ) , anxiety disorders ( auc = 0.807 ) , and mood disorders ( auc = 0.806 ) . in detail , the concordance was moderate ( auc : 0.70.8 ) for general anxiety disorder , ocd , other anxiety disorder , and psychosis . the concordance was almost perfect ( auc 0.9 ) for alcohol and substance use disorders and major depression , but fairly inadequate ( auc in the range : 0.60.7 ) for the remaining disorders ( bulimia nervosa ) . the concordance between the cidi and the scid for two individual disorders , bipolar disorders ( auc = 0.55 ) and anorexia nervosa ( auc = 0.50 ) was poor with very high sps and very poor sns . the majority of scid cases were detected by the cidi ( sn ) for major depression ( 69.6% ) , general anxiety disorders ( 58.6% ) , ocd ( 82.8% ) , substance dependence ( 87.0% ) , and psychosis screen ( 63.6% ) . the vast majority of cidi cases , in comparison , were confirmed by the scid ( ppv ) , including 83.3% ( 85.0100.0% ) with anxiety disorder , 96.7100% with mood disorder , and 98.3% with substance disorder . the sn values were particularly low for the diagnoses of bipolar disorder and eating disorder ( 0.040.0% ) . consistency of lifetime dsm - iv cidi and scid diagnoses with cross tabulation of findings ( n = 148 ) * other anxiety disorders : specific phobia disorder , social phobia , agoraphobia , panic disorder , ptsd . tn : true negatives ; fn : false - negatives ; fp : false - positives ; tp : true - positives ; or : odds ratio ; auc : area under the roc curve ; roc : receiver operating characteristic ; sn : sensitivity ; sp : specificity ; ppv : positive predictive value ; npv : negative predictive value . q : yules q ; se : standard error ; ptsd : posttraumatic stress disorder ; ocd : obsessive and compulsive disorder ; dsm - iv : diagnostic and statistical manual of mental disorders ; fourth edition ; scid : structured clinical interview for dsm - iv axis i disorders ; cidi : composite international diagnostic interview . the mean age of all patient participants was 33.5 14.2 years , 61.5% were females , and the mean educational year of the respondents was 10.1 years . within the subjects with mental disorders , women were somewhat overrepresented in the anxiety depression and eating groups and underrepresented in the substance use disorder group . table 2 shows the frequency of dsm - iv lifetime diagnoses identified in the scid - i and cidi interviews . in total , scid - i interviews recognized 46 cases with major depressive disorder , 20 with bipolar disorders , 59 with anxiety disorders , 20 with eating disorders , 33 with psychotic disorders , 20 with alcohol or drug disorders . demographic characteristics of patients with mental disorders and healthy people , n ( % ) frequency of dsm - iv lifetime diagnoses assigned by the clinician using scid - p and by the cidi 3.0 dsm - iv algorithms * 33 subjects was diagnosis as psychosis by clinicians ; other anxiety disorders : specific phobia disorder , social phobia , agoraphobia , panic disorder , ptsd . dsm - iv : diagnostic and statistical manual of mental disorders , fourth edition ; scid : structured clinical interview for dsm - iv axis i disorders ; cidi-3.0 : composite international diagnostic interview-3.0 ; ocd : obsessive and compulsive disorder . table 3 provides crosstab number of cidi - scid diagnose , auc , or , q , sns , sps , ppvs , and npvs for each diagnosis generated by the cidi . the separate disorder - specific cidi - scid comparisons of lifetime prevalence were made for major depression , bipolar disorder , general anxiety disorder , obsessive and compulsive disorder ( ocd ) , other anxiety disorders ( including panic disorder , phobias , posttraumatic stress disorder [ ptsd ] ) , alcohol abuse with or without dependence , anorexia nervosa , bulimia nervosa , binge eating disorder , and psychosis . the individual - level cidi - scid lifetime concordance for dsm - iv diagnosis was substantial to excellent for three disorders : substance use disorders ( auc = 0.926 ) , anxiety disorders ( auc = 0.807 ) , and mood disorders ( auc = 0.806 ) . in detail , the concordance was moderate ( auc : 0.70.8 ) for general anxiety disorder , ocd , other anxiety disorder , and psychosis . the concordance was almost perfect ( auc 0.9 ) for alcohol and substance use disorders and major depression , but fairly inadequate ( auc in the range : 0.60.7 ) for the remaining disorders ( bulimia nervosa ) . the concordance between the cidi and the scid for two individual disorders , bipolar disorders ( auc = 0.55 ) and anorexia nervosa ( auc = 0.50 ) was poor with very high sps and very poor sns . the majority of scid cases were detected by the cidi ( sn ) for major depression ( 69.6% ) , general anxiety disorders ( 58.6% ) , ocd ( 82.8% ) , substance dependence ( 87.0% ) , and psychosis screen ( 63.6% ) . the vast majority of cidi cases , in comparison , were confirmed by the scid ( ppv ) , including 83.3% ( 85.0100.0% ) with anxiety disorder , 96.7100% with mood disorder , and 98.3% with substance disorder . the sn values were particularly low for the diagnoses of bipolar disorder and eating disorder ( 0.040.0% ) . consistency of lifetime dsm - iv cidi and scid diagnoses with cross tabulation of findings ( n = 148 ) * other anxiety disorders : specific phobia disorder , social phobia , agoraphobia , panic disorder , ptsd . tn : true negatives ; fn : false - negatives ; fp : false - positives ; tp : true - positives ; or : odds ratio ; auc : area under the roc curve ; roc : receiver operating characteristic ; sn : sensitivity ; sp : specificity ; ppv : positive predictive value ; npv : negative predictive value . q : yules q ; se : standard error ; ptsd : posttraumatic stress disorder ; ocd : obsessive and compulsive disorder ; dsm - iv : diagnostic and statistical manual of mental disorders ; fourth edition ; scid : structured clinical interview for dsm - iv axis i disorders ; cidi : composite international diagnostic interview . the present study estimated the concordance of diagnoses based on the cidi-3.0 with diagnoses based on scid - i interviews . overall , our data suggested that the cidi-3.0 succeeds in validly eliciting diagnoses used in making dsm - iv diagnoses . there are several possible factors that may have contributed to the low concordance figures for the two disorders in this study . one major reason is the difference between the information - collecting methods of the cidi and scid . patients with anorexia nervosa were not more likely to endorse to some questions ( ea1 . this part of the interview is about problems you might have had either with eating or with your weight . because of the cultural differences in presentation of symptoms , some chinese patients do not admit the symptom of strongly feared being too fat or overweight , the study was used a clinical sample with the advantage of complete and comprehensive longitudinal symptom and diagnostic documentation . however , it is necessary to be cautious about generalizing the findings to nonclinical general population samples , for which one might expect more close - to - threshold conditions . another possibility is that in the current study , the clinical samples are known to have a considerable degree of comorbidity . however , this approach might also inflate the concordance estimations , due to the inclusions of the same subject in testing several diagnoses . similar risk existed in our choice of not using the optional dsm - iv / cidi-3.0 diagnostic hierarchy rules because it would considerably reduce the number of cidi-3.0 diagnoses assigned . finally , the small sample size did not allow for more extensive subgroup comparisons by characteristics of interviewers and subjects , but we could expect , based on similar previous studies , that the cidi-3.0 is quite robust in empirical validity . further , we focused on lifetime mental disorders , therefore , the validity of cidi in diagnosing 12-month or 1-month mental disorders needed to be examined in future studies . in conclusion , the validity study has shown that cidi - scid concordance in dsm - iv diagnoses is generally good , however , for specific mental disorders , such as bipolar and eating disorders , we should be cautious in using cidi . 201202022 ) , the national 11 5-year plan of ministry of science and technology of china ( no . 2007bai17b01 ) , and national key technology research and development program of the ministry of science and technology of china ( no . 201202022 ) , the national 11 5-year plan of ministry of science and technology of china ( no . 2007bai17b01 ) , and national key technology research and development program of the ministry of science and technology of china ( no .

autism spectrum disorders ( asd ) are a group of neurological disorders currently considered to manifest from a synaptic dysfunction or synaptopathy . in particular synapse formation and/or synaptic signal transduction and plasticity might be affected based on the identified candidate genes from large - scale genetic studies . however , most likely , environmental factors contribute to the etiology of asd . a strong association between imbalance in trace metal homeostasis and asd has been reported in numerous studies . for example , deficiencies for zn , ca , fe , mg , mn , and se as well as increased concentrations for al , as , cd , hg , and pb were noted in hair samples of autistic patients [ 4 , 5 ] and the burden of toxic metals in patients showed a correlation with the severity of the autism phenotype . this complex scenario prompted us to investigate the interplay and effects of a dyshomeostasis of different metal ions and the resultant pathological alterations of synapses . in the past , many studies have been carried out to investigate the essentiality and toxicity of trace metals , using cells in culture [ 712 ] . this research has identified various trace metals considered nowadays as essential ( biometals ) , neutral , or toxic for vertebrates . thus , the difference between toxic and essential elements is based on the narrow window of concentrations , where the physiological function of biometals is seen . however , trace metals do not act as separate entities influencing mechanisms or pathomechanisms in cells but exist in a careful orchestrated equilibrium . to study this equilibrium that not only involves biometals but also toxic metal ions an organism is exposed to , on cellular level in vitro , we have carried out experiments on metal ions such as aluminum ( al ) , cadmium ( cd ) , copper ( cu ) , iron ( fe ) , mercury ( hg ) , magnesium ( mg ) , lead ( pb ) , selenium ( se ) , and zinc ( zn ) , using primary hippocampal neurons . to test for downstream effects of imbalance of any one metal ion , which might lead to a domino effect and produces changes in all other metal ions , the present report describes the effects of heavy metal ions and the interaction among cd , cu , hg , pb , se , and al with zn in cultured cells . first , we analyzed the effect of metal overload of a single trace metal on various synaptic parameters by chronic treatment of primary rat neuronal cultures with metal chlorides . next , we determined the effect of metal overload of cd , cu , hg , and pb in combination since these metals were frequently described elevated in asd patients . we further analyzed whether the absence of zn and fe , a common feature in asd patients , modifies the effect of high cd , cu , hg , and pb levels . finally , we investigated whether zn supplementation is able to overcome synaptic defects induced by the trace metal profile characteristic for asd patients . zncl2 , cucl2 , cdcl2 , fecl2 , secl4 , alcl3 , mgcl2 , hgcl2 , and pbcl2 were purchased from sigma - aldrich . primary antibodies were purchased from sigma - aldrich ( map2 , glun1 , and shank1 for wb ) , synaptic systems ( bassoon , homer1b / c , shank3 ) , merck millipore ( glun2a and glun2b ) , and novus biological ( shank1 for if ) . the preparation of hippocampal cultures was performed essentially as described before from rat ( embryonic day 18 ; e18 ) . after preparation the hippocampal neurons were seeded on poly - l - lysine ( 0.1 mg / ml ; sigma - aldrich ) glass coverslips in a 24-well plate at a density of 3 10 cells / well or 10 cm petri dish at a density of 2.53 10 cells / dish . cells were grown in neurobasal ( nb ) medium ( life technologies ) , complemented with b27 supplement ( life technologies ) , 0.5 mm l - glutamine ( life technologies ) , and 100 u / ml penicillin / streptomycin ( life technologies ) and maintained at 37c in 5% co2 . all animal experiments were performed in compliance with the guidelines for the welfare of experimental animals issued by the federal government of germany and by the local ethics committee ( ulm university ) , i d number : 0.103 . for immunofluorescence , the primary cultures were fixed with 4% paraformaldehyde ( pfa)/1.5% sucrose / pbs at 4c for 20 min and processed for immunohistochemistry . after washing 2x 5 min with 1x pbs with 0.2% triton x-100 at rt , blocking was performed with 10% fbs/1x pbs for 1 h at rt , followed by the primary antibody at rt for 2 h. after a 3x 5 min washing - step with 1x pbs , incubation with the second antibody coupled with alexa488 , alexa568 , or alexa647 followed for 1 h at rt . the cells were washed again in 1x pbs for 10 min and counterstained with dapi and washed for 5 min with aqua bidest and mounted with vecta mount . cells were treated for 4 days with zncl2 ( 10200 m ) , cucl2 ( 10200 m ) , cdcl2 ( 110 m ) , alcl3 ( 10500 m ) , hgcl2 ( 110 m ) , mgcl2 ( 10200 m ) , pbcl2 ( 10500 m ) , secl4 ( 110 m ) and fecl2 ( 10400 m ) , or zncl2 ( 50 m ) and cucl2 ( 120 m ) combinations , zncl2 ( 50 m ) and cdcl2 ( 5 m ) combinations , zncl2 ( 50 m ) and hgcl2 ( 5 m ) combinations , zncl2 ( 50 m ) and pbcl2 ( 5 m ) combinations , or zncl2 ( 50 m ) and secl4 ( 6 m ) combinations . metal deficient neurobasal medium ( life technologies ) was generated using chelex 100 resin , 200400 dry mesh size , sodium form ( biorad ) . chelex 100 resin was used according to the manufacturer 's instructions ( batch method ) and the ph of the medium readjusted using hydrochloric acid . additionally , in some conditions , original metal concentrations of some or all chelated metals were reestablished according to the neurobasal medium ( 1x ) liquid media formulation using cacl2 , fe(no3)39h2o , mgcl2 , and znso47h2o . the efficacy of chelation of divalent metals was controlled by inductively coupled plasma mass spectrometry ( icp - ms ) ( table 1 ) . the trace metal concentration of growth media was measured by icp - ms at the spurenanalytisches laboratorium dr . baumann ( maxhtte - haidhof , germany ) . for fluorescent zn - staining of cultured neurons , coverslips were incubated with a solution of 5 m zinpyr1 in pbs for 1 h at rt . to obtain p2 fractions from hippocampal cultures , div 14 cells exposed to different compounds of interest for the indicated times were harvested and homogenized in homogenization buffer ( 320 mm sucrose , 10 mm hepes , ph 7.4 ) containing protease inhibitor mixture ( roche ) . cell debris and nuclei were removed by centrifugation at 3,200 rpm for 15 min resulting in supernatant s1 ( soluble fraction ) and pellet p1 ( membrane associated fraction ) . supernatants ( s1 ) were centrifuged for 20 min at 11,200 rpm , resulting in s2 ( soluble fraction ) and p2 ( crude synaptosomal fraction ) . the resulting pellet p2 was resuspended in homogenization buffer to perform bradford and analyzed by western blotting . immunoreactivity was visualized using hrp - conjugated secondary antibodies and the supersignal detection system ( pierce , upland , usa ) . first strand synthesis and quantitative real - time - pcr amplification were performed in a one - step , single - tube format using the quantifasttm sybr_green rt - pcr kit from qiagen according to the manufacturer 's protocol in a total volume of 20 l and gene specific quantitect primer assays ( qiagen ) . thermal cycling and fluorescent detection were performed using the rotor - gene q real - time pcr machine ( model 2-plex hrm ) ( qiagen ) . resulting data were analyzed using the hmbs gene as an internal standard to normalize transcript levels . acquisition and evaluation of all images were performed under blinded conditions . for cell culture fluorescence images were obtained using an upright axioscope microscope equipped with a zeiss ccd camera ( 16 bits ; 1280 1024 ppi ) using axiovision software ( zeiss ) and imagej 1.49i . statistical analysis was performed using microsoft excel for macintosh and tested for significance using t tests ( all values were normally distributed ) . three independent experiments were performed and blots imaged using a microchemi imaging system from biostep . all wb bands were normalized to -iii - tubulin or beta - actin and the ratios averaged and tested for significance with a level of significance set at 0.05 ( < 0.05 ; < 0.01 ; < 0.001 ) . relative quantification is based on internal reference genes to determine virtual mrna levels of target genes . cycle threshold ( ct ) values were calculated by the rotor - gene q software ( version 2.0.2 ) . ct values were transformed into virtual mrna levels according to the formula : virtual mrna level = 10 ( ( ct(target ) ct(standart))/slope of standard curve ) . in our previous study , we reported characteristic biometal profiles in neurological disorders . in asd , several trace metals are known to be either depleted or to occur in excess ( table 2 ) . therefore , to understand the consequences of all these alterations in trace metals for synapses , in a first set of experiments , we aimed to analyze first the influence of each trace metal on cell health and synapse numbers separately ( figures s1a and b in supplementary material available online at http://dx.doi.org/10.1155/2015/985083 and figure 1 ) . to that end , we treated hippocampal neuronal cell cultures between div 10 and div 14 with different concentrations of the trace metals al , cd , cu , fe , hg , mg , pb , se , and zn . as expected , a linear correlation between metal levels and the amount of cell death was found , especially for cd , cu , and zn ( figures 1(a ) and 1(b ) ) . we could not detect significant cell death over a wide spectrum of concentrations for al , fe , and pb . additionally , for hg and se , an exponential increase in cell death beyond a certain concentration was observed . exposure of cells to mg increased cell survival in a low concentration range and with higher concentrations leading to cell death ( figures 1(a ) and 1(b ) ) . treatment with mannitol was used to exclude the influence of osmotic stress on cell health ( figure s1c ) . based on the correlation between cell health and the concentration of a certain trace metal applied , ld50 concentrations were calculated for each metal ( figure s1d ) . glial cells that were present to a low extent in the neuronal cultures were much more resistant to alterations in trace metal concentrations and showed no signs of cell death in the toxic concentration range seen for neuronal cells ( figure s1e ) . furthermore , the number of primary , secondary , and tertiary dendrites of neurons after treatment was investigated . given that neurons show a fragmentation of dendrites , starting with branches more distal from the soma as first sign of affected cell health , a decrease in dendritic branching might reveal cells that did not show apoptotic nuclei yet already suffered from the exposure to trace metals . similar to the results shown above , high concentrations of metals that were shown to lead to cell death led to a reduction in dendritic branching starting from tertiary dendrites and also affecting secondary and primary in the most toxic cases ( figure 1(c ) ) . indeed , although , for some metal , no significant cell death in a certain concentrations was seen , already a fragmentation of dendrites is visible . interestingly , supplementation of mg and zn in a low concentration leads to a significant increase in dendritic branching restricted to tertiary dendrites ( figure 1(c ) ) . therefore , a decrease in synapse number is expected to correlate with the toxic effects of trace metals and the resulting cell death . indeed , the quantification of synapse density in treated neurons and compared to untreated control cells shows a correlation of toxic effects and synapse loss for metal ions , such as cd , cu , and se ( figure 1(d ) , figure s1f ) . however , an increase in synapse number can be seen for fe and lower concentrations of mg and zn ( figure 1(d ) , figure s1f ) . since , for some metal ions , we could determine an effect on synapses independent of a possible cellular toxicity ( figure 2 ) , a role of these metals in synapse formation and/or stabilization is likely . for example , mg shows only a weak correlation due to its nontoxic effects on neurons over a wide concentration range . additionally , supplementation of both zn and se , in low concentrations increased synapse density , which then can be found increased compared to untreated control despite ongoing cell death . based on the findings described above , it might be possible that the absence of a synaptogenic effect of some metals such as zn and the toxic effects of others act in combination in asd when deficiencies and overload of certain trace metals occur in parallel . therefore , we established a biometal profile resembling the assumed trace metal alterations in asd patients and evaluated the consequences on cell health and especially on synapses . hippocampal neurons were grown from div 10 to div 14 in cell culture media containing different sets of trace metals : control cells ( ctrl ) were grown in trace metal depleted neurobasal medium that was reconstituted for all depleted trace metals ( mg , ca , fe , and zn ) according to the manufacturer 's indicated metal concentrations . in none of the readouts , we could observe differences between original and reconstituted neurobasal medium ( figure s2 ) . additionally , cells were grown in neurobasal medium with addition of putative toxic metals ( 0.5 m cd , cu , hg , and 2 m pb ) found to be increased in asd patients ( asd1 ) . control cells for this condition were grown in neurobasal medium . in a third condition , cells were grown in trace metal depleted neurobasal medium that was reconstituted only for mg and ca , with addition of the putative toxic metals ( 0.5 m cd , cu , hg , and 2 m pb ) ( asd2 ) . asd2 growth medium , therefore , with fe and zn deficiency and overload of cd , cu , hg , and pb resembles the characteristic biometal profile found in asd . cells exposed to asd2 medium indeed displayed a significant reduction in cell health visible by an increased number of apoptotic nuclei and increased dendritic fragmentation ( figures 3(a ) and 3(b ) ) . the number of synapses per 10 m dendrite length assessed by quantification of bassoon and homer1b / c positive puncta was significantly reduced in cells growing in medium resembling the biometal profile found in asd patients ( asd2 ) ( figure 3(c ) ) . along with a reduction in the number of synapses , gene expression levels of synaptic receptors such as nmdar ( glun1 , glun2a , and glun2b ) as well as the zn dependent shank scaffold proteins ( shank1 , shank2 , and shank3 ) show significant alterations ( figure 3(d ) ) . while the presence of putative toxic metals increased in asd patients ( asd1 ) is sufficient to significantly increase glun2b expression levels , glun1 and glun2a and shank1 , shank2 , and shank3 mrna levels are only significantly altered in case of an additional zn and fe deficiency ( asd2 ) . fluorescent readouts for shank proteins show a reduction on protein level under asd1 conditions , while the remaining synapses under asd2 condition display normal shank levels ( figure 3(e ) ) . however , analysis using protein biochemistry shows that , overall , shank protein concentrations are reduced in the p2 fraction of cells growing under asd2 condition ( figure 3(f ) ) . additionally , similar to the decrease of mrna levels of nmdar subunits , we detected significantly less glun2a protein and a trend towards a decrease of glun2b protein levels in cells grown in asd2 medium . furthermore , znt-1 expression levels , a zn exporter recently described as being enriched at postsynapses , reacts very sensitively to changes in trace metal concentrations as those induced by application of asd2 medium ( figure 3(f ) ) . finally , in a last set of experiments , we wanted to determine , whether the addition of a single specific trace metal might lead to a rescue in the observed phenotype after inducing an asd like biometal profile in cell culture or whether all alterations need to be addressed separately . given that an antagonistic role of zn and pb or cu was proposed before [ 3 , 4 ] and that we could verify a potentially beneficial effect of zn on synapses , we next wanted to elucidate whether zn supplementation in combination with harmful metals ( asd2 ) is able to modify the toxicity of the trace metal on neurons in vitro . to that end however , asd2 medium ( neurobasal medium that was reconstituted only for mg and ca , with addition of the putative toxic metals ( 0.5 m cd , cu , hg , and 2 m pb ) ) was supplemented with 50 m zn . addition of zn was unable to rescue the observed cell death detected in cultures grown under asd2 conditions ( figure 4(a ) ) , although it led to a significant reduction in dendritic fragmentation ( figure 4(b ) ) . along with this , the reduction in the number of synapses per 10 m dendrite length assessed by quantification of bassoon and homer1b / c positive puncta seen under asd2 conditions was rescued after addition of zn ( asd2 + zn ) ( figure 4(c ) ) . supplementation of zn was also able to significantly increase the reduction in mrna levels of nmda receptor subunits and shank family members seen in cells growing under asd2 conditions ( figure 4(d ) ) . shank mrna expression levels were no longer significantly different from those of cells growing under control conditions . however , in case of nmda receptor subunits , mrna levels still remained significantly decreased compared to controls . the observed changes on protein level of nmda receptor subunits , the zn binding shank2 and shank3 , and znt-1 in turn were completely rescued after addition of zn ( asd2 + zn ) ( figure 4(f ) ) . immunofluorescence readouts revealed no changes in synaptic protein levels after supplementation of cells growing under asd2 conditions with zn ( figure 4(e ) , figure s2d ) . furthermore , we used icp - ms to measure the extracellular metal concentrations of hippocampal cell cultures with and without the presence of zn . a reduction in the uptake of cu , hg , and se was visible upon coapplication of zn , while an increase in uptake of pb was visible ( figure 4(g ) ) . when zncl2 ( 50 m ) was combined with either cdcl2 ( 5 m ) , cucl2 ( 120 m ) , hgcl2 ( 5 m ) , pbcl2 ( 5 m ) , or secl4 ( 6 m ) treatment , a slight decrease of neuronal cell death upon exposure to zncl2 and hgcl2 compared to the exposure to hgcl2 alone was observed . further , a significant improvement of neuronal cell viability was identified if zncl2 and cdcl2 were supplemented together . there , zinc could restore neuronal cell health status close to control levels ( figures s2e , f ) . the alterations observed can not be explained by changes in transsynaptic zn signaling given that presynapses in hippocampal cell culture have very low to absent levels of vesicular zn ( figure s2 g ) . additionally , the alterations caused under asd2 conditions although to high extent rescued by zn supplementation are not based on zn deficiency alone . a comparison between asd2 and pure zn deficient ( znd ) conditions reveals that zn deficiency without additional alterations in biometals does not lead to the significant reduction in cell health ( figures s3a , b ) and synapse number ( figure s3c ) such as seen under asd2 conditions . furthermore , the decrease in mrna expression levels of nmda receptor subunits is less pronounced and absent for shank family members under pure zn deficiency ( figure s3d ) . however , as reported before , zn deficiency significantly affects the synaptic levels of the zn binding shank family members shank2 and shank3 using immunofluorescent readouts ( figure s3e ) . protein biochemistry with p2 fractions ( figure s3f ) reveals that , on protein level , zn deficiency alone already affects nmdar subunit expression , but to a lesser extent the expression of shanks . an increasing number of reports of imbalanced trace metal homeostasis in children that suffer from neurodevelopmental disorders raise the need for a better understanding of how putative toxic metals affect neuronal cells . therefore , we investigated the effects of different essential and nonessential trace metals on neurons in vitro and further examined their relationship with zinc , one of most abundant trace metals in the brain . in a first set of experiments , we investigated the influence of overload with a single trace element . although trace metals such as mg , cu , fe , se , and zn are essential with a vital role in normal brain functions , increased levels in the brain may lead to severe neurological symptoms . however , we found a strong resistance of neurons against fecl2 induced toxicity with the absence of morphological changes even at very high concentrations ( up to 400 m ) and a slight dose - dependent synaptogenic effect . this absence of neuronal cell death is in line with the results from bishop et al . reporting that the exposure to 100 m of ferric ammonium citrate for 24 h to cultured neurons increased the intracellular fe content by factor 30 but did not affect cell viability . similarly , only few pathologically morphological changes of primary neurons could be seen after exposure to mgcl2 in the tested concentration range . similar findings were reported by regan et al . who treated primary cortical neurons with up to 3 mm mgcl2 without detecting any morphological evidence of cell injury . in our study , intermediate concentrations ( 100 m ) of mgcl2 increased the number of tertiary dendrites and slightly increased the number of synapses . in contrast , exposure of neurons to cucl2 leads to a dose - dependent cell death and a significant reduction of synapse numbers correlated to the reduced number of viable cells , which can be also observed in younger neurons at div 6 . se is an essential trace metal with a narrow range of concentrations between deficiency and toxicity . also , in this study , total neuronal cell death has been observed at 10 m secl4 , whereas 8 m secl4 were shown to increase synapse numbers . little is known about the toxic effect of secl4 on cultured neuronal cells due to the fact that most studies concentrated on the protective effect of se against glutamate induced excitotoxicity or against the toxicity of inorganic mercury . have reported that the exposure to 10 m sodium selenite decreased ht22 cell viability about 20% . these differences are likely due to the use of different cell culture systems and it is not surprising that an immortalized hippocampal cell line is much more resistant against se induced toxicity than primary neurons . zn is involved in a huge variety of cellular processes but an increased concentration of unbound zn ions has been reported to be especially harmful to the central nervous system and to kill cells in vitro . in this study , in contrast to essential trace metals , exposure to putative toxic metals is expected to be much more harmful . indeed , even low concentrations of cdcl2 had severe effects on neuronal cell health ( ld50 5 m ) . we observed a dose - dependent neuronal cell death and a linear correlated reduction of synapses . cd toxicity was shown to lead to impaired neurogenesis , reduced neuronal differentiation , and reduced axogenesis in vitro and even lower concentrations have been reported to repress dendritic and synaptic development [ 24 , 25 ] . despite fatal neuronal cell death , at low cd concentrations , glial cells seem to be more resistant which is in accordance with previous reports . although al is a well - accepted neurotoxin and discussed as factor for the development of neurodegeneration , for example , as seen in alzheimer 's disease ( ad ) , we did not observe adverse effects on neurons and synapses even at very high concentrations of alcl3 ( up to 500 m ) similar to a study by kawahara et al . . however , lipophilic aluminum species like aluminum acetylacetonate or aluminum maltolate might be more toxic than alcl3 . additionally , a possible precipitation of alcl3 in physiological ph might prevent al from reaching intracellular concentrations high enough to induce toxicity . it is possible that al does not affect the number of synapses but rather alters synaptic plasticity [ 30 , 31 ] or may act in addition to other predisposing factors . intriguingly , it was proposed that the genetic component of ad pathology might involve a susceptibility gene , yet , to be identified , that increases al absorption . thus it is not unexpected that elevated hg levels led to severe neuronal cell death already at low concentrations ( ld50 5 m ) accompanied with a dose - dependent reduction of synaptic density . hg toxicity has been reported to increase with incubation time ; thus , even lower concentrations of hgcl2 ( 25100 nm ) can lead to significantly reduced neuronal cell survival if exposure is expanded . pb has been known to be an environmental contaminant associated with several neurological diseases and has been reported to affect a child 's developing brain adversely [ 39 , 40 ] . however , in this study , pb seemed to act slightly beneficially on neuronal cell health and slightly increased synaptic number at higher concentrations . similarly , audesirk et al . have reported that pb showed the least effects on hippocampal neurons at intermediate concentrations ( 110 m ) . it might be speculated that the toxicity of pb is not caused by a direct influence on neurons but by interference with processes already outside the cns possibly due to the absence of certain receptors or competition with target proteins . a toxic interference , as reported , for example , for cu and zn , might happen already within the gastrointestinal system and not within the brain . it should be mentioned that many factors such as the use of fbs or b27 in the growth medium might influence the toxicity of certain metals . for example , bovine serum albumin ( bsa ) present in fetal bovine serum ( fbs ) and b27 supplement has high zn binding capacity and the actual concentration that reaches the neuron is hard to predict . indeed , it was shown that a concentration of more than 100 nm free based on this and the calculated value for zn in these experiments , it is likely that a high amount of trace metals will be buffered and bound to proteins immediately after application . already the manipulation of a single trace metal shows an influence on cell viability but also synapses . however , in patients such as those suffering from asd , a combination of trace metal alterations ( excess and deficiencies ) occurs that might establish a whole new environment for neurons and affect synapse number and function . thus , in a second set of experiments , we established a biometal profile that resembles the one reported in individuals with asd and assessed the effect on cell health and synapse density and composition . recently , a pathway at excitatory glutamatergic synapses was identified by large - scale genetic screens and the description of asd candidate genes that is centered around proteins of the shank family . mutations in the three family members shank1 , shank2 ( prosap1 ) , and shank3 ( prosap2 ) have been described to be associated with asd [ 46 , 47 ] and animal models with deletions of these genes displaying asd like phenotypes [ 48 , 49 ] . however , shank2 and shank3 are also proteins that are highly regulated by zn - binding and their amount reduced in animal models for zn deficiency or cu overload . therefore , these proteins might be promising candidates providing a link between trace metal imbalances and synaptic defects in asd . indeed , we observed a loss of synapses along with a reduction of p2 associated protein levels in shank family members . given that zn is one of the most abundant trace metals in the brain and acts at the postsynapse influencing synaptic proteins and plasticity [ 5255 ] , we investigated whether zn might be able to rescue the asd biometal profile induced phenotype . chronic zn supplementation was able to normalize the alterations in zn - binding shank2 and shank3 but did not further increase shank levels as reported to result from acute zn supplementation . thus , feedback mechanism might be involved that regulates shank levels over time limiting them to the level seen in controls . additionally , we were interested in levels of nmdar subunits since alterations such as a decrease of nmdars was described in shank3 deficient cells , shank2 knock - out mice , and prenatal zn deficient mice that also display a reduction of shank2 and shank3 [ 14 , 56 , 57 ] . indeed , similar to the expression of shank proteins , expression of nmdar subunits was sensitive to trace metal profiles such as those seen in asd patients , in particular increased levels of putative toxic metals in combination with zn and fe deficiency ( asd2 ) , on mrna and synaptic protein level . this again argues that alterations in trace metal status as seen in asd patients if present in a similar manner in the brain of patients might be sufficient to induce changes similar to those seen in genetic models of asd such as shank knock - out mice . in particular , a shift from nmdar subunit 2b ( glun2b ) containing synapses to glun2a as seen in normal development might be affected by the reduction in glun2a but not glun2b expression on mrna and protein level that happens only in the trace metal profile similar to that seen in asd patients . after zn supplementation , mrna levels of nmda receptor subunits remained significantly decreased compared to controls . in contrast , gene expression of shank family members is fully restored after zn supplementation . the mechanisms of how zn might influence nmdar and shank transcription are currently not well understood . however , myc - associated zinc finger protein ( maz ) binds a gc box element in the regulatory regions of glun1 , glun2a , glun2b , and glun2c . thus , availability of zn might affect , among many other transcription factors with zinc finger domains , maz , thereby affecting nmdar subunit expression rates . another transcription factor that is sensitive to trace metal levels , especially to zn , is mtf1 ( metal - regulatory transcription factor 1 ) . mtf1 binds to metal response elements ( mres ) within the promoter region of genes in presence of zn to increase their transcription rate [ 59 , 60 ] . however , whether gene regulation of shanks by zn is dependent on mtf1 has not been investigated so far . the obtained results from gene expression analyses did not always correlate with protein expression measured by western blot analysis and analysis of immunofluorescence intensity . it is thus possible that a decrease of mrna level might be reflected by a decrease in extrasynaptic proteins . additionally , the decrease on mrna levels might not have been present long enough to affect the concentration of stable proteins with slow turn - over rates . , almost complete loss of proteins leading to very low signal intensities below background level might not have been detected . furthermore , we analyzed the synaptic expression of znt-1 that was recently reported to be enriched at the psd and that is associated with nmdar . we found that protein levels of znt-1 react sensitively to the established trace metal profiles , again especially to asd2 . one might speculate that export of zn as synaptic acting signal , which might be decreased in zn depleted medium , is compensated by an increased number of znt-1 proteins at the synapse . however , given that znt-1 global expression usually is upregulated in response to increasing zn concentrations , a novel mechanism of synaptic znt-1 regulation is implied . intriguingly , the upregulation of zn - t1 is reduced after replenishment of asd2 medium with zn . although the alterations observed can not be explained by zn deficiency only , our data show that supplementation of asd2 medium with zn alone is already sufficient to rescue most of the effects caused by exposure to putative toxic trace metals along with zn and fe deficiency . addition of zn could partly restore cell health ( reduced branching of tertiary dendrites ) and synaptic loss . additionally , to an effect on synapses , the toxicity of pb , cd , cu , and hg might be reduced upon zn treatment due to an antagonistic relationship between zn and these trace metals . in accordance with the literature [ 62 , 63 ] , we could observe a strong and significant antagonistic behavior of zn and cd on neuronal cell health and synapse density . further , a slight reduction of cu and hg uptake has been seen upon coapplication of zncl2 that might be further increased if the zn concentration would not be equimolar to cu and hg but exceed their concentration . taken together , our results show that , independent of the important but so far unsolved question , whether these imbalances in trace elements seen in asd patients are cause or consequence , indeed an altered biometal status may have an influence on synapse composition and function . addressing the dysregulation of trace metals might be a novel approach in modifying the disease phenotype in patients and should be thoroughly investigated in vivo in future studies .

various recent studies revealed that biometal dyshomeostasis plays a crucial role in the pathogenesis of neurological disorders such as autism spectrum disorders ( asd ) . substantial evidence indicates that disrupted neuronal homeostasis of different metal ions such as fe , cu , pb , hg , se , and zn may mediate synaptic dysfunction and impair synapse formation and maturation . here , we performed in vitro studies investigating the consequences of an imbalance of transition metals on glutamatergic synapses of hippocampal neurons . we analyzed whether an imbalance of any one metal ion alters cell health and synapse numbers . moreover , we evaluated whether a biometal profile characteristic for asd patients influences synapse formation , maturation , and composition regarding nmda receptor subunits and shank proteins . our results show that an asd like biometal profile leads to a reduction of nmdar ( nr / grin / glun ) subunit 1 and 2a , as well as shank gene expression along with a reduction of synapse density . additionally , synaptic protein levels of glun2a and shanks are reduced . although zn supplementation is able to rescue the aforementioned alterations , zn deficiency is not solely responsible as causative factor . thus , we conclude that balancing zn levels in asd might be a prime target to normalize synaptic alterations caused by biometal dyshomeostasis .

1. Introduction 2. Material and Methods 3. Results 4. Discussion

autism spectrum disorders ( asd ) are a group of neurological disorders currently considered to manifest from a synaptic dysfunction or synaptopathy . in particular synapse formation and/or synaptic signal transduction and plasticity might be affected based on the identified candidate genes from large - scale genetic studies . for example , deficiencies for zn , ca , fe , mg , mn , and se as well as increased concentrations for al , as , cd , hg , and pb were noted in hair samples of autistic patients [ 4 , 5 ] and the burden of toxic metals in patients showed a correlation with the severity of the autism phenotype . this complex scenario prompted us to investigate the interplay and effects of a dyshomeostasis of different metal ions and the resultant pathological alterations of synapses . to study this equilibrium that not only involves biometals but also toxic metal ions an organism is exposed to , on cellular level in vitro , we have carried out experiments on metal ions such as aluminum ( al ) , cadmium ( cd ) , copper ( cu ) , iron ( fe ) , mercury ( hg ) , magnesium ( mg ) , lead ( pb ) , selenium ( se ) , and zinc ( zn ) , using primary hippocampal neurons . to test for downstream effects of imbalance of any one metal ion , which might lead to a domino effect and produces changes in all other metal ions , the present report describes the effects of heavy metal ions and the interaction among cd , cu , hg , pb , se , and al with zn in cultured cells . first , we analyzed the effect of metal overload of a single trace metal on various synaptic parameters by chronic treatment of primary rat neuronal cultures with metal chlorides . next , we determined the effect of metal overload of cd , cu , hg , and pb in combination since these metals were frequently described elevated in asd patients . we further analyzed whether the absence of zn and fe , a common feature in asd patients , modifies the effect of high cd , cu , hg , and pb levels . finally , we investigated whether zn supplementation is able to overcome synaptic defects induced by the trace metal profile characteristic for asd patients . primary antibodies were purchased from sigma - aldrich ( map2 , glun1 , and shank1 for wb ) , synaptic systems ( bassoon , homer1b / c , shank3 ) , merck millipore ( glun2a and glun2b ) , and novus biological ( shank1 for if ) . all animal experiments were performed in compliance with the guidelines for the welfare of experimental animals issued by the federal government of germany and by the local ethics committee ( ulm university ) , i d number : 0.103 . additionally , in some conditions , original metal concentrations of some or all chelated metals were reestablished according to the neurobasal medium ( 1x ) liquid media formulation using cacl2 , fe(no3)39h2o , mgcl2 , and znso47h2o . in our previous study , we reported characteristic biometal profiles in neurological disorders . therefore , to understand the consequences of all these alterations in trace metals for synapses , in a first set of experiments , we aimed to analyze first the influence of each trace metal on cell health and synapse numbers separately ( figures s1a and b in supplementary material available online at http://dx.doi.org/10.1155/2015/985083 and figure 1 ) . to that end , we treated hippocampal neuronal cell cultures between div 10 and div 14 with different concentrations of the trace metals al , cd , cu , fe , hg , mg , pb , se , and zn . as expected , a linear correlation between metal levels and the amount of cell death was found , especially for cd , cu , and zn ( figures 1(a ) and 1(b ) ) . we could not detect significant cell death over a wide spectrum of concentrations for al , fe , and pb . additionally , for hg and se , an exponential increase in cell death beyond a certain concentration was observed . based on the correlation between cell health and the concentration of a certain trace metal applied , ld50 concentrations were calculated for each metal ( figure s1d ) . glial cells that were present to a low extent in the neuronal cultures were much more resistant to alterations in trace metal concentrations and showed no signs of cell death in the toxic concentration range seen for neuronal cells ( figure s1e ) . similar to the results shown above , high concentrations of metals that were shown to lead to cell death led to a reduction in dendritic branching starting from tertiary dendrites and also affecting secondary and primary in the most toxic cases ( figure 1(c ) ) . interestingly , supplementation of mg and zn in a low concentration leads to a significant increase in dendritic branching restricted to tertiary dendrites ( figure 1(c ) ) . indeed , the quantification of synapse density in treated neurons and compared to untreated control cells shows a correlation of toxic effects and synapse loss for metal ions , such as cd , cu , and se ( figure 1(d ) , figure s1f ) . since , for some metal ions , we could determine an effect on synapses independent of a possible cellular toxicity ( figure 2 ) , a role of these metals in synapse formation and/or stabilization is likely . additionally , supplementation of both zn and se , in low concentrations increased synapse density , which then can be found increased compared to untreated control despite ongoing cell death . based on the findings described above , it might be possible that the absence of a synaptogenic effect of some metals such as zn and the toxic effects of others act in combination in asd when deficiencies and overload of certain trace metals occur in parallel . therefore , we established a biometal profile resembling the assumed trace metal alterations in asd patients and evaluated the consequences on cell health and especially on synapses . hippocampal neurons were grown from div 10 to div 14 in cell culture media containing different sets of trace metals : control cells ( ctrl ) were grown in trace metal depleted neurobasal medium that was reconstituted for all depleted trace metals ( mg , ca , fe , and zn ) according to the manufacturer 's indicated metal concentrations . additionally , cells were grown in neurobasal medium with addition of putative toxic metals ( 0.5 m cd , cu , hg , and 2 m pb ) found to be increased in asd patients ( asd1 ) . in a third condition , cells were grown in trace metal depleted neurobasal medium that was reconstituted only for mg and ca , with addition of the putative toxic metals ( 0.5 m cd , cu , hg , and 2 m pb ) ( asd2 ) . asd2 growth medium , therefore , with fe and zn deficiency and overload of cd , cu , hg , and pb resembles the characteristic biometal profile found in asd . cells exposed to asd2 medium indeed displayed a significant reduction in cell health visible by an increased number of apoptotic nuclei and increased dendritic fragmentation ( figures 3(a ) and 3(b ) ) . the number of synapses per 10 m dendrite length assessed by quantification of bassoon and homer1b / c positive puncta was significantly reduced in cells growing in medium resembling the biometal profile found in asd patients ( asd2 ) ( figure 3(c ) ) . along with a reduction in the number of synapses , gene expression levels of synaptic receptors such as nmdar ( glun1 , glun2a , and glun2b ) as well as the zn dependent shank scaffold proteins ( shank1 , shank2 , and shank3 ) show significant alterations ( figure 3(d ) ) . while the presence of putative toxic metals increased in asd patients ( asd1 ) is sufficient to significantly increase glun2b expression levels , glun1 and glun2a and shank1 , shank2 , and shank3 mrna levels are only significantly altered in case of an additional zn and fe deficiency ( asd2 ) . fluorescent readouts for shank proteins show a reduction on protein level under asd1 conditions , while the remaining synapses under asd2 condition display normal shank levels ( figure 3(e ) ) . however , analysis using protein biochemistry shows that , overall , shank protein concentrations are reduced in the p2 fraction of cells growing under asd2 condition ( figure 3(f ) ) . additionally , similar to the decrease of mrna levels of nmdar subunits , we detected significantly less glun2a protein and a trend towards a decrease of glun2b protein levels in cells grown in asd2 medium . finally , in a last set of experiments , we wanted to determine , whether the addition of a single specific trace metal might lead to a rescue in the observed phenotype after inducing an asd like biometal profile in cell culture or whether all alterations need to be addressed separately . given that an antagonistic role of zn and pb or cu was proposed before [ 3 , 4 ] and that we could verify a potentially beneficial effect of zn on synapses , we next wanted to elucidate whether zn supplementation in combination with harmful metals ( asd2 ) is able to modify the toxicity of the trace metal on neurons in vitro . to that end however , asd2 medium ( neurobasal medium that was reconstituted only for mg and ca , with addition of the putative toxic metals ( 0.5 m cd , cu , hg , and 2 m pb ) ) was supplemented with 50 m zn . addition of zn was unable to rescue the observed cell death detected in cultures grown under asd2 conditions ( figure 4(a ) ) , although it led to a significant reduction in dendritic fragmentation ( figure 4(b ) ) . along with this , the reduction in the number of synapses per 10 m dendrite length assessed by quantification of bassoon and homer1b / c positive puncta seen under asd2 conditions was rescued after addition of zn ( asd2 + zn ) ( figure 4(c ) ) . supplementation of zn was also able to significantly increase the reduction in mrna levels of nmda receptor subunits and shank family members seen in cells growing under asd2 conditions ( figure 4(d ) ) . however , in case of nmda receptor subunits , mrna levels still remained significantly decreased compared to controls . the observed changes on protein level of nmda receptor subunits , the zn binding shank2 and shank3 , and znt-1 in turn were completely rescued after addition of zn ( asd2 + zn ) ( figure 4(f ) ) . immunofluorescence readouts revealed no changes in synaptic protein levels after supplementation of cells growing under asd2 conditions with zn ( figure 4(e ) , figure s2d ) . furthermore , we used icp - ms to measure the extracellular metal concentrations of hippocampal cell cultures with and without the presence of zn . a reduction in the uptake of cu , hg , and se was visible upon coapplication of zn , while an increase in uptake of pb was visible ( figure 4(g ) ) . additionally , the alterations caused under asd2 conditions although to high extent rescued by zn supplementation are not based on zn deficiency alone . a comparison between asd2 and pure zn deficient ( znd ) conditions reveals that zn deficiency without additional alterations in biometals does not lead to the significant reduction in cell health ( figures s3a , b ) and synapse number ( figure s3c ) such as seen under asd2 conditions . furthermore , the decrease in mrna expression levels of nmda receptor subunits is less pronounced and absent for shank family members under pure zn deficiency ( figure s3d ) . however , as reported before , zn deficiency significantly affects the synaptic levels of the zn binding shank family members shank2 and shank3 using immunofluorescent readouts ( figure s3e ) . protein biochemistry with p2 fractions ( figure s3f ) reveals that , on protein level , zn deficiency alone already affects nmdar subunit expression , but to a lesser extent the expression of shanks . therefore , we investigated the effects of different essential and nonessential trace metals on neurons in vitro and further examined their relationship with zinc , one of most abundant trace metals in the brain . in a first set of experiments , we investigated the influence of overload with a single trace element . although trace metals such as mg , cu , fe , se , and zn are essential with a vital role in normal brain functions , increased levels in the brain may lead to severe neurological symptoms . similarly , only few pathologically morphological changes of primary neurons could be seen after exposure to mgcl2 in the tested concentration range . in contrast , exposure of neurons to cucl2 leads to a dose - dependent cell death and a significant reduction of synapse numbers correlated to the reduced number of viable cells , which can be also observed in younger neurons at div 6 . these differences are likely due to the use of different cell culture systems and it is not surprising that an immortalized hippocampal cell line is much more resistant against se induced toxicity than primary neurons . cd toxicity was shown to lead to impaired neurogenesis , reduced neuronal differentiation , and reduced axogenesis in vitro and even lower concentrations have been reported to repress dendritic and synaptic development [ 24 , 25 ] . although al is a well - accepted neurotoxin and discussed as factor for the development of neurodegeneration , for example , as seen in alzheimer 's disease ( ad ) , we did not observe adverse effects on neurons and synapses even at very high concentrations of alcl3 ( up to 500 m ) similar to a study by kawahara et al . thus it is not unexpected that elevated hg levels led to severe neuronal cell death already at low concentrations ( ld50 5 m ) accompanied with a dose - dependent reduction of synaptic density . however , in this study , pb seemed to act slightly beneficially on neuronal cell health and slightly increased synaptic number at higher concentrations . it might be speculated that the toxicity of pb is not caused by a direct influence on neurons but by interference with processes already outside the cns possibly due to the absence of certain receptors or competition with target proteins . a toxic interference , as reported , for example , for cu and zn , might happen already within the gastrointestinal system and not within the brain . it should be mentioned that many factors such as the use of fbs or b27 in the growth medium might influence the toxicity of certain metals . thus , in a second set of experiments , we established a biometal profile that resembles the one reported in individuals with asd and assessed the effect on cell health and synapse density and composition . mutations in the three family members shank1 , shank2 ( prosap1 ) , and shank3 ( prosap2 ) have been described to be associated with asd [ 46 , 47 ] and animal models with deletions of these genes displaying asd like phenotypes [ 48 , 49 ] . therefore , these proteins might be promising candidates providing a link between trace metal imbalances and synaptic defects in asd . indeed , we observed a loss of synapses along with a reduction of p2 associated protein levels in shank family members . given that zn is one of the most abundant trace metals in the brain and acts at the postsynapse influencing synaptic proteins and plasticity [ 5255 ] , we investigated whether zn might be able to rescue the asd biometal profile induced phenotype . chronic zn supplementation was able to normalize the alterations in zn - binding shank2 and shank3 but did not further increase shank levels as reported to result from acute zn supplementation . thus , feedback mechanism might be involved that regulates shank levels over time limiting them to the level seen in controls . additionally , we were interested in levels of nmdar subunits since alterations such as a decrease of nmdars was described in shank3 deficient cells , shank2 knock - out mice , and prenatal zn deficient mice that also display a reduction of shank2 and shank3 [ 14 , 56 , 57 ] . indeed , similar to the expression of shank proteins , expression of nmdar subunits was sensitive to trace metal profiles such as those seen in asd patients , in particular increased levels of putative toxic metals in combination with zn and fe deficiency ( asd2 ) , on mrna and synaptic protein level . this again argues that alterations in trace metal status as seen in asd patients if present in a similar manner in the brain of patients might be sufficient to induce changes similar to those seen in genetic models of asd such as shank knock - out mice . in particular , a shift from nmdar subunit 2b ( glun2b ) containing synapses to glun2a as seen in normal development might be affected by the reduction in glun2a but not glun2b expression on mrna and protein level that happens only in the trace metal profile similar to that seen in asd patients . after zn supplementation , mrna levels of nmda receptor subunits remained significantly decreased compared to controls . in contrast , gene expression of shank family members is fully restored after zn supplementation . however , myc - associated zinc finger protein ( maz ) binds a gc box element in the regulatory regions of glun1 , glun2a , glun2b , and glun2c . the obtained results from gene expression analyses did not always correlate with protein expression measured by western blot analysis and analysis of immunofluorescence intensity . furthermore , we analyzed the synaptic expression of znt-1 that was recently reported to be enriched at the psd and that is associated with nmdar . we found that protein levels of znt-1 react sensitively to the established trace metal profiles , again especially to asd2 . although the alterations observed can not be explained by zn deficiency only , our data show that supplementation of asd2 medium with zn alone is already sufficient to rescue most of the effects caused by exposure to putative toxic trace metals along with zn and fe deficiency . additionally , to an effect on synapses , the toxicity of pb , cd , cu , and hg might be reduced upon zn treatment due to an antagonistic relationship between zn and these trace metals . in accordance with the literature [ 62 , 63 ] , we could observe a strong and significant antagonistic behavior of zn and cd on neuronal cell health and synapse density . further , a slight reduction of cu and hg uptake has been seen upon coapplication of zncl2 that might be further increased if the zn concentration would not be equimolar to cu and hg but exceed their concentration . taken together , our results show that , independent of the important but so far unsolved question , whether these imbalances in trace elements seen in asd patients are cause or consequence , indeed an altered biometal status may have an influence on synapse composition and function . addressing the dysregulation of trace metals might be a novel approach in modifying the disease phenotype in patients and should be thoroughly investigated in vivo in future studies .

according to the taiwan department of health , the cumulative death rates from coronary artery disease , stroke , and diabetes are 152 cases per 100,000/yr , which nearly equals the death rate from malignancies . these ' modern diseases ' are a huge burden , not only to the patients themselves , but also to their families and society . this alarming trend is not unique to taiwan , and has been reported to occur in many other parts of the world . therefore , the prevention and early detection of coronary heart disease and diabetes has become a major public health issue . the well - known risk factors for these diseases include excess body weight , hypertension , hyperlipidemia , and hyperglycemia . indeed , the clustering of these risk factors was first noted in 1966 ( 1 ) . in 1988 , reaven introduced the term syndrome x , which consisted of hyperinsulinemia , hypertension , dyslipidemia , hyperglycemia , and resistance to insulin - mediated glucose uptake ( 2 ) . he suggested that insulin resistance ( ir ) plays an important role in the etiology and clinical course of patients with diabetes mellitus , hypertension , and coronary heart disease ( 3 ) . in 1998 , the world health organization ( who ) also recognized the importance of this clustering and further defined the clinical characteristics of the ' metabolic syndrome ' ( mets ) ( 4 ) . however , such criteria are not practical for routine clinical use because one of the major criteria , ir , requires measurement by a hyperinsulinemic euglycemic clamp . therefore , the national cholesterol education program adult treatment panel iii ( atp iii ) provided a simpler definition in 2001 , in the hope that it could be used even in general practice ( 5 ) . it had been shown that patients with mets , as defined by either who or atp iii criteria , have more severe ir as compared to the general population ( 6 , 7 ) . however , the cut - off values of these diagnostic criteria were originally determined arbitrarily , have never been stratified according to a weighted clinical effect , and may vary in different ethnic groups . moreover , it is unknown which of the five clinical characteristics amongst the atp iii criteria , if any , is related to more severe ir . in the current study , we measured ir directly by an insulin suppression test ( ist ) . subjects were placed into quartiles based on the level of each of the mets clinical characteristics . then , the steady state plasma glucose ( sspg ) level resulting from the ist was compared between the different quartiles representing one clinical characteristic as well as between all the different clinical characteristics . furthermore , factor analysis ( 8 , 9 ) , a multivariate statistical tool , could reduce a considerable number of inter - correlated variables to a smaller set that accounts for most of the variances between the data . by this mean , a set of dimensions ( sometimes called factors ) which are not easily observed in the original variable could be identified . therefore , it was also used to investigate which of mets factors are related to ir ( sspg levels).thus , we are able to determine the severity of ir between the quartiles representing each clinical characteristic , but also determine which clinical characteristic of mets was associated with the most severe ir when compared with other clinical characteristics within the chinese population . five hundred sixty four subjects , 250 males and 314 females aged 20 - 75 yr , were enrolled during routine health evaluations at the tri - service general hospital between 1995 and 1999 . subjects with a history of diabetes , hypertension , hyperlipidemia , or other significant medical or surgical diseases were excluded . subjects who were placed on medications which affect insulin sensitivity were also excluded from the study . the study was approved by the hospital ethics committee , and the nature , purpose , and potential risks of the study were explained to the patients before obtaining their consent to participate . since reaven suggested that the upper 25% in the general population are ir , we divided our subjects into four groups regarding each of five clinical characteristics of the mets and then evaluated the ir in each quartile ( 2 ) . the ability of insulin to dispose of a glucose load was estimated by modification of the ist , as described by shen et al . one catheter was used for the administration of a 180 min infusion of somatostatin ( 250 g / hr ) , insulin ( 25 mu / m / min ) , and glucose ( 240 mg / m / min ) . blood was collected every 30 min initially , and then at 10 min intervals from 150 - 180 min of the infusion , to determine the steady state plasma insulin ( sspi ) and sspg concentrations for each individual . the sspi concentrations were comparable in all individuals , thus the sspg concentrations provided the measure of efficacy of insulin in promoting disposal of the infused glucose load . the samples obtained at -5 and 0 min were analyzed for fasting plasma glucose ( fpg ) , fasting plasma insulin ( fpi ) , and lipid levels . plasma glucose levels were determined using the glucose oxidase method ( ysi 203 glucose analyzer ; scientific division , yellow spring instrument company , inc . , yellow spring , oh , u.s.a . ) . insulin levels were measured by a commercial solid phase radioimmunoassay kit ( 11 ; coat - a - count insulin kit ; diagnostic products corporation , los angeles , ca , u.s.a . ) . the intra- and inter - assay coefficients of variance for insulin were 3.3% and 2.5% , respectively . both triglyceride ( tg ) and total cholesterol ( tc ) levels were measured using the dry , multilayer analytical slide method in the fuji dri - chem 3000 analyzer ( fuji photo film corporation , minato - ku , tokyo , japan ) . serum high density lipoprotein cholesterol ( hdlc ) concentration was determined by an enzymatic , cholesterol assay method following dextran sulfate precipitation . analysis was performed using spss for windows , version 10.0 ( spss ; chicago , il , u.s.a . ) . data were tested for normal distribution using the kolmogorov - smirnov test and for homogeneity of variances with levene 's test . independent student 's t - test was used to evaluate gender - based physical and metabolic differences . the one - way anova with the bonferroni method as a post hoc test was also applied to compare differences between the clinical characteristics in each quartile . since age was considered as the confounding covariate , each variable of interest ( i.e. , body mass index [ bmi ] , tg , hdlc , fpg , and blood pressure ) was first adjusted for age by employing the analysis of covariance . the derived residuals ( adjusted variables ) were then again used for analyses after the unadjusted data were examined . all statistical tests were two - sided and p - values less than 0.05 were considered to indicate statistical significance . furthermore , exploratory factor analysis was conducted to examine the relationships among the variables which constituted mets . principle factor analysis was employed to transform the original variables into a new set of components which are independent of each other . then number of components to be retained was based on scree plot analysis ( factors above the break in the curve were retained ) and eigenvalue criteria ( 1.0 ) . such two modalities have been adopted to identify the minimum number of components ( 8 , 12 , 13 ) . five hundred sixty four subjects , 250 males and 314 females aged 20 - 75 yr , were enrolled during routine health evaluations at the tri - service general hospital between 1995 and 1999 . subjects with a history of diabetes , hypertension , hyperlipidemia , or other significant medical or surgical diseases were excluded . subjects who were placed on medications which affect insulin sensitivity were also excluded from the study . the study was approved by the hospital ethics committee , and the nature , purpose , and potential risks of the study were explained to the patients before obtaining their consent to participate . since reaven suggested that the upper 25% in the general population are ir , we divided our subjects into four groups regarding each of five clinical characteristics of the mets and then evaluated the ir in each quartile ( 2 ) . the ability of insulin to dispose of a glucose load was estimated by modification of the ist , as described by shen et al . one catheter was used for the administration of a 180 min infusion of somatostatin ( 250 g / hr ) , insulin ( 25 mu / m / min ) , and glucose ( 240 mg / m / min ) . blood was collected every 30 min initially , and then at 10 min intervals from 150 - 180 min of the infusion , to determine the steady state plasma insulin ( sspi ) and sspg concentrations for each individual . the sspi concentrations were comparable in all individuals , thus the sspg concentrations provided the measure of efficacy of insulin in promoting disposal of the infused glucose load . the samples obtained at -5 and 0 min were analyzed for fasting plasma glucose ( fpg ) , fasting plasma insulin ( fpi ) , and lipid levels . plasma glucose levels were determined using the glucose oxidase method ( ysi 203 glucose analyzer ; scientific division , yellow spring instrument company , inc . , yellow spring , oh , u.s.a . ) . insulin levels were measured by a commercial solid phase radioimmunoassay kit ( 11 ; coat - a - count insulin kit ; diagnostic products corporation , los angeles , ca , u.s.a . ) . the intra- and inter - assay coefficients of variance for insulin were 3.3% and 2.5% , respectively . both triglyceride ( tg ) and total cholesterol ( tc ) levels were measured using the dry , multilayer analytical slide method in the fuji dri - chem 3000 analyzer ( fuji photo film corporation , minato - ku , tokyo , japan ) . serum high density lipoprotein cholesterol ( hdlc ) concentration was determined by an enzymatic , cholesterol assay method following dextran sulfate precipitation . analysis was performed using spss for windows , version 10.0 ( spss ; chicago , il , u.s.a . ) . data were tested for normal distribution using the kolmogorov - smirnov test and for homogeneity of variances with levene 's test . independent student 's t - test was used to evaluate gender - based physical and metabolic differences . the one - way anova with the bonferroni method as a post hoc test was also applied to compare differences between the clinical characteristics in each quartile . since age was considered as the confounding covariate , each variable of interest ( i.e. , body mass index [ bmi ] , tg , hdlc , fpg , and blood pressure ) was first adjusted for age by employing the analysis of covariance . the derived residuals ( adjusted variables ) were then again used for analyses after the unadjusted data were examined . all statistical tests were two - sided and p - values less than 0.05 were considered to indicate statistical significance . furthermore , exploratory factor analysis was conducted to examine the relationships among the variables which constituted mets . principle factor analysis was employed to transform the original variables into a new set of components which are independent of each other . then number of components to be retained was based on scree plot analysis ( factors above the break in the curve were retained ) and eigenvalue criteria ( 1.0 ) . such two modalities have been adopted to identify the minimum number of components ( 8 , 12 , 13 ) . as expected , the male subjects in the current study had higher blood pressures and lower hdlc levels than the female subjects . the other mets clinical characteristics measured did not differ significantly between male and female subjects ( table 1 ) . we further divided the male and female subjects into quartiles according to the measured levels of each mets clinical characteristic , with subjects in the upper quartile having the highest levels and those in the lower quartile having the lowest levels . in the current study , we used two different methods to evaluate the importance of ir with respect to the mets . then , since there were six mean sspg values derived from each clinical characteristic ( i.e. , bmi , systolic blood pressure [ sbp ] , diastolic blood pressure [ dbp ] , fpg , tg , and hdlc ) , we further compared the sspg values derived from all clinical characteristics . the results of comparisons of the four quartiles for males and females are shown in fig . 1 , 2 , respectively . in males , the upper quartiles from the bmi and fpg clinical characteristics had the highest sspg levels as compared to the other three quartiles . the upper quartiles of the dbp and tg clinical characteristics were only greater than the lower two quartiles . in females , the sspg levels in the upper quartile of the bmi and tg clinical characteristics were significantly greater than the other lower three quartiles . while the sspg levels were highest in the sbp and fpg clinical characteristics , they were only higher than the third quartile ( fig . 2 ) . in order to evaluate which of the highest quartiles representing any of the mets clinical characteristics had the highest sspg levels , we further compared the upper quartiles of the bmi , sbp , dbp , fpg , hdlc , and tg clinical characteristics . 3 ) , except the sspg level was greater in the upper quartile of the bmi clinical characteristic than in the upper quartile of the hdlc clinical characteristic ( 12.370.47 vs. 9.520.52 finally , table 2 displays the results of factor analysis of core metabolic variables among the study subjects . 4 graphically depicts the same results with the percentage of variance explained by each factor . a two - factor solution , which was supported by the retention criteria described in the method , was obtained . these factors could be interpreted as 1 ) a " insulin resistance " factor , with a positive loading of bmi , fpg , tg , sspg and an inverse loading of hdlc , and 2 ) a " blood pressure " factor with a positive loading of bmi and both the sbp and dbp . the two - factor solution explained about 56% of the total variance in these subjects ( 28.4% factor 1 and 27.3% factor 2 ) . the data herein showed that , in general , the trend of higher sspg levels was associated with more mets clinical characteristics ( fig . 1 , 2 ) . however , only the bmi , fpg , and tg in both males and females , dpb in males , and sbp in females had significantly higher sspg levels in the upper quartile as compared to the lower quartiles . these results suggested that among the five clinical characteristics of the mets measured , bmi , fpg , and tg might be best related to ir and therefore , are the first clinical characteristics to be detected as abnormal . for example , cheal et al . found that among all the mets clinical characteristics , bmi and tg were best correlated with sspg ( 14 ) . by using factor analysis to evaluate the relationships between mets clinical characteristics and ir , meigs et al thus , he further suggested that ir should be the central focus in assessing the mets ( 8) . it has been long - recognized that ir is clearly associated with obesity , whether generalized or central ( 15 - 17 ) . the detrimental influence of central adiposity on ir is thought to be mediated by intra - abdominal fat deposition , in which hypertrophied adipocytes are resistant to anti - lipolytic action of insulin . as a consequence , elevated levels of free fatty acids may induce ir in the peripheral tissues and liver ( 18 ) . also , the preponderance of enlarged fat cells , as occur in abdominal obesity , may increase the risk of glucose intolerance and hypertriglyceridemia and thus account for the metabolic derangements ( 19 , 20 ) . it is interesting to note that the relationships between ir and some of the mets clinical characteristics were affected by gender . in our study , for example , the upper quartile of dbp in males had significantly higher sspg levels than the lower three quartiles . in contrast , instead of dbp , the sspg was higher in the upper quartile of sbp in females . these results suggested that the relationships between blood pressure and sspg were less consistent in our study . it is difficult to meaningfully compare this finding with other reports , since most of the other reports adjusted both sex and age when discussing the relationships between blood pressure and ir ( 17 , 21 , 22 ) . other than blood pressure , the relationships between ir and hdlc were also different between the genders . fig . 1 , 2 show that the levels of sspg did not differ between any quartiles representing the hdlc clinical characteristic . 3 , when comparing the upper quartiles of all the mets clinical characteristics , the upper quartile of the hdlc clinical characteristic had the lowest sspg level , but was only significant in males . it is well - recognized that ir is related to high tg and low hdlc ( 23 , 24 ) ; however , most of these reports showed that gender had no effect on the relationships involving ir . found that ir was more strongly related to hdlc in males than females , as evidenced by the intravenous glucose tolerance test ( 24 ) . this unique finding was further confirmed in another study conducted in subjects with morbid obesity . the correlations between ir and tg or hdlc were stronger in males than in females ( 25 ) . both hdlc and sbp were shown to be related to sspg levels in males , our data further imply that males might be more prone to develop mets when in middle age , as suggested by cook et al . at present , there are two possibilities to explain the metabolic differences which existed between males and females in our study . this in turn will further increase the risk of cardiovascular disease and diabetes ( 28 , 29 ) . second , male subjects tend to have more upper truncal adiposity ( i.e. , a higher waist - to - hip ratio [ whr ] ) than female subjects . in the current study , since we observed differences between each mets clinical characteristic , we did not adjust the effect of bmi on other risk factors , which is a departure from other studies ( 23 - 25 ) . it is generally agreed that there is a positive correlation between blood pressure and ir . using the euglycemic insulin clamp , this relationship has been repeatedly demonstrated in many different studies ( 17 , 21 , 22 ) . for instance , ferrannini et al . reported that male gender , age , and ir were independently associated with sbp , whereas dbp was related to age , ir , and fpi , but not to the bmi ( 17 ) . they also found that dbp had a higher correlation with ir than sbp ( r=0.34 and 0.18 , respectively ) . however , the same conclusion was not always reached in studies involving other ethnic groups ( 30 ) and the relationships did not exist when adjusted for body fat content ( 31 ) . the discrepancies of these results may be due to the fact that blood pressure per se is not clustered with ir . other possibilities such as the different ethnic groups or the methods used to measure adiposity might also play a role . in the ferrannini study , and as done herein , bmi was used to quantify adiposity . at the same time , toft et al . used whr and suggested that in subjects with the same bmi , changes in whr may still influence the results of variables associated with insulin sensitivity because the accumulation of visceral fat leads to altered insulin clearance ( 31 ) . in the peiris study , the clearance rates of insulin were similar between obese and non - obese subjects ( 32 ) ; however , it was inversely correlated with the whr in obese subjects . therefore , peiris et al . pointed out that whr is better associated with diminished insulin clearance than bmi ( 32 ) . it has been shown that the homeostasis model assessment of ir ( homa - ir ; 33 , 34 ) , which is calculated from fpg and fpi , is a useful surrogate for ir in healthy and diabetic subjects . it has proven to be highly correlated with the gold standard , the euglycemic hyperinsulinemic clamp ( 33 ) . at present , it is generally recognized that one of the main underlying pathophysiologies of glucose intolerance is ir . however , in subjects who already have ir , due to the compensatory increased secretion of the insulin , the fpg may persist within the normal range . that is why the study done by hollenbeck and reaven showed that there was no relationship found between fpg and ir ( 35 ) . on the contrary , surprisingly , in our study , the sspg seemed to be higher as the fpg increased . this finding suggested that even in the early stages before mets develops , fpg is already elevated in subjects with an elevated ir . the results of the factor analysis are similar to most of the other studies . in these studies , usually there are three to four factors being identified ; the " insulin resistant factor " , " obese factor " , " lipid factor " , and/or " blood pressure factor " ( 8 , 9 , 13 , 36 - 39 ) . to the present , one of the most important studies using factor analysis to explore the relationship between ir and other common risk factors for cardiovascular diseases was published by hanley et al . , they have used the intravenous glucose tolerance test to evaluate insulin sensitivity ( si ) . they have also identified two factors in the igt and ngt subjects : the " metabolic factor " ( comprising bmi , waist , si , tg , and hdlc ) ; and the " blood pressure factor " . the only differences are fpg was not loaded in factor 1 and bmi was not loaded in factor 2 . it could be noted that the clustering patterns in our study were not only similar to hanley 's study ( 40 ) but also to the most of other studies with different severities of impaired glucose metabolism and ethnic groups ( 13 , 36 , 38 , 39 ) . the clustering of the sspg , bmi , fpg and tg could further confirm that the ir is more correlated with lipids profile and bmi , but less with the blood pressure . it should be noted and emphasized that this was a hospital - based cohort study . however , the purpose of this study was to observe the relationships between ir and mets , thus , this drawback should not affect the conclusions drawn from the present study . our data suggested that adiposity , higher fpg , and hypertriglyceridemia are more strongly associated with ir in chinese subjects and the importance of these clinical characteristics should be reemphasized . when considering each of the mets clinical characteristics , subjects with a higher bmi may have the highest ir .

the impact the metabolic syndrome ( mets ) components on the severity of insulin resistance ( ir ) has not been reported . we enrolled 564 subjects with mets and they were divided into quartiles according to the level of each component ; and an insulin suppression test was performed to measure ir . in males , steady state plasma glucose ( sspg ) levels in the highest quartiles , corresponding to body mass index ( bmi ) and fasting plasma glucose ( fpg ) , were higher than the other three quartiles and the highest quartiles , corresponding to the diastolic blood pressure and triglycerides , were higher than in the lowest two quartiles . in females , sspg levels in the highest quartiles , corresponding to the bmi and triglycerides , were higher than in all other quartiles . no significant differences existed between genders , other than the mean sspg levels in males were greater in the highest quartile corresponding to bmi than that in the highest quartile corresponding to hdl - cholesterol levels . the factor analysis identified two underlying factors ( ir and blood pressure factors ) among the mets variables . the clustering of the sspg , bmi , triglyceride and hdl - cholesterol was noted . our data suggest that adiposity , higher fpg and triglyceride levels have stronger correlation with ir and subjects with the highest bmi have the highest ir .

INTRODUCTION MATERIALS AND METHODS Subjects Laboratory evaluation Statistical analysis RESULTS DISCUSSION

according to the taiwan department of health , the cumulative death rates from coronary artery disease , stroke , and diabetes are 152 cases per 100,000/yr , which nearly equals the death rate from malignancies . these ' modern diseases ' are a huge burden , not only to the patients themselves , but also to their families and society . this alarming trend is not unique to taiwan , and has been reported to occur in many other parts of the world . therefore , the prevention and early detection of coronary heart disease and diabetes has become a major public health issue . indeed , the clustering of these risk factors was first noted in 1966 ( 1 ) . he suggested that insulin resistance ( ir ) plays an important role in the etiology and clinical course of patients with diabetes mellitus , hypertension , and coronary heart disease ( 3 ) . in 1998 , the world health organization ( who ) also recognized the importance of this clustering and further defined the clinical characteristics of the ' metabolic syndrome ' ( mets ) ( 4 ) . however , such criteria are not practical for routine clinical use because one of the major criteria , ir , requires measurement by a hyperinsulinemic euglycemic clamp . therefore , the national cholesterol education program adult treatment panel iii ( atp iii ) provided a simpler definition in 2001 , in the hope that it could be used even in general practice ( 5 ) . it had been shown that patients with mets , as defined by either who or atp iii criteria , have more severe ir as compared to the general population ( 6 , 7 ) . however , the cut - off values of these diagnostic criteria were originally determined arbitrarily , have never been stratified according to a weighted clinical effect , and may vary in different ethnic groups . moreover , it is unknown which of the five clinical characteristics amongst the atp iii criteria , if any , is related to more severe ir . in the current study , we measured ir directly by an insulin suppression test ( ist ) . subjects were placed into quartiles based on the level of each of the mets clinical characteristics . then , the steady state plasma glucose ( sspg ) level resulting from the ist was compared between the different quartiles representing one clinical characteristic as well as between all the different clinical characteristics . furthermore , factor analysis ( 8 , 9 ) , a multivariate statistical tool , could reduce a considerable number of inter - correlated variables to a smaller set that accounts for most of the variances between the data . by this mean , a set of dimensions ( sometimes called factors ) which are not easily observed in the original variable could be identified . therefore , it was also used to investigate which of mets factors are related to ir ( sspg levels).thus , we are able to determine the severity of ir between the quartiles representing each clinical characteristic , but also determine which clinical characteristic of mets was associated with the most severe ir when compared with other clinical characteristics within the chinese population . five hundred sixty four subjects , 250 males and 314 females aged 20 - 75 yr , were enrolled during routine health evaluations at the tri - service general hospital between 1995 and 1999 . subjects with a history of diabetes , hypertension , hyperlipidemia , or other significant medical or surgical diseases were excluded . subjects who were placed on medications which affect insulin sensitivity were also excluded from the study . the study was approved by the hospital ethics committee , and the nature , purpose , and potential risks of the study were explained to the patients before obtaining their consent to participate . since reaven suggested that the upper 25% in the general population are ir , we divided our subjects into four groups regarding each of five clinical characteristics of the mets and then evaluated the ir in each quartile ( 2 ) . the ability of insulin to dispose of a glucose load was estimated by modification of the ist , as described by shen et al . one catheter was used for the administration of a 180 min infusion of somatostatin ( 250 g / hr ) , insulin ( 25 mu / m / min ) , and glucose ( 240 mg / m / min ) . blood was collected every 30 min initially , and then at 10 min intervals from 150 - 180 min of the infusion , to determine the steady state plasma insulin ( sspi ) and sspg concentrations for each individual . the sspi concentrations were comparable in all individuals , thus the sspg concentrations provided the measure of efficacy of insulin in promoting disposal of the infused glucose load . the samples obtained at -5 and 0 min were analyzed for fasting plasma glucose ( fpg ) , fasting plasma insulin ( fpi ) , and lipid levels . plasma glucose levels were determined using the glucose oxidase method ( ysi 203 glucose analyzer ; scientific division , yellow spring instrument company , inc . both triglyceride ( tg ) and total cholesterol ( tc ) levels were measured using the dry , multilayer analytical slide method in the fuji dri - chem 3000 analyzer ( fuji photo film corporation , minato - ku , tokyo , japan ) . analysis was performed using spss for windows , version 10.0 ( spss ; chicago , il , u.s.a . ) data were tested for normal distribution using the kolmogorov - smirnov test and for homogeneity of variances with levene 's test . independent student 's t - test was used to evaluate gender - based physical and metabolic differences . the one - way anova with the bonferroni method as a post hoc test was also applied to compare differences between the clinical characteristics in each quartile . , body mass index [ bmi ] , tg , hdlc , fpg , and blood pressure ) was first adjusted for age by employing the analysis of covariance . furthermore , exploratory factor analysis was conducted to examine the relationships among the variables which constituted mets . principle factor analysis was employed to transform the original variables into a new set of components which are independent of each other . then number of components to be retained was based on scree plot analysis ( factors above the break in the curve were retained ) and eigenvalue criteria ( 1.0 ) . five hundred sixty four subjects , 250 males and 314 females aged 20 - 75 yr , were enrolled during routine health evaluations at the tri - service general hospital between 1995 and 1999 . subjects with a history of diabetes , hypertension , hyperlipidemia , or other significant medical or surgical diseases were excluded . subjects who were placed on medications which affect insulin sensitivity were also excluded from the study . the study was approved by the hospital ethics committee , and the nature , purpose , and potential risks of the study were explained to the patients before obtaining their consent to participate . since reaven suggested that the upper 25% in the general population are ir , we divided our subjects into four groups regarding each of five clinical characteristics of the mets and then evaluated the ir in each quartile ( 2 ) . the ability of insulin to dispose of a glucose load was estimated by modification of the ist , as described by shen et al . one catheter was used for the administration of a 180 min infusion of somatostatin ( 250 g / hr ) , insulin ( 25 mu / m / min ) , and glucose ( 240 mg / m / min ) . blood was collected every 30 min initially , and then at 10 min intervals from 150 - 180 min of the infusion , to determine the steady state plasma insulin ( sspi ) and sspg concentrations for each individual . the sspi concentrations were comparable in all individuals , thus the sspg concentrations provided the measure of efficacy of insulin in promoting disposal of the infused glucose load . the samples obtained at -5 and 0 min were analyzed for fasting plasma glucose ( fpg ) , fasting plasma insulin ( fpi ) , and lipid levels . plasma glucose levels were determined using the glucose oxidase method ( ysi 203 glucose analyzer ; scientific division , yellow spring instrument company , inc . , yellow spring , oh , u.s.a . ) both triglyceride ( tg ) and total cholesterol ( tc ) levels were measured using the dry , multilayer analytical slide method in the fuji dri - chem 3000 analyzer ( fuji photo film corporation , minato - ku , tokyo , japan ) . analysis was performed using spss for windows , version 10.0 ( spss ; chicago , il , u.s.a . ) independent student 's t - test was used to evaluate gender - based physical and metabolic differences . the one - way anova with the bonferroni method as a post hoc test was also applied to compare differences between the clinical characteristics in each quartile . , body mass index [ bmi ] , tg , hdlc , fpg , and blood pressure ) was first adjusted for age by employing the analysis of covariance . all statistical tests were two - sided and p - values less than 0.05 were considered to indicate statistical significance . furthermore , exploratory factor analysis was conducted to examine the relationships among the variables which constituted mets . principle factor analysis was employed to transform the original variables into a new set of components which are independent of each other . then number of components to be retained was based on scree plot analysis ( factors above the break in the curve were retained ) and eigenvalue criteria ( 1.0 ) . as expected , the male subjects in the current study had higher blood pressures and lower hdlc levels than the female subjects . the other mets clinical characteristics measured did not differ significantly between male and female subjects ( table 1 ) . we further divided the male and female subjects into quartiles according to the measured levels of each mets clinical characteristic , with subjects in the upper quartile having the highest levels and those in the lower quartile having the lowest levels . in the current study , we used two different methods to evaluate the importance of ir with respect to the mets . then , since there were six mean sspg values derived from each clinical characteristic ( i.e. , bmi , systolic blood pressure [ sbp ] , diastolic blood pressure [ dbp ] , fpg , tg , and hdlc ) , we further compared the sspg values derived from all clinical characteristics . the results of comparisons of the four quartiles for males and females are shown in fig . in males , the upper quartiles from the bmi and fpg clinical characteristics had the highest sspg levels as compared to the other three quartiles . the upper quartiles of the dbp and tg clinical characteristics were only greater than the lower two quartiles . in females , the sspg levels in the upper quartile of the bmi and tg clinical characteristics were significantly greater than the other lower three quartiles . while the sspg levels were highest in the sbp and fpg clinical characteristics , they were only higher than the third quartile ( fig . in order to evaluate which of the highest quartiles representing any of the mets clinical characteristics had the highest sspg levels , we further compared the upper quartiles of the bmi , sbp , dbp , fpg , hdlc , and tg clinical characteristics . 3 ) , except the sspg level was greater in the upper quartile of the bmi clinical characteristic than in the upper quartile of the hdlc clinical characteristic ( 12.370.47 vs. 9.520.52 finally , table 2 displays the results of factor analysis of core metabolic variables among the study subjects . 4 graphically depicts the same results with the percentage of variance explained by each factor . a two - factor solution , which was supported by the retention criteria described in the method , was obtained . these factors could be interpreted as 1 ) a " insulin resistance " factor , with a positive loading of bmi , fpg , tg , sspg and an inverse loading of hdlc , and 2 ) a " blood pressure " factor with a positive loading of bmi and both the sbp and dbp . the two - factor solution explained about 56% of the total variance in these subjects ( 28.4% factor 1 and 27.3% factor 2 ) . the data herein showed that , in general , the trend of higher sspg levels was associated with more mets clinical characteristics ( fig . however , only the bmi , fpg , and tg in both males and females , dpb in males , and sbp in females had significantly higher sspg levels in the upper quartile as compared to the lower quartiles . these results suggested that among the five clinical characteristics of the mets measured , bmi , fpg , and tg might be best related to ir and therefore , are the first clinical characteristics to be detected as abnormal . found that among all the mets clinical characteristics , bmi and tg were best correlated with sspg ( 14 ) . by using factor analysis to evaluate the relationships between mets clinical characteristics and ir , meigs et al thus , he further suggested that ir should be the central focus in assessing the mets ( 8) . the detrimental influence of central adiposity on ir is thought to be mediated by intra - abdominal fat deposition , in which hypertrophied adipocytes are resistant to anti - lipolytic action of insulin . as a consequence , elevated levels of free fatty acids may induce ir in the peripheral tissues and liver ( 18 ) . also , the preponderance of enlarged fat cells , as occur in abdominal obesity , may increase the risk of glucose intolerance and hypertriglyceridemia and thus account for the metabolic derangements ( 19 , 20 ) . it is interesting to note that the relationships between ir and some of the mets clinical characteristics were affected by gender . in our study , for example , the upper quartile of dbp in males had significantly higher sspg levels than the lower three quartiles . in contrast , instead of dbp , the sspg was higher in the upper quartile of sbp in females . these results suggested that the relationships between blood pressure and sspg were less consistent in our study . it is difficult to meaningfully compare this finding with other reports , since most of the other reports adjusted both sex and age when discussing the relationships between blood pressure and ir ( 17 , 21 , 22 ) . other than blood pressure , the relationships between ir and hdlc were also different between the genders . 3 , when comparing the upper quartiles of all the mets clinical characteristics , the upper quartile of the hdlc clinical characteristic had the lowest sspg level , but was only significant in males . it is well - recognized that ir is related to high tg and low hdlc ( 23 , 24 ) ; however , most of these reports showed that gender had no effect on the relationships involving ir . found that ir was more strongly related to hdlc in males than females , as evidenced by the intravenous glucose tolerance test ( 24 ) . this unique finding was further confirmed in another study conducted in subjects with morbid obesity . the correlations between ir and tg or hdlc were stronger in males than in females ( 25 ) . both hdlc and sbp were shown to be related to sspg levels in males , our data further imply that males might be more prone to develop mets when in middle age , as suggested by cook et al . at present , there are two possibilities to explain the metabolic differences which existed between males and females in our study . this in turn will further increase the risk of cardiovascular disease and diabetes ( 28 , 29 ) . in the current study , since we observed differences between each mets clinical characteristic , we did not adjust the effect of bmi on other risk factors , which is a departure from other studies ( 23 - 25 ) . it is generally agreed that there is a positive correlation between blood pressure and ir . reported that male gender , age , and ir were independently associated with sbp , whereas dbp was related to age , ir , and fpi , but not to the bmi ( 17 ) . they also found that dbp had a higher correlation with ir than sbp ( r=0.34 and 0.18 , respectively ) . however , the same conclusion was not always reached in studies involving other ethnic groups ( 30 ) and the relationships did not exist when adjusted for body fat content ( 31 ) . the discrepancies of these results may be due to the fact that blood pressure per se is not clustered with ir . other possibilities such as the different ethnic groups or the methods used to measure adiposity might also play a role . in the ferrannini study , and as done herein , bmi was used to quantify adiposity . used whr and suggested that in subjects with the same bmi , changes in whr may still influence the results of variables associated with insulin sensitivity because the accumulation of visceral fat leads to altered insulin clearance ( 31 ) . in the peiris study , the clearance rates of insulin were similar between obese and non - obese subjects ( 32 ) ; however , it was inversely correlated with the whr in obese subjects . pointed out that whr is better associated with diminished insulin clearance than bmi ( 32 ) . it has been shown that the homeostasis model assessment of ir ( homa - ir ; 33 , 34 ) , which is calculated from fpg and fpi , is a useful surrogate for ir in healthy and diabetic subjects . it has proven to be highly correlated with the gold standard , the euglycemic hyperinsulinemic clamp ( 33 ) . at present , it is generally recognized that one of the main underlying pathophysiologies of glucose intolerance is ir . however , in subjects who already have ir , due to the compensatory increased secretion of the insulin , the fpg may persist within the normal range . that is why the study done by hollenbeck and reaven showed that there was no relationship found between fpg and ir ( 35 ) . on the contrary , surprisingly , in our study , the sspg seemed to be higher as the fpg increased . this finding suggested that even in the early stages before mets develops , fpg is already elevated in subjects with an elevated ir . the results of the factor analysis are similar to most of the other studies . in these studies , usually there are three to four factors being identified ; the " insulin resistant factor " , " obese factor " , " lipid factor " , and/or " blood pressure factor " ( 8 , 9 , 13 , 36 - 39 ) . to the present , one of the most important studies using factor analysis to explore the relationship between ir and other common risk factors for cardiovascular diseases was published by hanley et al . they have also identified two factors in the igt and ngt subjects : the " metabolic factor " ( comprising bmi , waist , si , tg , and hdlc ) ; and the " blood pressure factor " . the only differences are fpg was not loaded in factor 1 and bmi was not loaded in factor 2 . it could be noted that the clustering patterns in our study were not only similar to hanley 's study ( 40 ) but also to the most of other studies with different severities of impaired glucose metabolism and ethnic groups ( 13 , 36 , 38 , 39 ) . the clustering of the sspg , bmi , fpg and tg could further confirm that the ir is more correlated with lipids profile and bmi , but less with the blood pressure . however , the purpose of this study was to observe the relationships between ir and mets , thus , this drawback should not affect the conclusions drawn from the present study . our data suggested that adiposity , higher fpg , and hypertriglyceridemia are more strongly associated with ir in chinese subjects and the importance of these clinical characteristics should be reemphasized . when considering each of the mets clinical characteristics , subjects with a higher bmi may have the highest ir .

although injuries to the flexor tendons in the zone ii region look trivial , sustained commitment of the patient , the surgeon and the therapist is necessary to get a reasonable functional outcome . as our institute is situated in an industrial corridor of the city , most of our patients are manual workers with poor compliance . conforming to the established practice , we tried various early mobilization protocols after zone ii flexor tendon repair . as these protocols demanded a level of understanding and a degree of dedication from the patients , our results were suboptimal , with a high incidence of proximal interphalangeal joint ( pip ) joint flexion contractures and tendon ruptures . hence , in our institute immobilization became the norm for patients who were not expected to be compliant . to improve the results in such patients , we thought of implementing a new rehabilitation protocol that could entirely be under the control of the therapist , without the active participation of the patient . ultrasound therapy is being used safely after tendon repair , during the remodeling phase ( after three weeks ) as an adjunct to mobilization to improve the tendon gliding . we thought of using this mode of treatment during the earlier phase of tendon healing . such findings formed the basis , for the use of ultrasound to accelerate tendon healing and to prevent adhesions . though many clinical trials on animals are reported in the literature , there is no clinical study in the humans regarding the use of ultrasound during the early healing phase of the repaired tendons . encouraged by the in vitro and in vivo animal studies , we conducted the trial by selecting the patients at random and administered pulsed ultrasound therapy of different frequencies and intensities . the results of the ultrasound therapy in the three groups were analyzed and compared with each other and also with the immobilization group . this is a prospective study , done over a period of 5 years from january 2008 to january 2013 , involving a total of 100 patients and 139 digits with zone ii flexor tendon injuries . patients with isolated injury to the fds or fdp tendon , multiple level injuries of the flexor tendons , associated injury to the extensor apparatus and fractures were excluded . all the cases were operated as emergency procedures by senior residents and hence , for tendon repair technically less demanding two - strand modified kessler mason suture was used . after surgery , patients were allocated to ultrasound therapy , as demonstrated in the consort guideline flowchart [ figure 1 ] . the patients were asked to draw a card indiscriminately from an envelope containing a pack of cards labeled as ultrasound or immobilization in 2:1 ratio . thus , the treatment groups were randomized and the therapists had no control over the selection of the patients . after surgery , hands were immobilized for 3 weeks using dorsal slab with wrist kept in neutral position , mp joints in 70 degrees flexion and ip joints in extension . after 3 weeks , pop slab was removed and the patients were advised to attend therapy daily . from 3 to 6 weeks the therapy consisted of scar massage , active mobilization exercises , blocking exercises and place hold techniques . after 6 weeks , passive stretching and resisted exercises were added . after 8 weeks , the patients were allowed to lift weights and to join work . by this protocol after surgery hands were immobilized using dorsal slab with wrist in neutral , mp joints in 70 degrees flexion and ip joints in extension . with the dorsal splint in place , the dressings on the volar aspect were removed . the ultrasonic treatment head was placed over the site of tendon repair and gently moved in order to iron out the irregularities in the near field and to avoid standing waves due to reflection . the ultrasonic head was applied gently and moved continuously over the region of tendon repair to begin with , since january 2008 , ultrasound of 1-mhz frequency at an intensity of 0.7 w / cm was administered from the seventh postoperative day . twenty - six patients with involvement of a total of thirty eight digits were treated . twelve patients had associated digital nerve involvement . with the idea of further refining the technique , since august 2010 , we started ultrasound therapy of 1-mhz frequency at an intensity of 0.3 w / cm from the third postoperative day . we reduced the intensity due to fear of impaired skin healing as we had started the therapy earlier than the first group . as there was significant reduction in the percentage of excellent results in group 2 , from october 2011 , ultrasound therapy of 3 mhz frequency at an intensity of 0.5 w / cm was administered from the fifth postoperative day . due to cases of wound dehiscence this protocol was started for 27 patients with involvement of a total of 33 fingers . three patients developed wound dehiscence within 2 days of starting ultrasound therapy and were dropped from the study . out of 24 patients , 21 were males and 3 were females . the splint was removed after 3 weeks and mobilization programe was commenced in addition to the ultrasound therapy . active ranges of movements at pip and dip joints were measured after 3 weeks and at weekly intervals . after surgery , hands were immobilized for 3 weeks using dorsal slab with wrist kept in neutral position , mp joints in 70 degrees flexion and ip joints in extension . after 3 weeks , pop slab was removed and the patients were advised to attend therapy daily . from 3 to 6 weeks the therapy consisted of scar massage , active mobilization exercises , blocking exercises and place hold techniques . after 6 weeks , passive stretching and resisted exercises were added . after 8 weeks , the patients were allowed to lift weights and to join work . by this protocol after surgery hands were immobilized using dorsal slab with wrist in neutral , mp joints in 70 degrees flexion and ip joints in extension . with the dorsal splint in place , the dressings on the volar aspect were removed . the ultrasonic treatment head was placed over the site of tendon repair and gently moved in order to iron out the irregularities in the near field and to avoid standing waves due to reflection . the ultrasonic head was applied gently and moved continuously over the region of tendon repair to begin with , since january 2008 , ultrasound of 1-mhz frequency at an intensity of 0.7 w / cm was administered from the seventh postoperative day . twenty - six patients with involvement of a total of thirty eight digits were treated . twelve patients had associated digital nerve involvement . with the idea of further refining the technique , since august 2010 , we started ultrasound therapy of 1-mhz frequency at an intensity of 0.3 w / cm from the third postoperative day . we reduced the intensity due to fear of impaired skin healing as we had started the therapy earlier than the first group . one patient developed wound dehiscence and was dropped from the study . out of 18 patients , 15 were males and 3 were females . six patients had digital nerve involvement . as there was significant reduction in the percentage of excellent results in group 2 , from october 2011 , ultrasound therapy of 3 mhz frequency at an intensity of 0.5 w / cm this protocol was started for 27 patients with involvement of a total of 33 fingers . three patients developed wound dehiscence within 2 days of starting ultrasound therapy and were dropped from the study . out of 24 patients , 21 were males and 3 were females . in all the ultrasound therapy groups , the splint was removed after 3 weeks and mobilization programe was commenced in addition to the ultrasound therapy . to begin with , since january 2008 , ultrasound of 1-mhz frequency at an intensity of 0.7 w / cm was administered from the seventh postoperative day . twenty - six patients with involvement of a total of thirty eight digits were treated . with the idea of further refining the technique , since august 2010 , we started ultrasound therapy of 1-mhz frequency at an intensity of 0.3 w / cm from the third postoperative day . we reduced the intensity due to fear of impaired skin healing as we had started the therapy earlier than the first group . one patient developed wound dehiscence and was dropped from the study . out of 18 patients , 15 were males and 3 were females . as there was significant reduction in the percentage of excellent results in group 2 , from october 2011 , ultrasound therapy of 3 mhz frequency at an intensity of 0.5 w / cm was administered from the fifth postoperative day . due to cases of wound dehiscence this protocol was started for 27 patients with involvement of a total of 33 fingers . three patients developed wound dehiscence within 2 days of starting ultrasound therapy and were dropped from the study . out of 24 patients , 21 were males and 3 were females . the splint was removed after 3 weeks and mobilization programe was commenced in addition to the ultrasound therapy . active ranges of movements at pip and dip joints were measured after 3 weeks and at weekly intervals . the patients who were dropped from the study due to wound complications in the early phase were not included in the analysis of the results . comparison of results between various protocols by the end of 3 months is given in tables 1 and 2 . comparison of results between various protocols at 3 months ( original strickland ) comparison of excellent and good results in various protocols ( original strickland ) in ultrasound therapy group 1 protocol , out of 36 digits , 16 digits achieved pip + dip joint flexion range between 150 and 175 and 10 digits between 125 and 150 in 8 weeks time . the range in the remaining digits was between 90 and 120. extensor lag was nil in 34 digits by the end of 6 weeks . by the end of 8 weeks , none of the digits had extensor lag . grip strength of 95% compared to normal hand was achieved by 16 digits and 10 digits achieved grip strength of 80% compared to normal hand by the end of 12 weeks . in ultrasound therapy group 2 protocol , out of 27 digits , 13 digits achieved pip + dip joint flexion range between 150 and 175 by 8 weeks and 8 digits were in the range between 125 and 150 by 12 weeks . in remaining 6 digits the range was less than 90. extensor lag was nil in 18 digits and in 4 digits the extensor lag was between 10 and 40 by the end of 6 weeks . by 12 weeks , 3 digits presented with residual extensor lag between 10 and 30. grip strength was 90 % compared to normal hand for 21 digits in 12 weeks . in ultrasound therapy group 3 protocol , out of 30 digits , 23 digits achieved pip + dip joint flexion in the range of 125 -140 and 5 digits below 90 by the end of 8 weeks . in 2 digits tendons the degrees of extensor lag were between 10 and 25. extensor lag was nil in all digits except 1 by the end of 8 weeks , which was in the range of 15 -25. grip strength of 90% compared to normal hand was achieved by 23 digits in 12 weeks . in immobilization group , out of 40 digits , 10 digits achieved a pip + dip joint flexion in the range of 125 -150. and in 30 digits it was below 90 by the end of 12 weeks . in 6 digits , extensor lag was nil by the end of 6 weeks . remaining digits had extensor lag between 10 and 60. eighteen patients were left with residual pip joint flexion contracture in the range of 25 -45 by the end of 12 weeks . grip strength of 90% was achieved by 10 digits by the end of 12 weeks . as there are more than three groups of data to be compared and the data sets represent a continuous distribution , the one way anova test using the f distribution is chosen for the statistical analysis . the anova test was performed using the statistical tools available in origin 8.5.1 scientific data analysis software . this test compares the mean values of the distribution assuming equal variance for all the data [ table 3a , b ] . the result of this analysis shows that there is statistically significant improvement of score ( using p < 0.05 criteria ) in ultrasound protocols compared to immobilization . however , when only the three ultrasound methods are compared , the p value is 0.066 , which is slightly larger than the 0.05 criteria . hence , there is a 6.6% probability that the difference in the mean value between the ultrasound group 1 method and the other two methods may be due to chance factor . since the p value is only slightly larger than the 0.05 level , further investigations with a larger data set and more variation in parameters such as ultrasound intensity , duration of exposure , etc . may reveal their beneficial effects on the improvement of outcome . one - way mean anova ( f test ) one - way mean anova ( f test ) the table 3a reveals that the best mean score of 78.4 16.6 was achieved in ultrasound group 1 protocol . the mean scores of group 2 and group 3 protocols are 70.8 14.38 and 70.813.39 , respectively , which are more or less identical . a significant finding in the ultrasound ( group 1 ) treated patients was that , extensor lag was minimal in all cases , and only 2 patients required passive stretching after six weeks . the percentage of patients left with residual pip joint flexion contracture in all the ultrasound groups , is much less compared to the immobilization group [ table 4 ] . incidence of pip joint flexion contracture the scars became soft and supple in the ultrasound - treated patients much earlier than patients treated with the immobilization protocol [ figure 3 ] . the scars in the fingers and palm became soft and supple as early as 3 weeks . wound dehiscence was observed in 3 patients in ultrasound ( group 3 ) protocol and for 1 patient in ultrasound ( group 2 ) protocol . wound dehiscence was observed within 2 - 3 days of ultrasound therapy , and the therapy was discontinued immediately . tendon rupture was observed in 2 patients in ultrasound therapy ( group 3 ) protocol after 3 weeks . after this experience , in cases showing good results at 3 weeks itself , we did not increase the intensity further and adjusted the intensity according to the patient 's response . as there are more than three groups of data to be compared and the data sets represent a continuous distribution , the one way anova test using the f distribution is chosen for the statistical analysis . the anova test was performed using the statistical tools available in origin 8.5.1 scientific data analysis software . this test compares the mean values of the distribution assuming equal variance for all the data [ table 3a , b ] . the result of this analysis shows that there is statistically significant improvement of score ( using p < 0.05 criteria ) in ultrasound protocols compared to immobilization . however , when only the three ultrasound methods are compared , the p value is 0.066 , which is slightly larger than the 0.05 criteria . hence , there is a 6.6% probability that the difference in the mean value between the ultrasound group 1 method and the other two methods may be due to chance factor . since the p value is only slightly larger than the 0.05 level , further investigations with a larger data set and more variation in parameters such as ultrasound intensity , duration of exposure , etc . one - way mean anova ( f test ) one - way mean anova ( f test ) the table 3a reveals that the best mean score of 78.4 16.6 was achieved in ultrasound group 1 protocol . the mean scores of group 2 and group 3 protocols are 70.8 14.38 and 70.813.39 , respectively , which are more or less identical . a significant finding in the ultrasound ( group 1 ) treated patients was that , extensor lag was minimal in all cases , and only 2 patients required passive stretching after six weeks . the percentage of patients left with residual pip joint flexion contracture in all the ultrasound groups , is much less compared to the immobilization group [ table 4 ] . incidence of pip joint flexion contracture the scars became soft and supple in the ultrasound - treated patients much earlier than patients treated with the immobilization protocol [ figure 3 ] . the scars in the fingers and palm became soft and supple as early as 3 weeks . wound dehiscence was observed in 3 patients in ultrasound ( group 3 ) protocol and for 1 patient in ultrasound ( group 2 ) protocol . wound dehiscence was observed within 2 - 3 days of ultrasound therapy , and the therapy was discontinued immediately . tendon rupture was observed in 2 patients in ultrasound therapy ( group 3 ) protocol after 3 weeks . after this experience , in cases showing good results at 3 weeks itself , we did not increase the intensity further and adjusted the intensity according to the patient 's response . gan , huys et al . studied the effects of ultrasound on repaired flexor tendons of the chicken limbs . they found that administration of ultrasound resulted in increased range of movement , advancement of scar maturation and reduction of the amount of inflammatory infiltrate . there was no adverse effect on tensile strength and that early administration was more beneficial than late . though high - intensity ultrasound has tissue destructive effects , low intensities may enhance the healing process of the surgically repaired tendons . its effects on decreasing peritendinous adhesions , and enhancement of collagen fibrils maturation independent of the intensity applied can explain the improved results obtained in this study . the factors that determine the ultrasound energy delivered at the tissue level are the frequency , the pulse ratio , the intensity of the ultrasound and the duration of therapy . appropriate titration of these factors as done in this study will ensure the safety of this modality of treatment . the results of ultrasound therapy ( group 1 ) are better compared to other ultrasound therapy groups . starting of the ultrasound therapy from the seventh day is safer , as the tensile strength of the wound increases from the seventh post operative day and the repaired skin wound will be strong enough to withstand the effects of ultrasound . in our study , wound dehiscence was noticed in the other two groups , when ultrasound was started on the third or the fifth day . it has been reported by gelberman and his associates that peak fibronectin concentration and development of adhesions start by the seventh day . administration of ultrasound at this time would be ideal and safe to break down early adhesions before they get organized . relatively reduced percentage of excellent results was observed in the ultrasound therapy ( group 2 ) protocol . this can be explained by the fact that , lesser the frequency and intensity the lesser will be the absorption of ultrasound energy by the tissues . as the intensity was reduced to 0.3 w / cm , the effective energy delivered to the repaired flexor tendons might be less than optimal . the results of 3-mhz ultrasound ( group 3 ) are worse compared to 1-mhz ( group 1 and group 2 ) with nil percentage of excellent results and 7% of ruptures . when the frequency of ultrasound is high , there will be rapid absorption of ultrasound energy in the superficial tissues . the absorption coefficient of tendon is 1.12 decibels / cm for 1 mhz and 3.36 decibels / cm for 3 mhz . the repaired flexor tendon , being superficial and highly collagenous , might have been unable to withstand high energy absorption and concentration during the early phase of healing . nil excellent results in the ultrasound therapy ( group 3 ) protocol might be due to gap formation . tim watson ( www.electrotherapy.org ) suggested ideal frequencies and intensities of ultrasound for various anatomical regions and pathological conditions . but the safe dose of ultrasound therapy for recent surgical wounds has not been specified in the literature . ( 2003 ) demonstrated that ultrasound machines , delivering apparently the same treatment energy , give rise to different amounts of tissue heating and therefore the effects of ultrasound at the tissue level may not be accurately predictable . in this context , we like to stress that the optimal frequency and intensity of pulsed ultrasound can be determined only by clinical experience . but once the optimal parameters are defined , the intensity and duration can be well controlled by the therapist . as the patient 's active participation is less , the desired effect on the tendon healing can be achieved with minimal chance of rupture . tang after a 15 years review of reports of clinical outcomes associated with flexor tendon repair noted that the best functional results of 75% excellent to good results were reported after early active mobilization . he concluded that adhesion formation and stiffness of fingers remain frustrating problems and suggested modern biologic approaches as one of the means of improving the outcome . we suggest that early ultrasonic therapy can be one such approach as our results of 75% excellent to good results with rupture rate of 2% is comparable to mobilization protocols . . our study shows , starting ultrasound therapy with 1-mhz frequency on the seventh post operative day with intensity of 0.7 w / cm give high percentage of excellent - good results with no ruptures and pip joint flexion contractures . if 3-mhz frequency is used , the initial intensity must be set at a lower level , and adjusted according to the patient 's response after 3 weeks.pulsed ultrasound therapy can be used in patients who are not compliant for early mobilization protocols.the results of early ultrasound therapy in zone ii ftr are comparable with the results of established early mobilization protocols.as the ultrasound therapy is not interfering with mobilization protocols , it is possible to use it as an adjuvant to further improve the outcome.the ideal frequency and intensity can not be rigidly defined but can safely be arrived at by clinical experience . our study shows , starting ultrasound therapy with 1-mhz frequency on the seventh post operative day with intensity of 0.7 w / cm give high percentage of excellent - good results with no ruptures and pip joint flexion contractures . if 3-mhz frequency is used , the initial intensity must be set at a lower level , and adjusted according to the patient 's response after 3 weeks . pulsed ultrasound therapy can be used in patients who are not compliant for early mobilization protocols . the results of early ultrasound therapy in zone ii ftr are comparable with the results of established early mobilization protocols . as the ultrasound therapy is not interfering with mobilization protocols , it is possible to use it as an adjuvant to further improve the outcome . the ideal frequency and intensity can not be rigidly defined but can safely be arrived at by clinical experience .

background : in our institute , most of the patients treated for hand injuries were industrial workers with poor compliance . for rehabilitation after zone ii flexor tendon repair , we had tried various early mobilization protocols . as these protocols demanded a degree of commitment from the patients , our results were suboptimal . hence , to improve the results , we implemented a new rehabilitation protocol by administering the pulsed ultrasound therapy during the early phase of tendon healing.materials and methods : this is a prospective study done over a period of five years from january 2008 to january 2013 . a total of 100 patients and 139 digits with zone ii flexor tendon injuries were studied . after randomization , we administered pulsed ultrasound therapy of different frequencies and intensities for a total of 72 patients and 99 digits and formulated three groups . the results of ultrasound treated cases were compared with each other and with the results of cases treated by immobilization protocol . the results were analyzed using original strickland criteria.results:72% excellent - good results in ultrasound ( group 1 ) protocol , 75% excellent - good results in ultrasound ( group 2 ) protocol , and 77% excellent - good results in ultrasound ( group 3 ) protocol were achieved . there was no case of rupture in the first two groups . the rupture rate was 7% in ultrasound ( group 3 ) protocol . only 25% excellent - good results were obtained in the immobilization protocol.conclusion:after zone ii flexor tendon repair , pulsed ultrasound therapy during the early rehabilitation phase is safe and effective . the results are comparable to early mobilization protocols .

INTRODUCTION MATERIALS AND METHODS Immobilization protocol Ultrasound protocol Group 1 Group 2 Group 3 Assessments RESULTS Statistical analysis DISCUSSION CONCLUSION

although injuries to the flexor tendons in the zone ii region look trivial , sustained commitment of the patient , the surgeon and the therapist is necessary to get a reasonable functional outcome . as our institute is situated in an industrial corridor of the city , most of our patients are manual workers with poor compliance . conforming to the established practice , we tried various early mobilization protocols after zone ii flexor tendon repair . as these protocols demanded a level of understanding and a degree of dedication from the patients , our results were suboptimal , with a high incidence of proximal interphalangeal joint ( pip ) joint flexion contractures and tendon ruptures . hence , in our institute immobilization became the norm for patients who were not expected to be compliant . to improve the results in such patients , we thought of implementing a new rehabilitation protocol that could entirely be under the control of the therapist , without the active participation of the patient . ultrasound therapy is being used safely after tendon repair , during the remodeling phase ( after three weeks ) as an adjunct to mobilization to improve the tendon gliding . we thought of using this mode of treatment during the earlier phase of tendon healing . such findings formed the basis , for the use of ultrasound to accelerate tendon healing and to prevent adhesions . though many clinical trials on animals are reported in the literature , there is no clinical study in the humans regarding the use of ultrasound during the early healing phase of the repaired tendons . encouraged by the in vitro and in vivo animal studies , we conducted the trial by selecting the patients at random and administered pulsed ultrasound therapy of different frequencies and intensities . the results of the ultrasound therapy in the three groups were analyzed and compared with each other and also with the immobilization group . this is a prospective study , done over a period of 5 years from january 2008 to january 2013 , involving a total of 100 patients and 139 digits with zone ii flexor tendon injuries . patients with isolated injury to the fds or fdp tendon , multiple level injuries of the flexor tendons , associated injury to the extensor apparatus and fractures were excluded . all the cases were operated as emergency procedures by senior residents and hence , for tendon repair technically less demanding two - strand modified kessler mason suture was used . after surgery , patients were allocated to ultrasound therapy , as demonstrated in the consort guideline flowchart [ figure 1 ] . the patients were asked to draw a card indiscriminately from an envelope containing a pack of cards labeled as ultrasound or immobilization in 2:1 ratio . thus , the treatment groups were randomized and the therapists had no control over the selection of the patients . after surgery , hands were immobilized for 3 weeks using dorsal slab with wrist kept in neutral position , mp joints in 70 degrees flexion and ip joints in extension . after 3 weeks , pop slab was removed and the patients were advised to attend therapy daily . after 8 weeks , the patients were allowed to lift weights and to join work . with the dorsal splint in place , the dressings on the volar aspect were removed . the ultrasonic treatment head was placed over the site of tendon repair and gently moved in order to iron out the irregularities in the near field and to avoid standing waves due to reflection . the ultrasonic head was applied gently and moved continuously over the region of tendon repair to begin with , since january 2008 , ultrasound of 1-mhz frequency at an intensity of 0.7 w / cm was administered from the seventh postoperative day . twenty - six patients with involvement of a total of thirty eight digits were treated . twelve patients had associated digital nerve involvement . with the idea of further refining the technique , since august 2010 , we started ultrasound therapy of 1-mhz frequency at an intensity of 0.3 w / cm from the third postoperative day . we reduced the intensity due to fear of impaired skin healing as we had started the therapy earlier than the first group . as there was significant reduction in the percentage of excellent results in group 2 , from october 2011 , ultrasound therapy of 3 mhz frequency at an intensity of 0.5 w / cm was administered from the fifth postoperative day . due to cases of wound dehiscence this protocol was started for 27 patients with involvement of a total of 33 fingers . three patients developed wound dehiscence within 2 days of starting ultrasound therapy and were dropped from the study . out of 24 patients , 21 were males and 3 were females . the splint was removed after 3 weeks and mobilization programe was commenced in addition to the ultrasound therapy . after surgery , hands were immobilized for 3 weeks using dorsal slab with wrist kept in neutral position , mp joints in 70 degrees flexion and ip joints in extension . after 3 weeks , pop slab was removed and the patients were advised to attend therapy daily . after 8 weeks , the patients were allowed to lift weights and to join work . with the dorsal splint in place , the dressings on the volar aspect were removed . the ultrasonic treatment head was placed over the site of tendon repair and gently moved in order to iron out the irregularities in the near field and to avoid standing waves due to reflection . the ultrasonic head was applied gently and moved continuously over the region of tendon repair to begin with , since january 2008 , ultrasound of 1-mhz frequency at an intensity of 0.7 w / cm was administered from the seventh postoperative day . twenty - six patients with involvement of a total of thirty eight digits were treated . twelve patients had associated digital nerve involvement . with the idea of further refining the technique , since august 2010 , we started ultrasound therapy of 1-mhz frequency at an intensity of 0.3 w / cm from the third postoperative day . we reduced the intensity due to fear of impaired skin healing as we had started the therapy earlier than the first group . one patient developed wound dehiscence and was dropped from the study . out of 18 patients , 15 were males and 3 were females . six patients had digital nerve involvement . as there was significant reduction in the percentage of excellent results in group 2 , from october 2011 , ultrasound therapy of 3 mhz frequency at an intensity of 0.5 w / cm this protocol was started for 27 patients with involvement of a total of 33 fingers . three patients developed wound dehiscence within 2 days of starting ultrasound therapy and were dropped from the study . out of 24 patients , 21 were males and 3 were females . in all the ultrasound therapy groups , the splint was removed after 3 weeks and mobilization programe was commenced in addition to the ultrasound therapy . to begin with , since january 2008 , ultrasound of 1-mhz frequency at an intensity of 0.7 w / cm was administered from the seventh postoperative day . twenty - six patients with involvement of a total of thirty eight digits were treated . with the idea of further refining the technique , since august 2010 , we started ultrasound therapy of 1-mhz frequency at an intensity of 0.3 w / cm from the third postoperative day . we reduced the intensity due to fear of impaired skin healing as we had started the therapy earlier than the first group . one patient developed wound dehiscence and was dropped from the study . out of 18 patients , 15 were males and 3 were females . as there was significant reduction in the percentage of excellent results in group 2 , from october 2011 , ultrasound therapy of 3 mhz frequency at an intensity of 0.5 w / cm was administered from the fifth postoperative day . due to cases of wound dehiscence this protocol was started for 27 patients with involvement of a total of 33 fingers . three patients developed wound dehiscence within 2 days of starting ultrasound therapy and were dropped from the study . out of 24 patients , 21 were males and 3 were females . the splint was removed after 3 weeks and mobilization programe was commenced in addition to the ultrasound therapy . the patients who were dropped from the study due to wound complications in the early phase were not included in the analysis of the results . comparison of results between various protocols by the end of 3 months is given in tables 1 and 2 . comparison of results between various protocols at 3 months ( original strickland ) comparison of excellent and good results in various protocols ( original strickland ) in ultrasound therapy group 1 protocol , out of 36 digits , 16 digits achieved pip + dip joint flexion range between 150 and 175 and 10 digits between 125 and 150 in 8 weeks time . the range in the remaining digits was between 90 and 120. extensor lag was nil in 34 digits by the end of 6 weeks . by the end of 8 weeks , none of the digits had extensor lag . grip strength of 95% compared to normal hand was achieved by 16 digits and 10 digits achieved grip strength of 80% compared to normal hand by the end of 12 weeks . in ultrasound therapy group 2 protocol , out of 27 digits , 13 digits achieved pip + dip joint flexion range between 150 and 175 by 8 weeks and 8 digits were in the range between 125 and 150 by 12 weeks . in remaining 6 digits the range was less than 90. extensor lag was nil in 18 digits and in 4 digits the extensor lag was between 10 and 40 by the end of 6 weeks . in ultrasound therapy group 3 protocol , out of 30 digits , 23 digits achieved pip + dip joint flexion in the range of 125 -140 and 5 digits below 90 by the end of 8 weeks . in 2 digits tendons the degrees of extensor lag were between 10 and 25. extensor lag was nil in all digits except 1 by the end of 8 weeks , which was in the range of 15 -25. grip strength of 90% compared to normal hand was achieved by 23 digits in 12 weeks . in immobilization group , out of 40 digits , 10 digits achieved a pip + dip joint flexion in the range of 125 -150. and in 30 digits it was below 90 by the end of 12 weeks . in 6 digits , extensor lag was nil by the end of 6 weeks . remaining digits had extensor lag between 10 and 60. eighteen patients were left with residual pip joint flexion contracture in the range of 25 -45 by the end of 12 weeks . grip strength of 90% was achieved by 10 digits by the end of 12 weeks . as there are more than three groups of data to be compared and the data sets represent a continuous distribution , the one way anova test using the f distribution is chosen for the statistical analysis . this test compares the mean values of the distribution assuming equal variance for all the data [ table 3a , b ] . the result of this analysis shows that there is statistically significant improvement of score ( using p < 0.05 criteria ) in ultrasound protocols compared to immobilization . hence , there is a 6.6% probability that the difference in the mean value between the ultrasound group 1 method and the other two methods may be due to chance factor . since the p value is only slightly larger than the 0.05 level , further investigations with a larger data set and more variation in parameters such as ultrasound intensity , duration of exposure , etc . one - way mean anova ( f test ) one - way mean anova ( f test ) the table 3a reveals that the best mean score of 78.4 16.6 was achieved in ultrasound group 1 protocol . the mean scores of group 2 and group 3 protocols are 70.8 14.38 and 70.813.39 , respectively , which are more or less identical . a significant finding in the ultrasound ( group 1 ) treated patients was that , extensor lag was minimal in all cases , and only 2 patients required passive stretching after six weeks . the percentage of patients left with residual pip joint flexion contracture in all the ultrasound groups , is much less compared to the immobilization group [ table 4 ] . incidence of pip joint flexion contracture the scars became soft and supple in the ultrasound - treated patients much earlier than patients treated with the immobilization protocol [ figure 3 ] . the scars in the fingers and palm became soft and supple as early as 3 weeks . wound dehiscence was observed in 3 patients in ultrasound ( group 3 ) protocol and for 1 patient in ultrasound ( group 2 ) protocol . wound dehiscence was observed within 2 - 3 days of ultrasound therapy , and the therapy was discontinued immediately . tendon rupture was observed in 2 patients in ultrasound therapy ( group 3 ) protocol after 3 weeks . after this experience , in cases showing good results at 3 weeks itself , we did not increase the intensity further and adjusted the intensity according to the patient 's response . as there are more than three groups of data to be compared and the data sets represent a continuous distribution , the one way anova test using the f distribution is chosen for the statistical analysis . this test compares the mean values of the distribution assuming equal variance for all the data [ table 3a , b ] . the result of this analysis shows that there is statistically significant improvement of score ( using p < 0.05 criteria ) in ultrasound protocols compared to immobilization . hence , there is a 6.6% probability that the difference in the mean value between the ultrasound group 1 method and the other two methods may be due to chance factor . one - way mean anova ( f test ) one - way mean anova ( f test ) the table 3a reveals that the best mean score of 78.4 16.6 was achieved in ultrasound group 1 protocol . the mean scores of group 2 and group 3 protocols are 70.8 14.38 and 70.813.39 , respectively , which are more or less identical . a significant finding in the ultrasound ( group 1 ) treated patients was that , extensor lag was minimal in all cases , and only 2 patients required passive stretching after six weeks . the percentage of patients left with residual pip joint flexion contracture in all the ultrasound groups , is much less compared to the immobilization group [ table 4 ] . incidence of pip joint flexion contracture the scars became soft and supple in the ultrasound - treated patients much earlier than patients treated with the immobilization protocol [ figure 3 ] . the scars in the fingers and palm became soft and supple as early as 3 weeks . wound dehiscence was observed in 3 patients in ultrasound ( group 3 ) protocol and for 1 patient in ultrasound ( group 2 ) protocol . wound dehiscence was observed within 2 - 3 days of ultrasound therapy , and the therapy was discontinued immediately . tendon rupture was observed in 2 patients in ultrasound therapy ( group 3 ) protocol after 3 weeks . after this experience , in cases showing good results at 3 weeks itself , we did not increase the intensity further and adjusted the intensity according to the patient 's response . studied the effects of ultrasound on repaired flexor tendons of the chicken limbs . they found that administration of ultrasound resulted in increased range of movement , advancement of scar maturation and reduction of the amount of inflammatory infiltrate . there was no adverse effect on tensile strength and that early administration was more beneficial than late . though high - intensity ultrasound has tissue destructive effects , low intensities may enhance the healing process of the surgically repaired tendons . its effects on decreasing peritendinous adhesions , and enhancement of collagen fibrils maturation independent of the intensity applied can explain the improved results obtained in this study . the factors that determine the ultrasound energy delivered at the tissue level are the frequency , the pulse ratio , the intensity of the ultrasound and the duration of therapy . the results of ultrasound therapy ( group 1 ) are better compared to other ultrasound therapy groups . starting of the ultrasound therapy from the seventh day is safer , as the tensile strength of the wound increases from the seventh post operative day and the repaired skin wound will be strong enough to withstand the effects of ultrasound . in our study , wound dehiscence was noticed in the other two groups , when ultrasound was started on the third or the fifth day . administration of ultrasound at this time would be ideal and safe to break down early adhesions before they get organized . relatively reduced percentage of excellent results was observed in the ultrasound therapy ( group 2 ) protocol . this can be explained by the fact that , lesser the frequency and intensity the lesser will be the absorption of ultrasound energy by the tissues . the results of 3-mhz ultrasound ( group 3 ) are worse compared to 1-mhz ( group 1 and group 2 ) with nil percentage of excellent results and 7% of ruptures . when the frequency of ultrasound is high , there will be rapid absorption of ultrasound energy in the superficial tissues . the absorption coefficient of tendon is 1.12 decibels / cm for 1 mhz and 3.36 decibels / cm for 3 mhz . the repaired flexor tendon , being superficial and highly collagenous , might have been unable to withstand high energy absorption and concentration during the early phase of healing . nil excellent results in the ultrasound therapy ( group 3 ) protocol might be due to gap formation . tim watson ( www.electrotherapy.org ) suggested ideal frequencies and intensities of ultrasound for various anatomical regions and pathological conditions . but the safe dose of ultrasound therapy for recent surgical wounds has not been specified in the literature . ( 2003 ) demonstrated that ultrasound machines , delivering apparently the same treatment energy , give rise to different amounts of tissue heating and therefore the effects of ultrasound at the tissue level may not be accurately predictable . in this context , we like to stress that the optimal frequency and intensity of pulsed ultrasound can be determined only by clinical experience . but once the optimal parameters are defined , the intensity and duration can be well controlled by the therapist . as the patient 's active participation is less , the desired effect on the tendon healing can be achieved with minimal chance of rupture . tang after a 15 years review of reports of clinical outcomes associated with flexor tendon repair noted that the best functional results of 75% excellent to good results were reported after early active mobilization . he concluded that adhesion formation and stiffness of fingers remain frustrating problems and suggested modern biologic approaches as one of the means of improving the outcome . we suggest that early ultrasonic therapy can be one such approach as our results of 75% excellent to good results with rupture rate of 2% is comparable to mobilization protocols . . our study shows , starting ultrasound therapy with 1-mhz frequency on the seventh post operative day with intensity of 0.7 w / cm give high percentage of excellent - good results with no ruptures and pip joint flexion contractures . if 3-mhz frequency is used , the initial intensity must be set at a lower level , and adjusted according to the patient 's response after 3 weeks.pulsed ultrasound therapy can be used in patients who are not compliant for early mobilization protocols.the results of early ultrasound therapy in zone ii ftr are comparable with the results of established early mobilization protocols.as the ultrasound therapy is not interfering with mobilization protocols , it is possible to use it as an adjuvant to further improve the outcome.the ideal frequency and intensity can not be rigidly defined but can safely be arrived at by clinical experience . our study shows , starting ultrasound therapy with 1-mhz frequency on the seventh post operative day with intensity of 0.7 w / cm give high percentage of excellent - good results with no ruptures and pip joint flexion contractures . if 3-mhz frequency is used , the initial intensity must be set at a lower level , and adjusted according to the patient 's response after 3 weeks . pulsed ultrasound therapy can be used in patients who are not compliant for early mobilization protocols . the results of early ultrasound therapy in zone ii ftr are comparable with the results of established early mobilization protocols . as the ultrasound therapy is not interfering with mobilization protocols , it is possible to use it as an adjuvant to further improve the outcome . the ideal frequency and intensity can not be rigidly defined but can safely be arrived at by clinical experience .

i refer the reader to several fine reviews on avian skin epithelial stem cells ( chuong et al . , 2006 ) and other stem cells of the mammalian skin ( nishikawa and osawa , 2006 ; tiede et al . , 2007 ; waters et al . , 2007 ; fernandes et al . , 2008 ) the mammalian skin epidermis has been the paradigm for exploring homeostasis and injury repair in a stratified epithelium . the epidermis maintains a single inner ( basal ) layer of proliferative cells that adhere to an underlying basement membrane rich in ecm and growth factors ( fig . periodically , these cells withdraw from the cell cycle , commit to differentiate terminally , move outward , and are eventually shed from the skin surface . this magnificent architecture allows the epidermis to generate a self - perpetuating barrier that keeps harmful microbes out and essential body fluids in . the program of epidermal differentiation is shown in this schematic , illustrating the basement membrane at the base , the proliferative basal layer , and the three differentiation stages : spinous layer , granular layer , and outermost stratum corneum . at the right , key molecular markers are shown , which are described in the first section of this paper . upon commitment to terminally differentiate , an epidermal keratinocyte progresses through three distinct differentiation stages : spinous , granular , and stratum corneum ( fig . major changes in transcription , morphology , and function occur at the basal / spinous layer transition and again at the granular / stratum corneum transition such that differentiated cells reaching the skin surface are enucleated cellular skeletons that are packed with cables of keratin filaments encased by a -glutamyl--lysine cross - linked cornified envelope of proteins . an additional final step in the differentiation process is the extrusion of a lipid bilayer that creates a saran wrap seal to the body surface ( de guzman strong et al . , 2006 ; elias , 2007 ) . the process is in a dynamic flux so that surface cells are continually sloughed and replaced by inner cells differentiating and moving outward . in human epidermis , the self - renewing capacity of epidermal stem cells is enormous , and , within 4 wk , a basal cell has terminally differentiated and exited at the skin surface . in mice , the postnatal trunk epidermis becomes thinner , and proliferation slows substantially as the hair coat develops and becomes the primary line of protection . although researchers have known for years that stem cells exist within the basal layer of adult epidermis , it is still not clear whether all cells within the basal layer are stem cells or whether only a small number of stem cells exist within this layer . the epidermal proliferative unit ( epu ) has been architecturally defined as a bed of 10 tightly packed basal cells yielding a stack of increasingly larger and flatter cells that culminate with a single hexagonal surface cell ( potten , 1974 ) . this has led to the hypothesis that there is one self - renewing stem cell per epu and that the other basal cells are so - called transit - amplifying ( ta ) cells ( i.e. , committed cells that divide several times and then exit the basal layer and terminally differentiate ; potten , 1974 ; mackenzie , 1997 ) . in support of this notion are in vitro studies that show that human epidermal cells with the highest level of surface 1 integrins give rise to the largest colonies ( holoclones ) that can be passaged long term , whereas cells with lower levels of 1 integrins produce smaller meroclones that do not survive passaging ( barrandon and green , 1987 ; jones et al . , 1995 ) . 1 integrins are the transmembrane core components of focal adhesions ( fas ) , which are required for basement membrane assembly , cell substratum adhesion , and cell survival / proliferation . based largely on in vitro observations , it has long been surmised that basal epidermal cells divide symmetrically and give rise to equal daughter cells . in this scenario , a basal cell would progressively reduce its adhesiveness to the underlying basement membrane , delaminate , and commit to terminally differentiate ( fig . 2 ; watt et al . , 1984 ; watt and hogan , 2000 ) . in vivo , regional variations within the basement membrane and microenvironment have been described to suggest how distinct populations of integrin - rich stem cells and ta cells might arise within the basal layer ( lavker and sun , 1982 ; jensen et al . , 1999 ) . recent studies suggest that basal epidermal cells can divide asymmetrically , affording a different view of how one basal stem cell and one committed cell might arise ( lechler and fuchs , 2005 ; clayton et al . , 2007 ) . models for the generation of a single innermost ( basal ) layer of cells with proliferative potential and multiple layers of suprabasal cells . self - renewing stem cells ( scs ) exist in the basal layer of the epidermis . symmetrical divisions produce two stem cells , a process which can serve to replenish vacancies in the basal layer . in the three - step model , a transit - amplifying ( ta ) intermediate arises , which has been postulated to divide four to five times before delaminating ( straight arrows ) and entering into a terminal differentiation program . in the two - step model , a stem cell divides asymmetrically to preferentially partition proliferation - associated factors into the stem cell daughter while providing differentiation - inducing components to the other daughter , which is fated to become a spinous cell ( sp ) . depending on the orientation of the spindle , the divisions could either result in detachment of the sp daughter from the basement membrane or , if lateral , would then necessitate subsequent delamination of the committed sp daughter . the spinous cells enter a program of terminal differentiation as they move outward and are eventually sloughed from the skin surface ( see fig . are continually replaced by a flux of inner cells committing to terminally differentiate and moving outward . in the first study reporting asymmetrical divisions , at the onset of stratification , mouse embryonic basal cells were shown to shift their spindle orientation from a lateral to a more perpendicular orientation to undergo asymmetrical divisions ( lechler and fuchs , 2005 ) . asymmetrical divisions soon accounted for 70% of all basal cell mitoses , whereas 30% remained symmetric ( lechler and fuchs , 2005 ) . although the marked reduction in postnatal basal cell divisions posed technical difficulties in measuring asymmetrical divisions in most regions of adult mouse skin , ear skin as well as tongue displayed > 60% asymmetrical divisions . the asymmetrical divisions observed provide a natural means of maintaining one proliferative basal daughter cell rich in growth - promoting receptor tyrosine kinases ( rtks ) and integrins and one suprabasal daughter with reduced rtks and integrin levels ( fig . 2 ) . moreover , loss of function experiments demonstrated a role for 1 integrins and also -catenin ( a core member of cell cell adherens junctions ) in setting up the cell polarity required for proper spindle pole orientation and maintenance of homeostasis ( lechler and fuchs , 2005 ) . in a more recent study , lineage tracing experiments on adult mouse tail skin ( another area diminished in hair follicles ) also provided evidence that divisions in the basal layer are both asymmetric and symmetric , but , in this case , only 30% occurred at an angle of > 20 relative to the basement membrane ( clayton et al . , 2007 ) . in this case , it was proposed that asymmetrical divisions might leave both daughters physically adhering to their substratum but that the committed daughter cell asymmetrically inherits a stronger notch signal ( clayton et al . , 2007 ) , a key transcriptional determinant of the spinous cell fate ( discussed in more detail later in this section ; rangarajan et al . , 2001 ; pan et al . , 2004 ; the daughter inheriting the notch signal would differentiate , detach from the underlying basement membrane , and enter the spinous layer , whereas the daughter spared of the notch signal would remain a stem cell ( clayton et al . although additional studies will be needed to resolve these different models for basal cell divisions , they lead to distinct implications for the numbers of epidermal stem cells and the location of the epidermal stem cell niche . the epu model implies the existence of only a small number of basal stem cells , suggesting that within the basal layer , there should be spatially organized microenvironments that constitute a mini - niche for a single stem cell . in contrast , the asymmetrical division models suggest that a single progenitor population within the basal layer can give rise to committed cells by differentially partitioning proteins to the daughters . the lateral asymmetrical division model implies that asymmetrical divisions are intrinsic to the epider - mal stem cell , alleviating the need for a microenvironmental change to trigger commitment . the perpendicular asymmetrical division model suggests that the basement membrane itself may constitute the epidermal stem cell niche , naturally placing the committed cell into the spinous layer . the lateral symmetric divisions , which would yield two stem cells , might then provide a mechanism to replenish old or damaged basal stem cells ( in the case of perpendicular asymmetrical divisions ) or spinous cells ( after delamination in the case of lateral asymmetrical divisions ) . this perpendicular asymmetrical division model is analogous to that of drosophila melanogaster germ cell development , in which preservation of contact with the niche maintains stemness , whereas perpendicular asymmetrical divisions drive fate determination of the daughter cells that depart the niche ( fuller and spradling , 2007 ) . irrespective of the orchestrator of epidermal cell fate , the basement membrane demarcating the epidermis and dermis plays a critical role in governing the proliferative capacity of the epidermis . although its mechano - physical properties alone are likely to impact the proliferative properties of basal cells ( dobereiner et al . , 2005 ) , the basement membrane is also composed of a rich array of ecm polymers and growth factors that provide a complex repertoire of stimuli for basal cells . among them is laminin 5 , which promotes anchorage and signaling / migration through its respective abilities to act as ligand for 64-rich hemidesmosomes and 31-rich fas ( owens and watt , 2003 ; raghavan et al . 2004 ) . through downstream 31 integrin activation of fak , integrin - linked kinase , and the rtk src , integrin mapk pathway but also induces fa adhesion turnover and epidermal migration ( lorenz et al . , 2007 ; schober et al . , 2007 ) . notably , transgenic suprabasal expression of integrins promotes tumorigenesis ( carroll et al . , 1995 ) . conversely , conditional loss of fak in the skin of mice leads to an increased resistance to chemically induced skin tumorigenesis ( mclean et al . , 2004 ) , and , in vitro , fak - deficient keratinocytes exhibit defects in cell migration and fa turnover under conditions in which apoptosis and proliferation are unaffected ( schober et al . , 2007 ) . such studies underscore the importance of integrin / fak signaling in balancing homeostasis , wound repair , and tumorigenesis in the skin . the basement membrane is also rich in proteoglycans and other proteins that act as molecular sinks for growth factors , such as tgfs , which restrict epidermal proliferation , and tgf/egfs and insulin growth factors , which promote it ( for review see fuchs , 2007 ) . a recent study has shown that when tgf signaling is compromised through loss of the trii receptor , epidermal homeostasis is maintained , but the basal layer displays increased proliferation counterbalanced by increased apoptosis ( guasch et al . , 2007 ) . although homeostasis can be maintained , it is precarious : wounds heal faster , and with just one additional oncogenic mutation , the tissue transforms quickly to squamous cell carcinoma of the skin . interestingly , enhanced fak / integrin signaling and accelerated migration are also seen when tgf signaling is compromised in the epidermis ( guasch et al . conversely , elevated integrin signaling has been shown to suppress tgf signaling ( owens and watt , 2003 ) , suggesting that this molecular route may be a two - way street . although tgf signaling places the brake on epidermal proliferation and migration , egf receptor ( egfr ) signaling enhances proliferation and migration in the epidermis ( sibilia et al . , 2000 ) . a recent study shows that lrig1 , an inhibitory ligand for egfr signaling , is expressed within a less proliferative , 1-enriched subset of cells within the basal layer of human epidermis ( jensen and watt , 2006 ) . gene targeting has revealed that the epidermis of mice lacking lrig1 hyperproliferates ( suzuki et al . , 2002 ) , and human keratinocytes treated with lrig1 short hairpin rna produce larger colonies than normal . these observations have led to the hypothesis that lrig1 plays a role in basal stem cell quiescence ( jensen and watt , 2006 ) . mitogen - inducible gene 6 is also a suppressor of egfr signaling , and mice deficient in mitogen - inducible gene 6 also display epidermal hyperproliferation and increased tumor susceptibility ( ferby et al . , 2006 ) . one possible mechanism is through the ability of activated egfr to phosphorylate 4 integrin and promote hemidesmosome disassembly ( wilhelmsen et al . , 2007 ) . thus , through regulation of two opposing tyrosine kinase receptor pathways and two integrin structures , the basement membrane plays an integral role in controlling the ability of basal epidermal stem cells to maintain homeostasis under normal and injury conditions . when this regulation goes awry , there is an increased propensity to developing squamous cell carcinoma . as the roles for additional basement membrane constituents and their associates are uncovered , our understanding of the special features of the epidermal basement membrane and its ability to regulate epidermal stem cell biology should continue to unfold . the extrinsic signals received by the microenvironment and translated through transmembrane receptors are likely to couple with the intrinsic properties of the basal epidermal cells to define their ability to self - renew and undergo homeostasis and wound repair . one transcription factor that is likely to play a key role in regulating the self - renewal and long - term proliferative capacity of the stem cell is p63 , a member of the p53 family of protooncogenes . the n isoform of p63 is preferentially expressed in basal epidermal cells ( laurikkala et al . , 2006 ) . its importance in epidermal biology surfaced when two groups made the fortuitous discovery that mice lacking p63 are severely impaired in their ability to generate the epidermis ( mills et al . the groups differed in whether the thin epidermis was caused by a defect in stem cell renewal ( yang et al . , 1999 ) , an absence of lineage commitment , and/or an early block in epidermal differentiation ( mills et al . , 1999 ) , and recent studies still leave this issue unresolved . supporting a role for np63 in stem cell self - renewal are clonal analyses on cultured thymic epithelial and epidermal cells , which suggest that when p63 mrna is knocked down through small hairpin rnas , the cells form smaller colonies with reduced proliferation rates ( senoo et al . , 2007 ) . experiments on human epidermal raft cultures suggest that p63 's function in basal cells is mediated by its ability to inhibit its relative p53 , thereby promoting cell survival and longevity , whereas its effects on differentiation are p53 independent ( truong and khavari , 2007 ) . ( 2007 ) propose a role for p63 in triggering basal cells to switch from proliferation to terminal differentiation through the induction of ib kinase- , a putative regulator of differentiation ( hu et al . once cells become suprabasal , p63 is down - regulated by an underlying mechanism that is not yet clear . it has been reported that suprabasal signaling through notch transmembrane receptors leads to suprabasal repression of p63 and inhibition of proliferation ( nguyen et al . , 2006a ) . similarly , loss of function experiments on the basally expressed notch ligand , delta1 , results in hyperproliferation as well as the repression of differentiation , suggesting that delta1 functions by repressing epidermal proliferation as well as promoting differentiation ( estrach et al . , 2007 ) . that said , conditional ablation of all canonical notch signaling in the embryonic epidermis results in basal cell hypoproliferation and repression of spinous layer differentiation , with no obvious suprabasal expression of p63 ( blanpain et al . , 2006 ) ( 2007 ) suggest that the epidermal hyperproliferation seen when skin is partially compromised in notch signaling may arise from noncell autonomous changes in the underlying dermis . although the effects of notch on proliferation appear to be complex , the collective view emerging from the present studies is that notch signaling plays a key role in the transition between basal and suprabasal cells in the epidermis , where it functions in activating the basal to suprabasal fate switch ( fig . 3 ) . predicted roles of notch signaling in the three different lineages of the epidermis . notch ligands are often basal , whereas notch receptors are typically suprabasal ( pan et al . , 2004 ; blanpain et al . , 2006 ; estrach et al . , 2006 , 2007 ; nguyen et al . , 2006a ) . notch signaling results in the release of notch intracellular domain , which acts as a transcription cofactor for the dna - binding protein rbpj ( pan et al . , 2004 ) . downstream targets of notch intracellular domain / rbpj include hes1 and hey1 , which bind dna and often function as transcriptional repressors ( hurlbut et al . , 2007 ) . in the skin lineages , notch signaling , as indicated by either the expression of notch intracellular domain ( pan et al . , 2004 ) , notch target genes hes1 and hey1 ( blanpain et al . , 2006 ) , or loss of function studies ( blanpain et al . , 2006 ; estrach et al . , 2006 , 2007 ) , is particularly prominent at the transition of fates from proliferative to differentiating . the arrows denote molecular steps along the pathways for each of the three major epithelial lineages of the skin . immunofluorescence microscopy was used on frozen mouse skin sections of e18.5 ( epidermis ) and postnatal day 28 ( hair follicle bulb ) to illustrate the localization of notch3 , hes1 , and hey1 . all samples were counterstained with dapi ( blue ) to mark the nuclei . where indicated , antibodies against 4 integrin ( int ) were used to mark the dermo - epidermal boundary , and keratin 14 ( ker14 ) was used to denote the basal layer of the ors . the top two panels illustrate the coexpression of notch3 and hes1 , which is particularly strong at the basal to suprabasal transition of the epidermis . the bottom two panels show that hes1 is prominent in the differentiating cells of the irs ( left ) , whereas hey1 is more broadly expressed in both the precortex and irs ( right ) . the inset in the image in the bottom right frame shows a close - up view of hair shaft precursor cells that have been labeled with antibodies against hair keratins in green and hey1 in red to show that nuclear hey1 colocalizes with cells that generate the hair shaft . note that the highly proliferative matrix cells at the base of the hair bulb are negative for signs of notch signaling . for further information and similar images ( courtesy of c. blanpain and w.e . overall , the basal expression of notch ligands such as delta1 and the suprabasal expression of notch receptors and notch downstream target genes such as hes1 are consistent with a role for notch signaling in controlling this switch . a role for notch as the gatekeeper of the commitment of basal epidermal cells to terminally differentiate is particularly intriguing in light of a notch inhibitor , numb , which functions as an intrinsic cell fate determinant that is segregated into only one of the two daughter cells during neural precursor division in drosophila neuroblasts ( for review see yu et al . , numb localization is determined by opposing signals from the par3atypical pkc complex , and this machinery is also present in basal epidermal cells and is present at the right time and place to control asymmetrical divisions ( lechler and fuchs , 2005 ; clayton et al . , 2007 ) . although much still remains to be learned about how basal epidermal cells become activated to withdraw from the cell cycle and commit to terminally differentiate , the action appears to converge at the base of the epidermis between the basal and first suprabasal layers . one of the most remarkable features of the vertebrate epidermis is its extraordinary ability to generate elaborate appendages . the scales of a fish , the horn of a rhinocerous , the claws of an eagle , the mane of a lion , and the sweat ( eccrine ) and oil ( sebaceous ) glands of our skin are all epidermal appendages . knowledge of how epidermal appendages develop has been gained by studying the embryonic skin at a stage when it exists as a single - layered epithelium . hair follicle morphogenesis begins early in embryonic development as dermal cells populate the skin , and signals from the epithelium induce the formation of dermal condensates . dermal condensates are the precursors to the dermal papillae ( dp ) , which are the permanent mesenchymal component of the hair follicle . at approximately embryonic day ( e ) 14.5 of mouse development , mesenchymal epithelial interactions result in the formation of the first wave of hair placodes , which appear as small epidermal invaginations into the dermis ( fig . 4 ) . once initiated , these placodes undergo marked proliferation and downgrowth to produce first the hair germs ( e15.5 ) and then hair pegs ( e16.517.5 ) . the follicle cells at the leading front ( matrix ) remain highly proliferative , and , through contact with the dp , they engage in a program of gene expression that is distinct from the root . the outer root sheath ( ors ) maintains contact with the basement membrane , whereas the inner root sheath ( irs ) provides the channel for the emerging hair ( e18.5 ) . at birth , the most mature hairs begin to break the skin surface , and the sebaceous gland ( sg ) precursor cells become established in the upper segment of the root . 4 ; for review see schmidt - ullrich and paus , 2005 ) . there are four waves of follicle morphogenesis , with the large primary guard hairs forming first ( e14.5 ) and the bulk of the hair coat follicles initiating within a few days after this . the process of follicle morphogenesis occurs relatively late in embryonic development . in the mouse , discrete waves of placodes are seen beginning at approximately e14.5 and ending near birth . once initiated , newly formed follicles continue to mature postnatally until approximately day 9 , at which time matrix ( ta ) cells at the base of the follicle continue to rapidly proliferate and differentiate , generating hair growth . for a comprehensive analysis of the regional , temporal , and strain - specific variations in the timing of hair follicle morphogenesis , see the reviews muller - rover et al . , 2001 andschmidt - ullrich and paus , 2005 . one dermal cue that appears to be critical is the bone morphogenetic protein ( bmp ) inhibitory protein noggin , whose absence severely impairs hair follicle morphogenesis ( botchkarev et al . ectodermal wnt signals , which include wnt10b , are thought to act in concert with dermal signals to instruct the epidermal cells to grow downward ( botchkarev et al . , 1999 ; dasgupta and fuchs , 1999 ; reddy et al . , 2001 ; andl et al . , 2002 ) . at the crux of this signaling cross talk is the bipartite transcription factor complex composed of lef1 and stabilized -catenin , which is critical for hair follicle morphogenesis ( van genderen et al . , 1994 ; zhou et al . , 1995 ; gat et al . , 1998 ) and whose activity can be monitored by the wnt reporter gene topgal ( dasgupta and fuchs , 1999 ; millar , 2002 ) . inhibition of bmp signaling is permissive for lef1 expression and also promotes the stabilization of -catenin ( jamora et al . , 2003 ; kobielak et al . , 2003 ; andl et al . , 2004 ; zhang et al . , 2006 ) , whereas wnts are well - established instigators of -catenin stabilization ( for review see clevers , 2006 ) . interestingly , although this paradigm serves the bulk of follicle morphogenesis , the first wave of follicle morphogenesis , resulting in formation of the large primary guard hairs , diverges somewhat , particularly in the external cues guiding these internal regulatory processes . this first wave is uniquely dependent on a tnf - like ligand receptor complex composed of ectodysplasin ( eda ) and eda receptor , which , instead of inducing noggin , induces two different bmp inhibitors . 2001 ) , and signs of wnt reporter gene activity and lef1/-catenin are present by e14.5 , when the first wave of follicle morphogenesis occurs ( dasgupta and fuchs , 1999 ; pummila et al . studies over the past decade indicate that it is indeed central to the decision - making process . thus , when excess -catenin is stabilized in the epidermis of transgenic mice , ectopic hair follicles appear within the interfollicular epidermis , generating super - furry mice ( gat et al . , 1998 ) , and , when the wnt inhibitor dickoff 1 ( dkk1 ) is expressed ectopically or when -catenin is conditionally targeted for ablation , hair follicle morphogenesis is blocked altogether ( huelsken and birchmeier , 2001 ; andl et al . , 2002 ) . recently , ito et al . ( 2007 ) showed that when the skin of mice is severely wounded , endogenous wnt signaling is elevated in the skin , and this leads to the induction of hair follicle formation from epidermal stem cells . collectively , these findings underscore the role for stabilized -catenin in governing the choice of whether to become epidermis or hair follicle . once the placode forms , signaling events downstream of wnts / bmps drive the downgrowth and maturation of hair follicles . sonic hedgehog ( shh ) is an early gene expressed downstream of wnt / bmp receptor ( bmpr ) signaling ( and eda / eda receptors in the case of guard hairs ) and placode formation ( dahmane et al . , 1997 ; oro et al . , 1997 ; gat et al . , 1998 ; shh plays a critical role in organizing the dermal cells into a condensate , or dp , which thereafter becomes a permanent component of the hair follicle ( hardy , 1992 ; st - jacques et al . , 1998 ; oro and higgins , 2003 ; overall , the formation of the hair bud involves many changes in gene expression as exemplified by the differences between the transcriptional profile of placode cells and their epidermal counterparts ( rhee et al . , 2006 ) . among the early steps is the activation of additional key transcription factors ( e.g. , lhx2 and sox9 ) whose absence results in a failure to maintain follicle stem cell behavior in mice ( vidal et al . additional changes involve down - regulation of some adhesion proteins ( e.g. , epithelial cadherin [ e - cadherin ] ) and up - regulation of others ( jamora et al . , 2003 ) . the field is still in the midst of elucidating how multiple signaling pathways converge on the ectoderm to elicit changes in transcription factors that then subsequently change the expression and dynamics of the extracellular matrix , cytoskeleton , and cell matrix and cell cell junctions that remodel the epithelium from its single layer to a hair bud . once understood , this process is likely to be relevant to other morphogenetic processes ranging from development of the mammary gland to that of the tooth . matrix cells have been referred to as ta cells because they proliferate rapidly during the growth ( anagen ) phase of the cycle but then suddenly undergo apoptosis by a mechanism that is still poorly understood . during this 34-d destructive phase ( catagen ) , the hair bulb and root shrivel to an epithelial strand , which pulls the dp upward to rest at the base of the permanent , noncycling portion of the follicle . in mice , backskin follicles remain dormant for 12 d in the first resting phase ( telogen ) but then rest for > 3 wk in the second telogen . old hairs can remain in their socket through several bouts of hair cycling , rendering the hair coat impervious to this cyclic behavior . at the onset of each new anagen , the cycling portion of the follicle regenerates , a process that necessitates a reservoir of follicle stem cells . these stem cells reside in the lowest permanent portion of the follicle , within the ors , which bulges around the existing old hair pocket to enable the new hair to emerge through the same orifice at the skin surface . the cells within the bulge of the ors cycle less frequently than other epithelial cells within the skin ( cotsarelis et al . , a cluster of stem cells at the base of the bulge become activated to proliferate . grafting and lineage tracing experiments have shown that some bulge cells are stem cells and that offspring of a single bulge stem cell can form sgs and epidermis as well as new hair follicles ( blanpain et al . , 2004 ; morris et al . , 2004 ; claudinot et al . , 2005 ) . that said , although follicle stem cells are multipotent , unless the skin is wounded , follicle stem cells only function in hair follicle homeostasis and do not contribute to interfollicular epidermis ( ito et al . , 2005 ; , 2005 , 2007 ) . to understand the special features of the bulge , researchers have conducted microarray profiling . approximately 150 genes are preferentially expressed in the bulge relative to the proliferating basal cells of the epidermis ( blanpain et al . the purification of bulge stem cells has been accomplished by facs based on either ( 1 ) bulge cell surface markers 6 and cd34 coupled with k14-gfp expression ( blanpain et al . , 2004 ) , 2004 ) , or ( 3 ) a fluorescent histone pulse - chase experiment in mice expressing a tetracycline - inducible transcriptional regulatory repressor ( tet ) under the control of a k5 promoter and histone h2b - gfp under the control of a tetracycline regulatory element ( tumbar et al . , 2004 ) . although each of these different procedures purifies slightly different cell populations , the array data are in quite good agreement , enabling researchers to exploit this information to learn more about follicle stem cells . like the embryonic epidermis , bulge cells are wnt responsive , as judged by their expression of two lef1-related dna - binding proteins , tcf3 and tcf4 , and several wnt receptor proteins ( fzds ) . however , at least in telogen , most bulge cells appear to be in a state of wnt inhibition , as judged by the array data and by the lack of detectable nuclear -catenin or topgal activity ( dasgupta and fuchs , 1999 ) . these findings are consistent with a recent study showing that tcf3 can function as a repressor when -catenin is absent or underrepresented and that tcf3 on its own functions to repress the differentiation of skin stem cells ( nguyen et al . , 2006b ) . although wnt inhibition seems to be a feature of the dormant stem cell niche in the skin , stabilized -catenin is a feature of activated stem cells . by transgenically elevating the levels of stabilized -catenin , hair follicles precociously conditional loss of bmpr1a in telogen follicles also results in the precocious entry of follicles into anagen and the stabilization of -catenin , and , at least in part , this appears to be caused by the activation of pi3 kinase signaling , which , in turn , phosphorylates and inactivates gsk3 , the kinase that otherwise targets -catenin for phosphorylation and ubiquitin - mediated degradation ( zhang et al . , 2006 ; kobielak et al . , 2007 ) . these studies further clarify how the inhibition of bmp signaling and activation of wnt signaling converge to regulate stem cell activation . once follicle stem cells are activated , wnt3 signaling appears to be required for hair shaft differentiation , whereas bmp signaling is required for irs and directly or indirectly is also required for hair shaft differentiation ( millar , 2002 ; kobielak et al . thus , when -catenin is conditionally targeted for depletion in adult skin when follicles are in telogen phase , the bulge stem cell compartment can not be maintained , and the new hair cycle does not reinitiate ( lowry et al . , 2005 ) ; when stabilized -catenin is constitutively activated , tumors develop that are composed of a center of hair shaft cells surrounded by matrix - like cells expressing nuclear lef1/-catenin ( gat et al . , 1998).when the gene encoding bmpr1a is conditionally targeted for depletion in adult follicles in telogen phase , bulge stem cells become activated , as judged by their loss of brdu label retention and their entry into s phase ( andl et al . , 2004 ; kobielak et al . , 2007 ) . however , the cells can not terminally differentiate , and , within weeks , tumors develop that display markers such as lhx2 , sox4 , sox9 , and shh , which are features of activated stem cells . despite the fact that they are no longer slow cycling and do not express appreciable cd34 , the follicle cells lacking bmpr1a are still able to repair epidermis in a wound response , and they generate what appear to be long - lived tumors ( zhang et al . , 2006 ; these studies are tantalizing in that they suggest that quiescence may not be an essential feature of follicle stem cells . overall , these data suggest that bmp signaling plays an important role in follicle stem cell quiescence . consistent with this view are immunofluorescence studies that detect phosphorylated smad1 , which is reflective of active bmp signaling , and the behavior of cultured bulge cells exposed to bmp6 , which results in their transient withdrawal from the cell cycle ( blanpain et al . , 2004 ; kobielak et al . , 2007 ) . tgf signaling also appears to be up - regulated in bulge cells ( tumbar et al . , 2004 ) and is known to dampen the cell cycle ( massague , 2007 ) . however , conditional ablation of the tgf receptor did not appear to compromise the slow - cycling capability of the follicle stem cells in the bulge ( guasch et al . although studies in recent years have led to major advances in our knowledge of bulge stem cells and the signaling pathways involved in their activation at the start of the hair cycle ( for review see fuchs , 2007 ) , the precise nature of the signals and where they come from are still poorly understood . thus , despite the established role for -catenin stabilization in follicle stem cell activation , documentation for the involvement of a specific wnt is still lacking . similarly , although the condensate of dp cells typically rests at the base of the bulge in telogen follicles and expresses several bmp inhibitory factors and wnts that are candidates for follicle stem cell activation , it is not clear precisely what this inductive signal is . further complicating the issue is that in the whisker cycle of rodents , the dp never reaches the bulge before the start of a new anagen . it has been proposed that stem cells from the bulge therefore migrate along the ors to the base of the follicle , where they become activated ( oshima et al . although further studies are required , most studies to date are consistent with the notion that this may also happen in backskin follicles . if so , it would imply that sox9 , tcf3 , and lhx2 may be markers of follicle stem cells irrespective of whether they are quiescent or proliferative and that there are likely to be factors beyond dp signals that are required to activate the hair cycle . appendages of the hair follicle , sgs , are located above the bulge and just below the hair shaft orifice at the skin surface . the major role of the gland is to generate terminally differentiated sebocytes , which degenerate to release lipids and sebum and lubricate the skin surface . sg homeostasis necessitates a population of progenitor cells that gives rise to a continual flux of proliferating and differentiating cells that are sloughed through the hair canal . lineage tracing by retroviral - mediated gene transfer suggests that a small population of cells near or at the base of the sg might be stem cells ( ghazizadeh and taichman , 2001 ) . recently , the transcriptional repressor protein blimp1 was identified in a genetic screen for hair follicle transcription factors , and it was shown to mark a small population of cells at the sg base ( horsley et al . , 2006 ) . these blimp1-positive cells appeared to be in close association with the basement membrane surrounding the gland and are contiguous with the bm that demarcates the dermal epidermal border and surrounds the hair follicle . genetic lineage tracing experiments reveal that the blimp1-positive sg cells can regenerate the entire gland , including the sebocytes . the likely explanation stems from blimp1 's role in b lymphocytes , in which it transcriptionally represses c - myc ( chang et al . , 2000 ) , a gene known to induce sg hyperplasia and sebocyte differentiation at the expense of hair follicle differentiation ( arnold and watt , 2001 ; waikel et al . , 2001 ) . however , if blimp1 truly marks the progenitor / stem cell population of the sg , why do n't the enlarged glands eventually recede in blimp1 's absence even if hyperplasia is the initial outcome ? although further studies are needed , the underlying explanation could be rooted in the ability of the bulge to become mobilized when the sg progenitor cell population is depleted . as judged by brdu pulse and pulse - chase experiments , blimp1-negative bulge cells show signs of active cycling and reduced label retention in the absence of blimp1 ( horsley et al . , 2006 ) . in this regard , the behavior of bulge stem cells in response to a loss of blimp1 in sg progenitors resembles their response to the epidermis lost upon injury in normal mice . such precursor - product relation between the bulge and other skin stem cell populations has also been documented by engraftment experiments with isolated bulge stem cells ( blanpain et al . , 2004 ; claudinot et al . , 2005 ) . an emerging view of skin stem cells is that there appears to be at least three distinct niches for skin stem cells : the follicle bulge , the base of the sg , and the basal layer of the epidermis . it is not yet resolved whether the basal layer contains a subset of stem cells as originally posited by potten ( 1974 ) or whether the basal compartment is composed of a single progenitor population , as more recently proposed by clayton et al . are there unifying features of keratinocyte stem cells and their activation mechanisms that guide them along specific lineages ? although the answer to this question remains unknown , it seems reasonable to predict that certain features of keratinocyte stem cells and stem cell activation must be similar given the similarities in stem cell niches . although p63 has also been implicated in differentiation , p63 remains one of the best candidates to date for a gatekeeper of proliferation in epithelial stem cells ( truong et al . cells within all three progenitor pools also express e - cadherin and display elevated levels of adherens junctions and reduced levels of desmosomes . a reduction in e - cadherin appears to be an essential feature in mobilizing embryonic epidermal cells to invaginate to form a hair follicle ( jamora et al . , 2003 ) , and reductions in -catenin and p120-catenin have also been linked to epithelial cancers , a less organized form of invagination ( scott and yap , 2006 ; reynolds , 2007 ) . such studies make it tempting to speculate that the mobilization of stem cells to exit their niches and either form a hair follicle or repair wounds may entail the remodeling of intercellular junctions . additionally , the three progenitor compartments are typified by their proximity to an underlying basement membrane , and all three types of progenitors express integrins , including 64 and 31 , as well as their associated proteins . the higher the integrin level , the greater the proliferative potential seems to be , as judged by the early studies of jones et al . one intriguing aspect is the up - regulation of 6 integrin and the presence of the v6 integrin ligand , tenascin c , in the bulge stem cells as they transit from telogen to anagen ( tumbar et al . , 2004 ) . similar changes are seen in the epidermal basal layer in response to injury ( fassler et al . , 1996 ) , leading to the speculation that these changes might be important in understanding how stem cells become activated to migrate from their niche and embark upon their specific mission . thus , although none of these markers is exclusive to the three progenitor populations , they do appear to be general features of keratinocytes that maintain their proliferative potential , whether sustained or transient . it is still formally possible that expression of these markers empowers keratinocytes to retain or maintain their proliferative potential and their stem - ness , in which case down - regulation of these markers might define a point of no return along a stem cell lineage . as future studies are conducted the similarities in keratinocyte stem cell behavior may extend to shared principles and pathways , even if the precise players may differ . one example is blimp1 , which appears to mark sg progenitors and repress c - myc expression ( horsley et al . , 2006 ) , whereas miz1 seems to play a similar c - myc repressive function in the epidermal basal layer ( gebhardt et al . , 2006 ) . the outcome of c - myc expression is keratinocyte proliferation in both the sg and epidermis , leading to the postulate that c - myc might control the conversion of stem cells to so - called ta ( i.e. , committed ) cells ( waikel et al . , 2001 ; frye et al . , 2003 ) . in this regard , it is intriguing that later in the epidermal lineage , ovol1 , yet another transcriptional repressor of c - myc , is expressed suprabasally , where it may act to prevent proliferation after ta cells commit to terminally differentiate ( nair et al . , 2006 ) . another signaling pathway likely to impact on more than one keratinocyte stem cell niche is notch , which controls selective cell fate determination through close - range interactions not only in the epidermis but also in the hair follicle and the sg ( yamamoto et al . , 2003 ; pan et al . , 2004 ; vauclair et al . , 2005 ; blanpain et al . , , 2006 ; nguyen et al . , 2006a ) . the studies to date support a role for notch in activating the keratinocyte switch from the undifferentiated to differentiated fate ( fig . although notch signaling appears to affect the transition from proliferation to differentiation throughout keratinocyte populations in the skin , other signaling pathways seem to have more potent effects on one lineage over another . thus , in the follicle bulge , stem cells enter the new hair cycle when threshold levels of stabilized -catenin are reached ( gat et al . , 1998 ; huelsken et al . , 2001 ; van mater et al . , 2003 ; lo celso et al conversely , a negative role for wnt signaling has been proposed for sg fate determination because sg hyperplasia occurs when a truncated version of lef1 is expressed that is unable to associate with -catenin ( merrill et al . , 2001 ; niemann et al . , although epidermal stem cells are seemingly unaffected by the loss of -catenin ( huelsken et al . , 2001 ) , they adopt a hair follicle cell fate when -catenin is stabilized through either genetic manipulation or wounding ( gat et al . , 1998 ; as outlined in this paper , growth factor receptor signaling also appears to affect different keratinocyte stem cell populations in distinct ways . when taken with the fact that cell fate signaling pathways are often interdependent on one another , such differences can have a potent impact on lineage determination , as recently demonstrated by estrach et al . in the precortex of the hair follicle , where wnt signaling is particularly high ( dasgupta and fuchs , 1999 ) , the wnt target gene jag1 is expressed , leading to active notch signaling in these cells , promoting proliferation and differentiation to produce hair ( estrach et al . , 2006 ) . conversely , in the absence of jag1 , hair follicles fail to form , but the epidermis is spared . thus , by activating the expression of other signaling genes , wnt signaling can accentuate its effects on a particular lineage within the skin . in closing , the existence of different skin stem cell compartments and the ability of these niches to respond differentially to environmental cues has been an exciting new development in the field . increasing knowledge about these stem cell populations over the past 5 yr has begun to shed light on the similarities and differences among these niches . are these different stem cell populations multipotent , as recent experiments by ito et al . ( 2007 ) suggest ? is the multipotency of stem cells in response to wounding a reflection of mechanical disruption of the niche or a response to growth / differentiation factors released in a wound response ? to what extent do the gene expression programs of other progenitor populations overlap with that of follicle stem cells , and do these similarities reflect their self - renewing , undifferentiated features ? when do these different proliferative epithelial compartments form during skin development , and to what extent are their differences in gene expression patterns dependent on their distinct in vivo microenvironments ? what factors control the differences in the slow - cycling ability of different stem cell populations within the skin ? the answers to these questions will bring us ever closer in the strife to understanding how stem cells are maintained in their undifferentiated , growth - restricted state and what prompts them to become activated , exit their niches , and embark upon distinct lineages .

the skin epidermis and its appendages provide a protective barrier that is impermeable to harmful microbes and also prevents dehydration . to perform their functions while being confronted with the physicochemical traumas of the environment , these tissues undergo continual rejuvenation through homeostasis , and , in addition , they must be primed to undergo wound repair in response to injury . the skin 's elixir for maintaining tissue homeostasis , regenerating hair , and repairing the epidermis after injury is its stem cells , which reside in the adult hair follicle , sebaceous gland , and epidermis . stem cells have the remarkable capacity to both self - perpetuate and also give rise to the differentiating cells that constitute one or more tissues . in recent years , scientists have begun to uncover the properties of skin stem cells and unravel the mysteries underlying their remarkable capacity to perform these feats . in this paper , i outline the basic lineages of the skin epithelia and review some of the major findings about mammalian skin epithelial stem cells that have emerged in the past five years .

The epidermis The hair follicle The hair cycle and hair follicle stem cells SGs Common themes in skin stem cells

i refer the reader to several fine reviews on avian skin epithelial stem cells ( chuong et al . , 2006 ) and other stem cells of the mammalian skin ( nishikawa and osawa , 2006 ; tiede et al . , 2008 ) the mammalian skin epidermis has been the paradigm for exploring homeostasis and injury repair in a stratified epithelium . the epidermis maintains a single inner ( basal ) layer of proliferative cells that adhere to an underlying basement membrane rich in ecm and growth factors ( fig . periodically , these cells withdraw from the cell cycle , commit to differentiate terminally , move outward , and are eventually shed from the skin surface . this magnificent architecture allows the epidermis to generate a self - perpetuating barrier that keeps harmful microbes out and essential body fluids in . at the right , key molecular markers are shown , which are described in the first section of this paper . major changes in transcription , morphology , and function occur at the basal / spinous layer transition and again at the granular / stratum corneum transition such that differentiated cells reaching the skin surface are enucleated cellular skeletons that are packed with cables of keratin filaments encased by a -glutamyl--lysine cross - linked cornified envelope of proteins . an additional final step in the differentiation process is the extrusion of a lipid bilayer that creates a saran wrap seal to the body surface ( de guzman strong et al . in human epidermis , the self - renewing capacity of epidermal stem cells is enormous , and , within 4 wk , a basal cell has terminally differentiated and exited at the skin surface . this has led to the hypothesis that there is one self - renewing stem cell per epu and that the other basal cells are so - called transit - amplifying ( ta ) cells ( i.e. in support of this notion are in vitro studies that show that human epidermal cells with the highest level of surface 1 integrins give rise to the largest colonies ( holoclones ) that can be passaged long term , whereas cells with lower levels of 1 integrins produce smaller meroclones that do not survive passaging ( barrandon and green , 1987 ; jones et al . in this scenario , a basal cell would progressively reduce its adhesiveness to the underlying basement membrane , delaminate , and commit to terminally differentiate ( fig . self - renewing stem cells ( scs ) exist in the basal layer of the epidermis . symmetrical divisions produce two stem cells , a process which can serve to replenish vacancies in the basal layer . in the three - step model , a transit - amplifying ( ta ) intermediate arises , which has been postulated to divide four to five times before delaminating ( straight arrows ) and entering into a terminal differentiation program . in the two - step model , a stem cell divides asymmetrically to preferentially partition proliferation - associated factors into the stem cell daughter while providing differentiation - inducing components to the other daughter , which is fated to become a spinous cell ( sp ) . in the first study reporting asymmetrical divisions , at the onset of stratification , mouse embryonic basal cells were shown to shift their spindle orientation from a lateral to a more perpendicular orientation to undergo asymmetrical divisions ( lechler and fuchs , 2005 ) . in a more recent study , lineage tracing experiments on adult mouse tail skin ( another area diminished in hair follicles ) also provided evidence that divisions in the basal layer are both asymmetric and symmetric , but , in this case , only 30% occurred at an angle of > 20 relative to the basement membrane ( clayton et al . although additional studies will be needed to resolve these different models for basal cell divisions , they lead to distinct implications for the numbers of epidermal stem cells and the location of the epidermal stem cell niche . the epu model implies the existence of only a small number of basal stem cells , suggesting that within the basal layer , there should be spatially organized microenvironments that constitute a mini - niche for a single stem cell . in contrast , the asymmetrical division models suggest that a single progenitor population within the basal layer can give rise to committed cells by differentially partitioning proteins to the daughters . the lateral symmetric divisions , which would yield two stem cells , might then provide a mechanism to replenish old or damaged basal stem cells ( in the case of perpendicular asymmetrical divisions ) or spinous cells ( after delamination in the case of lateral asymmetrical divisions ) . this perpendicular asymmetrical division model is analogous to that of drosophila melanogaster germ cell development , in which preservation of contact with the niche maintains stemness , whereas perpendicular asymmetrical divisions drive fate determination of the daughter cells that depart the niche ( fuller and spradling , 2007 ) . irrespective of the orchestrator of epidermal cell fate , the basement membrane demarcating the epidermis and dermis plays a critical role in governing the proliferative capacity of the epidermis . conversely , conditional loss of fak in the skin of mice leads to an increased resistance to chemically induced skin tumorigenesis ( mclean et al . , 2004 ) , and , in vitro , fak - deficient keratinocytes exhibit defects in cell migration and fa turnover under conditions in which apoptosis and proliferation are unaffected ( schober et al . such studies underscore the importance of integrin / fak signaling in balancing homeostasis , wound repair , and tumorigenesis in the skin . the basement membrane is also rich in proteoglycans and other proteins that act as molecular sinks for growth factors , such as tgfs , which restrict epidermal proliferation , and tgf/egfs and insulin growth factors , which promote it ( for review see fuchs , 2007 ) . although homeostasis can be maintained , it is precarious : wounds heal faster , and with just one additional oncogenic mutation , the tissue transforms quickly to squamous cell carcinoma of the skin . although tgf signaling places the brake on epidermal proliferation and migration , egf receptor ( egfr ) signaling enhances proliferation and migration in the epidermis ( sibilia et al . as the roles for additional basement membrane constituents and their associates are uncovered , our understanding of the special features of the epidermal basement membrane and its ability to regulate epidermal stem cell biology should continue to unfold . the extrinsic signals received by the microenvironment and translated through transmembrane receptors are likely to couple with the intrinsic properties of the basal epidermal cells to define their ability to self - renew and undergo homeostasis and wound repair . one transcription factor that is likely to play a key role in regulating the self - renewal and long - term proliferative capacity of the stem cell is p63 , a member of the p53 family of protooncogenes . supporting a role for np63 in stem cell self - renewal are clonal analyses on cultured thymic epithelial and epidermal cells , which suggest that when p63 mrna is knocked down through small hairpin rnas , the cells form smaller colonies with reduced proliferation rates ( senoo et al . predicted roles of notch signaling in the three different lineages of the epidermis . notch signaling results in the release of notch intracellular domain , which acts as a transcription cofactor for the dna - binding protein rbpj ( pan et al . in the skin lineages , notch signaling , as indicated by either the expression of notch intracellular domain ( pan et al . the arrows denote molecular steps along the pathways for each of the three major epithelial lineages of the skin . where indicated , antibodies against 4 integrin ( int ) were used to mark the dermo - epidermal boundary , and keratin 14 ( ker14 ) was used to denote the basal layer of the ors . the top two panels illustrate the coexpression of notch3 and hes1 , which is particularly strong at the basal to suprabasal transition of the epidermis . the bottom two panels show that hes1 is prominent in the differentiating cells of the irs ( left ) , whereas hey1 is more broadly expressed in both the precortex and irs ( right ) . the inset in the image in the bottom right frame shows a close - up view of hair shaft precursor cells that have been labeled with antibodies against hair keratins in green and hey1 in red to show that nuclear hey1 colocalizes with cells that generate the hair shaft . a role for notch as the gatekeeper of the commitment of basal epidermal cells to terminally differentiate is particularly intriguing in light of a notch inhibitor , numb , which functions as an intrinsic cell fate determinant that is segregated into only one of the two daughter cells during neural precursor division in drosophila neuroblasts ( for review see yu et al . one of the most remarkable features of the vertebrate epidermis is its extraordinary ability to generate elaborate appendages . hair follicle morphogenesis begins early in embryonic development as dermal cells populate the skin , and signals from the epithelium induce the formation of dermal condensates . dermal condensates are the precursors to the dermal papillae ( dp ) , which are the permanent mesenchymal component of the hair follicle . at approximately embryonic day ( e ) 14.5 of mouse development , mesenchymal epithelial interactions result in the formation of the first wave of hair placodes , which appear as small epidermal invaginations into the dermis ( fig . the follicle cells at the leading front ( matrix ) remain highly proliferative , and , through contact with the dp , they engage in a program of gene expression that is distinct from the root . at birth , the most mature hairs begin to break the skin surface , and the sebaceous gland ( sg ) precursor cells become established in the upper segment of the root . for a comprehensive analysis of the regional , temporal , and strain - specific variations in the timing of hair follicle morphogenesis , see the reviews muller - rover et al . interestingly , although this paradigm serves the bulk of follicle morphogenesis , the first wave of follicle morphogenesis , resulting in formation of the large primary guard hairs , diverges somewhat , particularly in the external cues guiding these internal regulatory processes . studies over the past decade indicate that it is indeed central to the decision - making process . thus , when excess -catenin is stabilized in the epidermis of transgenic mice , ectopic hair follicles appear within the interfollicular epidermis , generating super - furry mice ( gat et al . , 1998 ) , and , when the wnt inhibitor dickoff 1 ( dkk1 ) is expressed ectopically or when -catenin is conditionally targeted for ablation , hair follicle morphogenesis is blocked altogether ( huelsken and birchmeier , 2001 ; andl et al . ( 2007 ) showed that when the skin of mice is severely wounded , endogenous wnt signaling is elevated in the skin , and this leads to the induction of hair follicle formation from epidermal stem cells . collectively , these findings underscore the role for stabilized -catenin in governing the choice of whether to become epidermis or hair follicle . , 1998 ; shh plays a critical role in organizing the dermal cells into a condensate , or dp , which thereafter becomes a permanent component of the hair follicle ( hardy , 1992 ; st - jacques et al . , 1998 ; oro and higgins , 2003 ; overall , the formation of the hair bud involves many changes in gene expression as exemplified by the differences between the transcriptional profile of placode cells and their epidermal counterparts ( rhee et al . the field is still in the midst of elucidating how multiple signaling pathways converge on the ectoderm to elicit changes in transcription factors that then subsequently change the expression and dynamics of the extracellular matrix , cytoskeleton , and cell matrix and cell cell junctions that remodel the epithelium from its single layer to a hair bud . matrix cells have been referred to as ta cells because they proliferate rapidly during the growth ( anagen ) phase of the cycle but then suddenly undergo apoptosis by a mechanism that is still poorly understood . at the onset of each new anagen , the cycling portion of the follicle regenerates , a process that necessitates a reservoir of follicle stem cells . these stem cells reside in the lowest permanent portion of the follicle , within the ors , which bulges around the existing old hair pocket to enable the new hair to emerge through the same orifice at the skin surface . the cells within the bulge of the ors cycle less frequently than other epithelial cells within the skin ( cotsarelis et al . grafting and lineage tracing experiments have shown that some bulge cells are stem cells and that offspring of a single bulge stem cell can form sgs and epidermis as well as new hair follicles ( blanpain et al . that said , although follicle stem cells are multipotent , unless the skin is wounded , follicle stem cells only function in hair follicle homeostasis and do not contribute to interfollicular epidermis ( ito et al . approximately 150 genes are preferentially expressed in the bulge relative to the proliferating basal cells of the epidermis ( blanpain et al . these findings are consistent with a recent study showing that tcf3 can function as a repressor when -catenin is absent or underrepresented and that tcf3 on its own functions to repress the differentiation of skin stem cells ( nguyen et al . although wnt inhibition seems to be a feature of the dormant stem cell niche in the skin , stabilized -catenin is a feature of activated stem cells . by transgenically elevating the levels of stabilized -catenin , hair follicles precociously conditional loss of bmpr1a in telogen follicles also results in the precocious entry of follicles into anagen and the stabilization of -catenin , and , at least in part , this appears to be caused by the activation of pi3 kinase signaling , which , in turn , phosphorylates and inactivates gsk3 , the kinase that otherwise targets -catenin for phosphorylation and ubiquitin - mediated degradation ( zhang et al . however , the cells can not terminally differentiate , and , within weeks , tumors develop that display markers such as lhx2 , sox4 , sox9 , and shh , which are features of activated stem cells . consistent with this view are immunofluorescence studies that detect phosphorylated smad1 , which is reflective of active bmp signaling , and the behavior of cultured bulge cells exposed to bmp6 , which results in their transient withdrawal from the cell cycle ( blanpain et al . however , conditional ablation of the tgf receptor did not appear to compromise the slow - cycling capability of the follicle stem cells in the bulge ( guasch et al . although studies in recent years have led to major advances in our knowledge of bulge stem cells and the signaling pathways involved in their activation at the start of the hair cycle ( for review see fuchs , 2007 ) , the precise nature of the signals and where they come from are still poorly understood . it has been proposed that stem cells from the bulge therefore migrate along the ors to the base of the follicle , where they become activated ( oshima et al . appendages of the hair follicle , sgs , are located above the bulge and just below the hair shaft orifice at the skin surface . the major role of the gland is to generate terminally differentiated sebocytes , which degenerate to release lipids and sebum and lubricate the skin surface . sg homeostasis necessitates a population of progenitor cells that gives rise to a continual flux of proliferating and differentiating cells that are sloughed through the hair canal . lineage tracing by retroviral - mediated gene transfer suggests that a small population of cells near or at the base of the sg might be stem cells ( ghazizadeh and taichman , 2001 ) . these blimp1-positive cells appeared to be in close association with the basement membrane surrounding the gland and are contiguous with the bm that demarcates the dermal epidermal border and surrounds the hair follicle . in this regard , the behavior of bulge stem cells in response to a loss of blimp1 in sg progenitors resembles their response to the epidermis lost upon injury in normal mice . such precursor - product relation between the bulge and other skin stem cell populations has also been documented by engraftment experiments with isolated bulge stem cells ( blanpain et al . an emerging view of skin stem cells is that there appears to be at least three distinct niches for skin stem cells : the follicle bulge , the base of the sg , and the basal layer of the epidermis . are there unifying features of keratinocyte stem cells and their activation mechanisms that guide them along specific lineages ? although the answer to this question remains unknown , it seems reasonable to predict that certain features of keratinocyte stem cells and stem cell activation must be similar given the similarities in stem cell niches . although p63 has also been implicated in differentiation , p63 remains one of the best candidates to date for a gatekeeper of proliferation in epithelial stem cells ( truong et al . one intriguing aspect is the up - regulation of 6 integrin and the presence of the v6 integrin ligand , tenascin c , in the bulge stem cells as they transit from telogen to anagen ( tumbar et al . similar changes are seen in the epidermal basal layer in response to injury ( fassler et al . the outcome of c - myc expression is keratinocyte proliferation in both the sg and epidermis , leading to the postulate that c - myc might control the conversion of stem cells to so - called ta ( i.e. another signaling pathway likely to impact on more than one keratinocyte stem cell niche is notch , which controls selective cell fate determination through close - range interactions not only in the epidermis but also in the hair follicle and the sg ( yamamoto et al . thus , in the follicle bulge , stem cells enter the new hair cycle when threshold levels of stabilized -catenin are reached ( gat et al . , 1998 ; as outlined in this paper , growth factor receptor signaling also appears to affect different keratinocyte stem cell populations in distinct ways . in the precortex of the hair follicle , where wnt signaling is particularly high ( dasgupta and fuchs , 1999 ) , the wnt target gene jag1 is expressed , leading to active notch signaling in these cells , promoting proliferation and differentiation to produce hair ( estrach et al . conversely , in the absence of jag1 , hair follicles fail to form , but the epidermis is spared . increasing knowledge about these stem cell populations over the past 5 yr has begun to shed light on the similarities and differences among these niches . is the multipotency of stem cells in response to wounding a reflection of mechanical disruption of the niche or a response to growth / differentiation factors released in a wound response ? to what extent do the gene expression programs of other progenitor populations overlap with that of follicle stem cells , and do these similarities reflect their self - renewing , undifferentiated features ? the answers to these questions will bring us ever closer in the strife to understanding how stem cells are maintained in their undifferentiated , growth - restricted state and what prompts them to become activated , exit their niches , and embark upon distinct lineages .

senile plaques of amyloid- ( a ) and neurofibrillary tangles ( nft ) of tau are pathophysiological markers of alzheimer s disease ( ad ) . tau proteins include six different intracellular , intrinsically disordered isoforms consisting of two functional domains . the projection domain mediates interactions of microtubules with neural plasma membrane and cytoskeletal elements and is involved in signal transduction , while the tau microtubule binding pseudorepeat domain ( mtbr ) regulates microtubule assembly from tubulin ( for a comprehensive review , see ref ( 1 ) ) . aggregation of tau into nft is promoted in cases of hyperphosphorylation or deficiency in dephosphorylation . more than 37 mutations of tau isoforms have been shown to induce neurodegenerative diseases ( e.g. , frontotemporal dementia ) ; however , there are no tau mutations directly linked to any known familial form ad . while a loss of function hypothesis is often invoked to explain the role of tau aggregation in ad , the latter does not fully account for the etiology of the disease , and does not address the role of a in ad , or its possible interaction with tau . unlike tau which resides in the neurons , the a peptide is produced in the extracellular space from the proteolytic cleavage of a large transmembrane amyloid -protein precursor protein ( app ) . an amyloid cascade hypothesis has been proposed that stipulates that ad is caused by the aggregation of a , with -rich toxic oligomers or fibrillar aggregates signaling cell apoptosis or decreasing synaptic plasticity . however , this hypothesis has been challenged given the lack of strong correlation between the amount of a produced , or senile plaques deposited , and neuronal loss or cognitive impairment . the observation that a monomers or soluble oligomers permeate cell membranes and form highly functional multimeric aggregates capable of interfering with normal cellular activities is offering a new research direction in ad . yet another hypothesis , and the one that we will focus on in this work , is that the interaction of a and tau may be significant in ad . indeed , while the a peptide is produced in the extracellular space , soluble a oligomers have been shown to exist in the intracellular space as well and interact with a variety of proteins including tau . exposure of tau to a oligomers could lead to loss of microtubule integrity , formation of new , insoluble aggregates , or an enhancement of nft formation . tau has been shown to induce a toxicity in hippocampal neurons , while a-mediated tau phosphorylation has been discovered in both hippocampal and cholinergic neurons . the full length a is a 3943-residue peptide with a(140 ) and a(142 ) being the two most abundant isoforms . the full length tau , depending on its isoforms , can be as long as 400 residues , and studying complexes of a and tau poses a real challenge , both from an experimental and computational standpoint . we then turn in this work to the study of a model system that captures the essential features of a and tau assembly . we use a combination of ion - mobility mass spectrometry ( im - ms ) , atomic force microscopy ( afm ) , and computational modeling to investigate the interactions between an a and a tau fragment : a(2535 ) and tau(273284 ) . these two peptide fragments contain important regions of the full a and tau proteins , and we and others have studied early oligomer conformations and aggregation propensities of both peptides . a(2535 ) is an amphipathic peptide with similar aggregation propensity and toxicity to the full length a(142 ) . takashima et al . have shown that embryonic rat hippocampal neurons undergo progressive degeneration and that tau phosphorylation is enhanced after exposure to a(2535 ) , similar to the result obtained with a(140 ) by busciglio and co - workers . tau(273284 ) is in the second repeat ( r2 ) of mtbr and encompasses the phf6 * hexapeptide ( vqiink ) , one of the two hexapeptides known to play an important role in tau aggregation . it has also been shown experimentally that tau binds between the middle and c - terminal regions of a(142 ) ( the region of app inserted into the cell membrane ) . a recent theoretical work by nussinov and co - workers also suggests that a oligomers interact more strongly with r2 of mtbr than r3 or r4 . although r2 appears in some but not all tau isoforms ( encoded only by exon 10 of the mapt protein ) , changing the ratio of four - repeat tau to three - repeat tau is sufficient to cause serious degeneration in microtubule assembly . of note is that both peptides contain a hydrophobic stretch of six - residue sequence , i.e. , phf6 * in tau(273284 ) and gaiigl from a(2535 ) . these two hexapeptides have been shown to adopt steric zipper motifs that facilitate amyloid fibril formation . ( a ) full length four - repeat tau and location of phf6 * and the tau(273284 ) construct used in this study ( see larini et al . the longest tau isoforms contain either three or four imperfect repeats in the microtubule - binding pseudorepeat domain ( mtbr ) . ( b ) full length a(142 ) with different regions and the peptide sequence of a(2535 ) . all peptides studied here have acetylated n - termini and amidated c - termini . lyophilized samples of ac - tau(273284)-nh2 were purchased from genscript ( piscataway , nj , usa ) . the peptide was purified by reverse - phase high - performance liquid chromatography ( hplc ) and characterized by mass spectrometry and amino acid analysis to confirm the purity and integrity of the peptide ( > 95% purity ) . working stock solutions ( 2 mm for the ac - tau(273284)-nh2 and 500 m for ac - a(2535)-nh2 ) were prepared by dissolving the lyophilized peptide in filtered deionized water . this stock was divided into several tubes , flash - frozen in liquid nitrogen , and stored at 80 c until use . the concentration of peptide samples in im - ms and afm experiments is 200 m . louis , mo , usa ) and stocked at 1 mm concentration in water at room temperature . in im - ms experiments , stock solutions were diluted in water to a desired concentration , loaded into gold coated nano - electrospray ionization ( esi ) capillaries , and electrosprayed on a home - built instrument . ions were pulled through a 200 cm long , helium filled drift cell under the influence of a weak electrical field and the drag force created from collisions with buffer gas ions . particular species with specific m / z can be mass - selected , and their arrival time distributions can be measured at different pressure to drift voltage ratios ( p / v ) , allowing the determination of reduced mobility k0 and experimental collision cross - sections . afm images were collected using an asylum mfp-3d - sa system ( asylum research , santa barbara , ca , usa ) . images were processed using igor pro software and were modified by masking fibrils and then applying a first - order flatten to height images ( magic mask in mfp3d software ) . all height and length measurements of single fibrils and granular aggregates were conducted using igor pro , and each reported value is the average of 20 measurements . temperature - based replica exchange molecular dynamics ( t - remd ) simulations were performed using the gromacs 4.6.3 package with a combination of the opls - aa force field and tip3p water . in these simulations , the initial conformation of ac - a(2535)-nh2 is a -hairpin obtained from t - remd simulations of the same peptide using the same force field ( data not shown ) , and also consistent with a previous simulation of free terminal a(2535 ) by larini and shea . an initial ac - tau(273284)-nh2 was chosen from the t - remd simulations described in ref ( 30 ) . structures obtained from these simulations were minimized in the gas phase to mimic the dehydration process that occurred when solution - phase structures enter a solvent - free environment . their collision cross - sections were computed using the trajectory method available from the mobcal package . all im - ms experiments are performed on a home - built im - ms instrument described previously . the mass spectra of ac - a(2535)-nh2 , ac - tau(273284)-nh2 , and a mixture of both at 1:1 ratio are shown in figure 2 . water is used in place of buffer to slow down aggregation kinetics , as well as minimize the charge screening effect of buffer ions that may affect aggregation . the mass spectrum of ac - a(2535)-nh2 ( figure 2a ) contains two major peaks at 550 m / z ( n / z 1/2 ) and 1101 m / z ( n / z 1/1 ) , where n is the a(2535 ) oligomer size and z is the charge . less intense peaks are observed at 734 m / z ( n / z 2/3 ) , 826 m / z ( n / z 3/4 ) , and 1407 m / z ( the arrival time distributions ( atds ) at m / z other than 1101 contain either a single oligomer species or a single family of conformations ( see supporting information figure s1a c , e ) . atds of the n / z 1/1 peak contain multiple features from dimer to hexamer ( supporting information figure s1d ) . the progression of oligomer cross - sections as a function of oligomer size n ( see figure 3a ) shows a positive deviation from the isotropic oligomer model starting at the trimer stage , indicating the formation of extended oligomers . based on our earlier studies on free terminal a(2535 ) formation of -rich oligomers is consistent with the fibril formation propensity of the peptide probed by afm ( see the next section ) . mass spectra of ( a ) ac - a(2535)-nh2 , ( b ) ac - tau(273284)-nh2 , and ( c , d ) a mixture of the two peptides at 1:1 ratio . each peak is annotated with [ n + k]/z where n is the oligomer number of ac - a(2535)-nh2 , k is the oligomer number of ac - tau(273284)-nh2 , and z is the net charge of the complex . a representative atd of the peak at 705 m / z ( k / z 1/2 ) is shown in panel b to illustrate that dimer is the largest tau oligomer formed under these conditions . ( a ) experimental cross - section of ac - a(2535)-nh2 ( exp , ) as a function of n. ( b ) correlated cross - section of ac - a(2535)-nh2 + ac - tau(273 - 284)-nh2 ( corr , ) obtained from the experimental cross - section of heteroligomers . the isotropic cross - sections of ac - a(2535)-nh2 are shown by the dashed line . the mass spectrum of ac - tau(273284)-nh2 ( figure 2b ) consists of two peaks at 470 m / z ( k / z 1/3 ) and 705 m / z ( k / z 1/2 ) where k is the oligomer number of ac - tau(273284)-nh2 . peaks at higher m / z are not observed , consistent with the fact that peptide oligomerization occurs at a much slower rate compared to the same peptide in buffer solution . the dimer is the largest oligomer observed ( the atd of the peak at 705 m / z is shown in the inset of figure 2b ) . in the absence of aggregation - promoting factors ( e.g. , heparin ) , this peptide is unable to form any structural aggregates . furthermore , previous simulations and experimental cross - sections ( exp = 310313 for z = + 2 and + 3 ) suggest that the tau monomer is in a relatively compact conformation ( i.e. , -turn or -hairpin ) . simulations suggest that aggregation is controlled by a conformational transition of the monomers which later associate to form extended dimers . the mass spectrum of the mixture ( figure 2c ) shows strong peaks for ac - tau(273284)-nh2 monomer , but the peaks associated with ac - a(2535)-nh2 are strongly suppressed , replaced by several new peaks composed of peptides from both fragments . these mass spectral peaks are annotated with [ n + k]/z where n is the oligomer number of ac - a(2535)-nh2 , k is the oligomer number of ac - tau(273284)-nh2 , and z is the net charge of the complex . figure 3b qualitatively represents the correlated cross - section of ac - a(2535)-nh2 monomer and oligomers extracted from the experimental cross - section of the heteroligomers ( see supporting information section s2.1 and table s1 ) , assuming that the tau conformation is relatively compact ( as indicated from simulation ) . we note that corr ( n ) can be considered as representative values for the ratios exp(n , k)/iso(n , k ) between the experimental cross - sections of heteroligomers and the isotropic cross - section model of heteroligomers . hence if exp(n , k ) iso(n , k ) , we expect a large deviation of corr(n ) from the isotropic cross - section of a(2535 ) oligomer , iso(n ) , indicating that the heteroligomers are nonisotropic . under this assumption , we observe that the cross - section deviation occurs as early as heterodimers ( n = 1 , k = 1 ) and the trend continues for larger oligomers ( n = 3 , k = 2 and n = 2 , k = 3 ) . this conclusion is generally true regardless of our assumption that the tau conformation is compact . however , the largest heteroligomers observed in this im - ms experiment are hexamers , whereas , in the pure ac - a(2535)-nh2 sample , hexamers and nonamers can be unambiguously detected . thus , it appears that ac - tau(273284)-nh2 can trap ac - a(2535)-nh2 in smaller oligomeric forms . the significant intensities of the heterodimer peaks ( i.e. , [ 1 + 1]/4 at 627 m / z and [ 1 + 1]/3 at 837 m / z ) indicate that the tau fragment has a high affinity to bind ac - a(2535)-nh2 monomer relative to forming homo - tau dimers ( kd in micromolar range ; see supporting information section s2.2 ) . t - remd simulation of the heterodimer reveals the existence of at least two major families of structures : compact structures ( 40% , in which both tau and a peptide chains are relatively compact ) and extended structures ( 10% , where both chains are extended in antiparallel configuration ) and other structures in between ( see supporting information figure s3 ) . in terms of cross - sections , two populated clusters of extended antiparallel heterodimers have average cross - sections of theory = 553 and 525 ( clusters f and h shown in supporting information figure s3 , respectively ) , which are in good agreement with the [ 1 + 1]/4 species with the cross - section of exp = 559 . similarly , the most populated compact heterodimer clusters have average cross - sections of theory = 496 and 493 ( clusters a and b , respectively ) , which agree well with the [ 1 + 1]/3 species , exp = 492 . we note that the theoretical cross - section obtained from the trajectory method available from the mobcal package , from a specific structure , is good to less than 5% . figure 4a shows a schematic representation of the alignment of two tau(273284 ) peptides , a mixed tau(273284)/a(2535 ) , and two a(2535 ) peptides within a single - layer ideal -sheet . in the context of a fragment such as tau(273284 ) or full length tau , the hydrophobic phf6 * region is blocked between k274 at one end and k280/281 at the other end , making associations between the hydrophobic phf6 * cores more restricted . in the ac - tau(273284)-nh2 extended dimer conformation probed in the simulation of larini et al . , the two phf6 * segments are shifted away from each other . the extended dimer is stabilized by a weak hydrogen bonding network between backbone atoms of hydrophobic residues such as isoleucine and hydrophilic charged residues such as lysine , consistent with the scheme shown in figure 4a . proposed sequence alignments of the peptides in ( a ) single extended -sheet and ( b ) steric zipper conformation . since the two phf6 * segments are not within an optimal distance to create a dry interface , the extended dimer structure does not support steric zipper formation optimal for fibrillization . on the other hand , the gaiigl segment of ac - a(2535)-nh2 can behave similarly to phf6 * , and the adjacent residues around this segment are less charged than those of tau . in addition , since ac - a(2535)-nh2 is one residue shorter than ac - tau(273284)-nh2 and the gaiigl segment is near the c - terminus , figure 4a also proposes that a better alignment between gaiigl and phf6 * can be achieved in antiparallel fashion . simulations suggest that both the homodimer of tau(273284 ) and of a(2535 ) and the heterodimer of tau(273284 ) and a(2535 ) can adopt antiparallel , extended structures with the hydrogen bonding networks shown in figure 4a . the distributions of distances between hydrophobic cores ( between one phf6 * and another phf6 * in homo - tau(273284 ) dimer , and between phf6 * and gaiigl in the heterodimer ) suggest that the hydrophobic cores in the heterodimer can interact more strongly ( i.e. , within a 5.0 distance ) than phf6 * in the homo - tau(273284 ) dimer ( see supporting information figure s2 and figure 5c ) . according to figure 4b , the interactions between two -sheets within a heterodimer steric zipper maintains interactions at the dry interface similar to when phf6 * is considered alone , with no charged residues in the dry interface . the homo - tau(273284 ) steric zipper , on the other hand , is much less stable , since it has to accommodate charged residues such as lysine and aspartic acid within the dry interface and hydrophobic residues such as isoleucine and leucine at the wet interface . therefore , it is reasonable to believe that interactions between ac - a(2535)-nh2 and ac - tau(273284)-nh2 can promote -rich oligomers better than when ac - tau(273284)-nh2 is incubated separately . representative structures of the most populated extended clusters obtained from the simulations of ( a ) blocked terminal homo - tau(273284 ) dimers ( see ref ( 30 ) ) , ( b ) free terminal homo- a(2535 ) trimers ( see ref ( 31 ) ) and , ( c ) blocked terminal hetero - tau(273284)/a(2535 ) dimers . the interacting hydrophobic cores are highlighted using the same color codes shown in figure 4 . the isoleucine pairs within the hydrophobic stretches are shown in licorice . as previously examined from both experiment and theory , the aggregation process of a(2535 ) involves a structural transition from dimer to trimer stages , in which a compact dimer composed of mainly -hairpin monomers converts into extended -rich structures , as shown in figure 5b . in these populated -sheet oligomers ( trimer or larger ) the hydrophobic cores are nkgaii whose locations are more toward the middle of the chains , providing access to steric zipper formation , and eventually fibril formation . nkgaii -sheets can interact in both face - to - face and back - to - back fashions ( protein data bank ( pdb ) i d 3q2x ) . due to the versatility of these interaction motifs , it is likely that these nkgaii segments can provide a strong steric zipper interface for a(2535 ) to quickly aggregate into fibrils . the gaiigl steric zipper ( pdb i d 3pzz ) is composed of antiparallel -sheets interacting face - to - back . this class 6 zipper is often found in weakly aggregating systems such as the islet amyloid polypeptide fragment nflvhss ( pdb i d 3fth ) , and the enkephalin mutants yvvfv ( 4olr ) and yvvfl ( 4onk ) . thus , we can predict that the heterosystem of a(2535 ) and tau(273284 ) stabilized by phf6*/gaiigl interactions would self - assemble with slower kinetics into a different aggregate morphology as compared to pure a(2535 ) . in figure 2c , two types of heteroligomers larger than dimers are observed : ( 1 ) those with more a than tau peptide chains , which will be referred to as hetero - a oligomers and ( 2 ) those with more tau than a , referred to as hetero - tau oligomers . the hetero - a oligomer peaks ( i.e. , [ 2 + 1]/4 at 903 m / z , [ 3 + 2]/7 at 875 m / z , and [ 3 + 2]/6 at 1016 m / z ) are abundant in intensity indicating that ac - tau(273284)-nh2 can effectively replace ac - a(2535)-nh2 peptide chains to form dimer , trimer , and pentamer , and such processes are favorable . similar hetero - tau oligomers are also observed ( i.e. , [ 1 + 2]/4 at 979 m / z and [ 2 + 3]/5 at 1280 m / z ) . at first glance , the compositions of these hetero - tau oligomers appear to be similar to those of hetero - a oligomers , which may suggest that the a and tau peptides can randomly associate to form large oligomers . however , as seen in figure 6a , c , the atds of hetero - a oligomers ( i.e. , trimer and pentamer ) present multiple features and a variety of compact and extended conformations with similar intensities . on the other hand , the atds of hetero - tau oligomers contain a dominant feature with few minor features ( figure 6b , d ) . a possible explanation is that since homoligomers of ac - tau(273284)-nh2 larger than dimers are not stable and populated in water , the number of possible arrangements that hetero - tau oligomers can adopt should always be less than that of hetero - a oligomers . representative atds of hetero - a and hetero - tau trimer ( a , b ) and pentamer ( c , d ) peaks . each feature is annotated with an experimental cross - section ( , ) . possible configurations of the heteroligomers are shown with ac - a(2535)-nh2 in red and ac - tau(273284)-nh2 in blue . the hetero - a trimer atd has two peaks with cross - sections of exp = 673 and 706 which mechanistically can be assigned to a trimer composed of two a monomers and a tau monomer intercalating in between ( i.e. , aba ) and a trimer of an a dimer and a tau monomer ( i.e. , aab configuration where a is an ac - a(2535)-nh2 and b is an ac - tau(273284)-nh2 monomer ) . it is worth noting that if both aba and aab adopt similar conformations ( i.e. , extended structures ) then the cross - section (aab ) should be larger than that of (aba ) , since the former structure would have more tau side chains exposed to the outside ( tau is larger in size than a ) , which increases the collision cross - section . both features are populated since the a monomer and dimer in the sample are abundant ( see supporting information figure s1 ) . on the other hand , the hetero - tau trimer has three features , but the shortest and longest arrival time features are very minor . statistically the bba / abb structures are more likely than the bab structures ( i.e. , the bba / abb can be formed starting with either ab heterodimer or bb homodimer , whereas the bab can only be formed from the heterodimer ) suggesting this is the structure of the major peak , but this can not be determined with certainty . global stabilization of each quaternary structure often involves not only strong pairwise interactions between adjacent chains but also a network of noncovalent contacts among different subunits . consequently , the preceding mechanistic description of heteroligomers appears to be too simple . however , the t - remd simulations of hetero - a and hetero - tau trimers qualitatively support such a description . as seen in figure 7a , the radial distribution function ( rdf g(r ) ) computed for the distances between the a monomers within the hetero - a trimers populates several peaks near 0.5 and 1.0 nm . the distance of 0.5 nm indicates that the two ac - a(2535)-nh2 peptides are adjacent to each other ( aab conformations ) , whereas that of 1.0 nm suggests the tau peptide is intercalating in between ( aba conformations ) . clustering analysis shows that the conformation of each chain within a structure type can be either compact or extended , which gives rise to a diverse set of conformations . the representative structures and the theoretical cross - sections of the major clusters obtained from the simulation of hetero - a trimer are shown in supporting information figure s4 . while the tau monomer is relatively extended , the two ac - a(2535)-nh2 chains can adopt either -hairpin - like or extended structures . although the largest experimental cross - section of the hetero - a trimer is 10% larger than the reported theoretical cross - sections , if we consider the broad distributions of theoretical cross - sections , the theory and experiment are in good agreement kinetically stable oligomers have been observed and discussed in our recent study of free terminal a(2535 ) aggregation . radial distribution functions rdfs g(r ) computed for the distances r ( nm ) ( a ) between the ac - a(2535)-nh2 monomers within the hetero - a trimer and ( b ) between the ac - tau(273284)-nh2 monomers within the hetero - tau trimer . for the hetero - tau trimer , all of the dominant peaks of g(r ) are located near 0.5 nm , suggesting that the bba conformations are dominant , in agreement with the assignment made in figure 6b . clustering analysis of the hetero - tau trimers obtained from t - remd reveals that a majority of extended structures is of bba type . some compact , disordered structures are also observed ( see supporting information figure s5 ) . as the size of the heteroligomers grows , a clear distinction between hetero - a and hetero - tau oligomers is observed in the im - ms data . for the heteropentamer , those of a contain multiple features in the atds corresponding to different species ( i.e. , there are six possible configurations of hetero - a pentamer ) . since the size of the systems is expensive for high - level md simulations , we constructed a series of fully extended hetero - a pentamers corresponding to the configurations shown schematically in figure 6 ( see supporting information figure s6 for the structures obtained from simulation and their theoretical cross - sections ) . these antiparallel -sheet models have cross - sections in good agreement with the experiment cross - sections for both charge states ( z = + 6 and + 7 ) , suggesting the hetero - a pentamers adopt extended , -sheet - like structures . in contrast , the formation of the single hetero - tau pentamer is conformationally restricted , as manifested in the atd containing only one narrow feature . this feature has a smaller cross - section and lower charge state than the hetero - a pentamers , suggesting that the conformation is relatively more compact . both the im - ms data and the t - remd simulations support extended conformations of both ac - a(2535)-nh2 and ac - tau(273284)-nh2 as early as the heterodimer stage . figure 8 shows the differences in quantities and morphologies of aggregates in the pure a(2535 ) , tau(273284 ) , and mixture samples . from these data , we conclude that the formation of heteroligomers does not lead to enhanced fibril formation . the behavior of ac - tau(273284)-nh2 is similar to an inhibitor affecting the aggregation process of ac - a(2535)-nh2 , in which tau monomer ( and some dimer ) competitively binds to ac - a(2535)-nh2 monomer and oligomers . for the sample of pure ac - a(2535)-nh2 , abundant fibrillar aggregates are observed ( figure 8a and supporting information figure s7c f ) , whereas less fibrils but more granular aggregates are seen in the mixture sample ( figure 8b ) and finally no aggregates are found in the pure tau(273284 ) sample ( figure 8c ) . this raises two questions : ( 1 ) what is the nature of the granular aggregates and fibril - like structures observed in the afm image of the mixture ? ( 2 ) through which mechanisms does ac - tau(273284)-nh2 limit the strong aggregation of ac - a(2535)-nh2 and why do the extended conformations of heteroligomers , observed by experiment and by theoretical modeling , not promote abundant formation of fibrils ? representative afm images of ( a , b ) 1 week incubated 200 m ac - a(2535)-nh2 in water without ac - tau(273284)-nh2 and with ac - tau(273284)-nh2 at the ratio of 1:1 and ( c ) 2 week incubated 200 m ac - tau(273284)-nh2 in water . to answer the first question the granular aggregates can be either ( a ) homo - tau aggregates , ( b ) early globular aggregates of homo - a(2535 ) , or ( c ) a type of new aggregate produced by the heteroligomers . granular aggregates have been observed as intermediates in some tau fragment aggregation but not specifically for ac - tau(273284)-nh2 in the absence of aggregation - promoting factor ( see figure 8c ) . in the presence of 11 kda polydisperse heparin at 4:1 peptide : heparin ratio , aggregation of ac - tau(273284)-nh2 in water occurs within 1 h to form abundant well - defined aggregates ( see supporting information figure s7a , b ) and no granular aggregates are detected . thus , the granular aggregates observed in the mixture sample should not be from the tau itself ; this eliminates option a of homo - tau oligomers . we note that , in our experiment , fresh ac - a(2535)-nh2 forms large , -rich oligomers within a short time after sample preparation and forms fibrillar aggregates within an hour ( supporting information figure s7c , d ) , suggesting that there is not a noticeable morphology transition occurring during the aggregation of ac - a(2535)-nh2 . a few small aggregates are sparsely distributed at the edges of the afm slide ( supporting information figure s7g , h ) . however , these aggregates vary widely in length and have on average a lower height than the granular aggregates observed in the mixture sample which suggests a different composition ( see table 1 ) . we consider the possibility that ac - tau(273284)-nh2 acts as an inhibitor to efficiently disassemble ac - a(2535)-nh2 fibrils into globular aggregates . in other words , the interactions between ac - tau(273284)-nh2 and ac - a(2535)-nh2 must be significantly stronger than a(2535 ) with itself in order to successfully compete with existing interactions between a chains inside the fibrils . to examine whether ac - tau(273284)-nh2 can disaggregate ac - a(2535)-nh2 fibrils , fresh tau was added to a week - old sample containing extensive fibrillar aggregates of ac - a(2535)-nh2 . it is clear that ac - tau(273284)-nh2 does not disassemble ac - a(2535)-nh2 aggregates , which eliminates b as an option ( see supporting information figure s8 ) . finally , we turn to option c , the possibility of forming mixed aggregates . as seen in im - ms and in simulations it is hence reasonable to assume that ac - tau(273284)-nh2 can decorate existing ac - a(2535)-nh2 oligomers and protofibrils by occasionally adding on the growing aggregate in place of new a chains . the more a that are replaced by tau , the less prone the oligomers are to grow further since there are structural differences between hetero - a and hetero - tau oligomers . compact hetero - tau and some hetero - a oligomers can trap ac - a(2535)-nh2 oligomers in -hairpin or -turn conformations , leading to the formation of granular aggregates . since ac - tau(273284)-nh2 monomer is larger than ac - a(2535)-nh2 monomer in size , the fibrils grown from heteroligomers would be more likely to have larger dimensions than the pure ac - a(2535)-nh2 fibrils . additional afm measurements show that both heights and lengths of the fibrils and granular features formed in the mixture sample are greater than those of the pure ac - a(2535)-nh2 ( see table 1 ) . in these measurements , only the dimension of individual fibrils is taken into account , and the larger features which are probably clusters of several fibrils are not considered . for the second question , we first consider the mechanisms through which a(2535 ) can aggregate . the simulations of ac - a(2535)-nh2 by wei et al . show the existence of a v - shaped parallel dimer , an out of register antiparallel dimer , and an intertwined -hairpin conformation at small populations as potential seeds for amyloid formation . the major driving force for the first two families of structures is hydrophobic interactions between gaiigl segments . larini and shea , in simulations of a(2535 ) , suggest that the transition from -hairpin a(2535 ) monomer to extended structures of oligomers is regulated by a competition between electrostatics and hydrophobic effects . electrostatics dominate for dimer formation to produce mainly compact structures composed of two tilted -hairpin monomers , whereas hydrophobic interactions facilitate the -sheet trimers and tetramers . they also propose the role of -hairpin structure in a kinetic growth model as a means to stabilize flat -sheets and prevent newly formed oligomers from hydrophobic collapse . when ac - tau(273284)-nh2 and ac - a(2535)-nh2 form extended dimers , or ac - tau(273284)-nh2 intercalates between ac - a(2535)-nh2 hairpins at the trimer or pentamer , tau limits the influences of -hairpin structure in the growth of a(2535 ) fibrils and subsequently decreases the aggregation propensity . by replacing the hydrophobic core of nkgaii / nkgaii with phf6*/gaiigl , and limiting the role of a(2535 ) hairpins , the hetero - a oligomers are relatively extended , but not as aggregation - prone as the homo - a oligomers , due to the mismatches in side - chain interdigitations and their heterogeneity . the presence of a few tau chains within the heterocomplexes may decrease the ability to self - associate , but the system is still able to grow further into fibrils , which is consistent with what has been observed in the afm image of the mixture ( figure 8b ) . it has been long predicted that heterotypic -sheet and steric zipper structures can exist . eisenberg and co - workers have shown that the macroscopic morphologies and abundances of fibrillar aggregates formed in binary mixtures of different a segments are distinct from the component segments . however , no x - ray crystal structures of heterotypic -sheet and steric zipper have been solved . one of the possible explanations is that even in a binary system of two short peptides , the tendency of homotypic structure formation is dominant , and crystallization is highly dependent upon the kinetics and growth conditions which are very selective . the two factors often promote the crystallization of one type or the other , but not heterotypic crystals . thus , im - ms appears to be a versatile technique to approach this problem . previous work has shown that the experimental cross - sections of aggregating peptide oligomers are in good agreement with x - ray crystal structures . furthermore , even a small amount of heteroligomers can be detected with this technique . ( 1 ) using a combination of experimental and theoretical techniques , we are able to identify the interactions promoting and stabilizing ac - a(2535)-nh2/ac - tau(273284)-nh2 heteroligomers . the formation of heteroligomers triggers conformation transitions from compact to extended forms for both peptides as early as the dimer stage . ac - tau(273284)-nh2 monomer prefers to associate with an ac - a(2535)-nh2 monomer over another tau monomer as shown by the populations of heterodimer and homo - tau dimer in the mixture . the intercalation of ac - tau(273284)-nh2 monomers between ac - a(2535)-nh2 monomers ( and oligomers ) is a probable mechanism allowing large heteroligomers to grow with a wide range of configurations and conformations . ( 2 ) the heterogeneity of the heteroligomers and mismatches in side - chain interdigitations limit ( but do not fully eliminate ) aggregation . the hetero - tau oligomers can be trapped at relatively small sizes , producing granular aggregates . on the other hand , the architectures of hetero - a oligomers contain -rich content , which later can grow into heterofibrils , as illustrated in figure 9 . ( 3 ) a possible source of toxicity of early a oligomers may lie in their ability to bind tau monomers , thereby decreasing the population of tau needed to regulate microtubule dynamics . other possible pathways to toxicity lie in the structures of the heteroligomers formed by the interactions of different tau to a fragments , which awaits a further systematic investigation . ( 4 ) im - ms can probe conformations and abundances of heteroligomers , demonstrating its capacity as a promising screening method to identify important segments from different aggregating proteins ( e.g. , a , tau , prion , islet amyloid polypeptide ) that can interact with each other to form heterotypic structures . these studies will provide insights into synergy effects of protein markers responsible for aggregation - related diseases , which are currently challenging to unravel . illustration of how fibrillar and granular aggregates can be formed in the mixture of ac - a(2535)-nh2 and ac - tau(273284)-nh2 . the morphologies of the aggregates are determined by the population of the individual peptides .

we have investigated at the oligomeric level interactions between a(2535 ) and tau(273284 ) , two important fragments of the amyloid- and tau proteins , implicated in alzheimer s disease . we are able to directly observe the coaggregation of these two peptides by probing the conformations of early heteroligomers and the macroscopic morphologies of the aggregates . ion - mobility experiment and theoretical modeling indicate that the interactions of the two fragments affect the self - assembly processes of both peptides . tau(273284 ) shows a high affinity to form heteroligomers with existing a(2535 ) monomer and oligomers in solution . the configurations and characteristics of the heteroligomers are determined by whether the population of a(2535 ) or tau(273284 ) is dominant . as a result , two types of aggregates are observed in the mixture with distinct morphologies and dimensions from those of pure a(2535 ) fibrils . the incorporation of some tau into -rich a(2535 ) oligomers reduces the aggregation propensity of a(2535 ) but does not fully abolish fibril formation . on the other hand , by forming complexes with a(2535 ) , tau monomers and dimers can advance to larger oligomers and form granular aggregates . these heteroligomers may contribute to toxicity through loss of normal function of tau or inherent toxicity of the aggregates themselves .

Introduction Materials and Methods Results and Discussion Summary and Conclusions

senile plaques of amyloid- ( a ) and neurofibrillary tangles ( nft ) of tau are pathophysiological markers of alzheimer s disease ( ad ) . while a loss of function hypothesis is often invoked to explain the role of tau aggregation in ad , the latter does not fully account for the etiology of the disease , and does not address the role of a in ad , or its possible interaction with tau . yet another hypothesis , and the one that we will focus on in this work , is that the interaction of a and tau may be significant in ad . we use a combination of ion - mobility mass spectrometry ( im - ms ) , atomic force microscopy ( afm ) , and computational modeling to investigate the interactions between an a and a tau fragment : a(2535 ) and tau(273284 ) . these two peptide fragments contain important regions of the full a and tau proteins , and we and others have studied early oligomer conformations and aggregation propensities of both peptides . a(2535 ) is an amphipathic peptide with similar aggregation propensity and toxicity to the full length a(142 ) . tau(273284 ) is in the second repeat ( r2 ) of mtbr and encompasses the phf6 * hexapeptide ( vqiink ) , one of the two hexapeptides known to play an important role in tau aggregation . , phf6 * in tau(273284 ) and gaiigl from a(2535 ) . ( b ) full length a(142 ) with different regions and the peptide sequence of a(2535 ) . the peptide was purified by reverse - phase high - performance liquid chromatography ( hplc ) and characterized by mass spectrometry and amino acid analysis to confirm the purity and integrity of the peptide ( > 95% purity ) . in these simulations , the initial conformation of ac - a(2535)-nh2 is a -hairpin obtained from t - remd simulations of the same peptide using the same force field ( data not shown ) , and also consistent with a previous simulation of free terminal a(2535 ) by larini and shea . the mass spectrum of ac - a(2535)-nh2 ( figure 2a ) contains two major peaks at 550 m / z ( n / z 1/2 ) and 1101 m / z ( n / z 1/1 ) , where n is the a(2535 ) oligomer size and z is the charge . the progression of oligomer cross - sections as a function of oligomer size n ( see figure 3a ) shows a positive deviation from the isotropic oligomer model starting at the trimer stage , indicating the formation of extended oligomers . based on our earlier studies on free terminal a(2535 ) formation of -rich oligomers is consistent with the fibril formation propensity of the peptide probed by afm ( see the next section ) . mass spectra of ( a ) ac - a(2535)-nh2 , ( b ) ac - tau(273284)-nh2 , and ( c , d ) a mixture of the two peptides at 1:1 ratio . simulations suggest that aggregation is controlled by a conformational transition of the monomers which later associate to form extended dimers . the mass spectrum of the mixture ( figure 2c ) shows strong peaks for ac - tau(273284)-nh2 monomer , but the peaks associated with ac - a(2535)-nh2 are strongly suppressed , replaced by several new peaks composed of peptides from both fragments . figure 3b qualitatively represents the correlated cross - section of ac - a(2535)-nh2 monomer and oligomers extracted from the experimental cross - section of the heteroligomers ( see supporting information section s2.1 and table s1 ) , assuming that the tau conformation is relatively compact ( as indicated from simulation ) . hence if exp(n , k ) iso(n , k ) , we expect a large deviation of corr(n ) from the isotropic cross - section of a(2535 ) oligomer , iso(n ) , indicating that the heteroligomers are nonisotropic . under this assumption , we observe that the cross - section deviation occurs as early as heterodimers ( n = 1 , k = 1 ) and the trend continues for larger oligomers ( n = 3 , k = 2 and n = 2 , k = 3 ) . however , the largest heteroligomers observed in this im - ms experiment are hexamers , whereas , in the pure ac - a(2535)-nh2 sample , hexamers and nonamers can be unambiguously detected . , [ 1 + 1]/4 at 627 m / z and [ 1 + 1]/3 at 837 m / z ) indicate that the tau fragment has a high affinity to bind ac - a(2535)-nh2 monomer relative to forming homo - tau dimers ( kd in micromolar range ; see supporting information section s2.2 ) . in terms of cross - sections , two populated clusters of extended antiparallel heterodimers have average cross - sections of theory = 553 and 525 ( clusters f and h shown in supporting information figure s3 , respectively ) , which are in good agreement with the [ 1 + 1]/4 species with the cross - section of exp = 559 . figure 4a shows a schematic representation of the alignment of two tau(273284 ) peptides , a mixed tau(273284)/a(2535 ) , and two a(2535 ) peptides within a single - layer ideal -sheet . in the context of a fragment such as tau(273284 ) or full length tau , the hydrophobic phf6 * region is blocked between k274 at one end and k280/281 at the other end , making associations between the hydrophobic phf6 * cores more restricted . on the other hand , the gaiigl segment of ac - a(2535)-nh2 can behave similarly to phf6 * , and the adjacent residues around this segment are less charged than those of tau . simulations suggest that both the homodimer of tau(273284 ) and of a(2535 ) and the heterodimer of tau(273284 ) and a(2535 ) can adopt antiparallel , extended structures with the hydrogen bonding networks shown in figure 4a . the distributions of distances between hydrophobic cores ( between one phf6 * and another phf6 * in homo - tau(273284 ) dimer , and between phf6 * and gaiigl in the heterodimer ) suggest that the hydrophobic cores in the heterodimer can interact more strongly ( i.e. according to figure 4b , the interactions between two -sheets within a heterodimer steric zipper maintains interactions at the dry interface similar to when phf6 * is considered alone , with no charged residues in the dry interface . the homo - tau(273284 ) steric zipper , on the other hand , is much less stable , since it has to accommodate charged residues such as lysine and aspartic acid within the dry interface and hydrophobic residues such as isoleucine and leucine at the wet interface . representative structures of the most populated extended clusters obtained from the simulations of ( a ) blocked terminal homo - tau(273284 ) dimers ( see ref ( 30 ) ) , ( b ) free terminal homo- a(2535 ) trimers ( see ref ( 31 ) ) and , ( c ) blocked terminal hetero - tau(273284)/a(2535 ) dimers . as previously examined from both experiment and theory , the aggregation process of a(2535 ) involves a structural transition from dimer to trimer stages , in which a compact dimer composed of mainly -hairpin monomers converts into extended -rich structures , as shown in figure 5b . this class 6 zipper is often found in weakly aggregating systems such as the islet amyloid polypeptide fragment nflvhss ( pdb i d 3fth ) , and the enkephalin mutants yvvfv ( 4olr ) and yvvfl ( 4onk ) . thus , we can predict that the heterosystem of a(2535 ) and tau(273284 ) stabilized by phf6*/gaiigl interactions would self - assemble with slower kinetics into a different aggregate morphology as compared to pure a(2535 ) . in figure 2c , two types of heteroligomers larger than dimers are observed : ( 1 ) those with more a than tau peptide chains , which will be referred to as hetero - a oligomers and ( 2 ) those with more tau than a , referred to as hetero - tau oligomers . at first glance , the compositions of these hetero - tau oligomers appear to be similar to those of hetero - a oligomers , which may suggest that the a and tau peptides can randomly associate to form large oligomers . on the other hand , the atds of hetero - tau oligomers contain a dominant feature with few minor features ( figure 6b , d ) . possible configurations of the heteroligomers are shown with ac - a(2535)-nh2 in red and ac - tau(273284)-nh2 in blue . on the other hand , the hetero - tau trimer has three features , but the shortest and longest arrival time features are very minor . the distance of 0.5 nm indicates that the two ac - a(2535)-nh2 peptides are adjacent to each other ( aab conformations ) , whereas that of 1.0 nm suggests the tau peptide is intercalating in between ( aba conformations ) . as the size of the heteroligomers grows , a clear distinction between hetero - a and hetero - tau oligomers is observed in the im - ms data . since the size of the systems is expensive for high - level md simulations , we constructed a series of fully extended hetero - a pentamers corresponding to the configurations shown schematically in figure 6 ( see supporting information figure s6 for the structures obtained from simulation and their theoretical cross - sections ) . both the im - ms data and the t - remd simulations support extended conformations of both ac - a(2535)-nh2 and ac - tau(273284)-nh2 as early as the heterodimer stage . figure 8 shows the differences in quantities and morphologies of aggregates in the pure a(2535 ) , tau(273284 ) , and mixture samples . from these data , we conclude that the formation of heteroligomers does not lead to enhanced fibril formation . the behavior of ac - tau(273284)-nh2 is similar to an inhibitor affecting the aggregation process of ac - a(2535)-nh2 , in which tau monomer ( and some dimer ) competitively binds to ac - a(2535)-nh2 monomer and oligomers . for the sample of pure ac - a(2535)-nh2 , abundant fibrillar aggregates are observed ( figure 8a and supporting information figure s7c f ) , whereas less fibrils but more granular aggregates are seen in the mixture sample ( figure 8b ) and finally no aggregates are found in the pure tau(273284 ) sample ( figure 8c ) . this raises two questions : ( 1 ) what is the nature of the granular aggregates and fibril - like structures observed in the afm image of the mixture ? ( 2 ) through which mechanisms does ac - tau(273284)-nh2 limit the strong aggregation of ac - a(2535)-nh2 and why do the extended conformations of heteroligomers , observed by experiment and by theoretical modeling , not promote abundant formation of fibrils ? to answer the first question the granular aggregates can be either ( a ) homo - tau aggregates , ( b ) early globular aggregates of homo - a(2535 ) , or ( c ) a type of new aggregate produced by the heteroligomers . granular aggregates have been observed as intermediates in some tau fragment aggregation but not specifically for ac - tau(273284)-nh2 in the absence of aggregation - promoting factor ( see figure 8c ) . in the presence of 11 kda polydisperse heparin at 4:1 peptide : heparin ratio , aggregation of ac - tau(273284)-nh2 in water occurs within 1 h to form abundant well - defined aggregates ( see supporting information figure s7a , b ) and no granular aggregates are detected . thus , the granular aggregates observed in the mixture sample should not be from the tau itself ; this eliminates option a of homo - tau oligomers . a few small aggregates are sparsely distributed at the edges of the afm slide ( supporting information figure s7g , h ) . however , these aggregates vary widely in length and have on average a lower height than the granular aggregates observed in the mixture sample which suggests a different composition ( see table 1 ) . in other words , the interactions between ac - tau(273284)-nh2 and ac - a(2535)-nh2 must be significantly stronger than a(2535 ) with itself in order to successfully compete with existing interactions between a chains inside the fibrils . as seen in im - ms and in simulations it is hence reasonable to assume that ac - tau(273284)-nh2 can decorate existing ac - a(2535)-nh2 oligomers and protofibrils by occasionally adding on the growing aggregate in place of new a chains . compact hetero - tau and some hetero - a oligomers can trap ac - a(2535)-nh2 oligomers in -hairpin or -turn conformations , leading to the formation of granular aggregates . additional afm measurements show that both heights and lengths of the fibrils and granular features formed in the mixture sample are greater than those of the pure ac - a(2535)-nh2 ( see table 1 ) . larini and shea , in simulations of a(2535 ) , suggest that the transition from -hairpin a(2535 ) monomer to extended structures of oligomers is regulated by a competition between electrostatics and hydrophobic effects . when ac - tau(273284)-nh2 and ac - a(2535)-nh2 form extended dimers , or ac - tau(273284)-nh2 intercalates between ac - a(2535)-nh2 hairpins at the trimer or pentamer , tau limits the influences of -hairpin structure in the growth of a(2535 ) fibrils and subsequently decreases the aggregation propensity . by replacing the hydrophobic core of nkgaii / nkgaii with phf6*/gaiigl , and limiting the role of a(2535 ) hairpins , the hetero - a oligomers are relatively extended , but not as aggregation - prone as the homo - a oligomers , due to the mismatches in side - chain interdigitations and their heterogeneity . the presence of a few tau chains within the heterocomplexes may decrease the ability to self - associate , but the system is still able to grow further into fibrils , which is consistent with what has been observed in the afm image of the mixture ( figure 8b ) . eisenberg and co - workers have shown that the macroscopic morphologies and abundances of fibrillar aggregates formed in binary mixtures of different a segments are distinct from the component segments . ( 1 ) using a combination of experimental and theoretical techniques , we are able to identify the interactions promoting and stabilizing ac - a(2535)-nh2/ac - tau(273284)-nh2 heteroligomers . ac - tau(273284)-nh2 monomer prefers to associate with an ac - a(2535)-nh2 monomer over another tau monomer as shown by the populations of heterodimer and homo - tau dimer in the mixture . the intercalation of ac - tau(273284)-nh2 monomers between ac - a(2535)-nh2 monomers ( and oligomers ) is a probable mechanism allowing large heteroligomers to grow with a wide range of configurations and conformations . ( 2 ) the heterogeneity of the heteroligomers and mismatches in side - chain interdigitations limit ( but do not fully eliminate ) aggregation . on the other hand , the architectures of hetero - a oligomers contain -rich content , which later can grow into heterofibrils , as illustrated in figure 9 . ( 3 ) a possible source of toxicity of early a oligomers may lie in their ability to bind tau monomers , thereby decreasing the population of tau needed to regulate microtubule dynamics . other possible pathways to toxicity lie in the structures of the heteroligomers formed by the interactions of different tau to a fragments , which awaits a further systematic investigation . illustration of how fibrillar and granular aggregates can be formed in the mixture of ac - a(2535)-nh2 and ac - tau(273284)-nh2 . the morphologies of the aggregates are determined by the population of the individual peptides .

alzheimer 's disease ( ad ) is the most common age - related neurodegenerative disorder , clinically manifested by a progression from episodic memory problems to a slow global decline of cognitive function that leaves patients with end - stage ad bedridden and dependent on custodial care , with death occurring on average 9 years after diagnosis . the two distinctive key hallmarks of ad consist of senile plaques ( sp ) , composed of extracellular deposits of amyloid- peptides ( a ) , and intracellular neurofibrillary tangles ( nft ) , formed by accumulation of abnormal filaments of protein tau , in brain regions that serve memory and cognition [ 2 , 3 ] . although numerous studies have been performed on both a and tau biology , the exact molecular mechanisms behind the pathology are still not completely elucidated . current therapies used to treat ad patients are aimed to ameliorate symptoms for a limited period . at this point , there is no approved treatment with a proven disease - modifying effect [ 1 , 4 ] . early studies mainly focused on the a biology , in part because several genetic mutations leading to early onset familial ad were identified in the genes encoding the -amyloid precursor protein ( app ) and presenilins , components of the -secretase complex , which are involved in the formation of a peptides . over the past decade , however , tau biology has received increasing attention . in 1998 , for instance , it was demonstrated that mutations in tau were responsible for another neurodegenerative disease , frontotemporal dementia and parkinsonism linked to chromosome 17 ( ftdp-17 ) [ 68 ] . these mutations unequivocally proved that tau malfunctioning , in itself , can result in neurodegeneration and cognitive decline . in addition , tau is implicated in neurodegeneration in various other diseases , such as pick 's disease and progressive supranuclear palsy ( psp ) , which , similar to ad and ftdp-17 , are all characterized by the appearance of intraneuronal inclusions of aggregated tau proteins [ 911 ] . tauopathies and strongly suggest that tau malfunction is a major factor underlying neurodegeneration in all these diseases . in this respect , many studies concerning ad have indicated an intimate link between a and tau pathology , whereby a accumulation would constitute an upstream event , triggering tau pathology and neurodegeneration [ 12 , 13 ] . indeed , amyloid deposition was demonstrated to precede tau tangle formation in a triple transgenic mouse model of alzheimer 's disease . furthermore , administration of a42 , the most fibrillogenic form of a peptide , has been shown to induce the formation of tau - containing filaments , both in tissue culture , as in p301l transgenic mice . in cultured hippocampal neurons , tau knockout resulted in the loss of neurodegeneration in the presence of a . in addition , reducing endogenous tau was shown to ameliorate a-induced behavioral deficits in an ad mouse model , without altering their high a levels . thus , all these data indicate that , while a accumulation may represent the prime trigger in the onset of ad , tau pathology likely constitutes the crucial effector of neurodegeneration in this disease . key questions that remain to be solved involve the molecular nature of the toxic tau species as well as the molecular mechanisms leading to neurodegeneration . tau , also known as microtubule associated protein tau ( mapt ) , is predominantly expressed in neurons where its main function seems to be the stabilization of microtubules ( mts ) , particularly in axons . the mapt gene is located on chromosome 17 and consists of 16 exons [ 19 , 20 ] . in the central nervous system , alternative splicing of exons 2 , 3 , and 10 yields six tau isoforms ( figure 1 ) . the isoforms differ by the presence or absence of one or two acidic inserts at the n - terminal domain , and whether they contain three or four repeats of a conserved tubulin binding motif at the c - terminal . the tubulin - binding repeat region is the central part of the microtubule - binding domain ( mtbd ) , and tau isoforms with 4 repeats ( 4r - tau ) bind microtubules with greater affinity than isoforms with three repeats ( 3r - tau ) . in normal adult human tissue , the ratio of 4r- to 3r - tau is 1 [ 21 , 22 ] . the mt - binding affinity of tau is posttranslationally regulated primarily by serine / threonine directed phosphorylation , which can effectively modulate the binding affinity of tau for mts . through its ability to modulate mt dynamics , tau plays a vital role in regulating the appropriate morphology of neurons . in addition , as the mt network is key to the sophisticated transport machinery allowing for transport of molecules and organelles ( e.g. , mitochondria and vesicles ) along the axons , tau clearly can have profound effects on axonal transport and , hence , on the function and viability of neurons and their highly extended processes . under normal physiological conditions , tau is in a constant dynamic equilibrium , on and off the mts . as mt - binding of tau is largely controlled by its phosphorylation status , cellular morphology and axonal transport are critically dependent on the balance of the activities of tau kinases and phosphatases . interestingly , although the primary function of tau appears to be the stabilization of microtubules , it has been shown that tau can also interact , either directly or indirectly , with actin and affect actin polymerization as well as the interaction of actin filaments with microtubules [ 2631 ] . furthermore , tau interacts with the plasma membrane and with several proteins involved in signal transduction , in most part via its n - terminal projection domain [ 3239 ] . the binding of tau to signalling molecules implies that tau is either a downstream substrate or a regulator of the activity of the proteins it binds , or even both . for instance , tau is not only phosphorylated by the fyn kinase [ 40 , 41 ] , it also modulates fyn activity . interestingly , fyn is shown to play a crucial role in the recruitment of both a and tau into lipid rafts [ 43 , 44 ] . the importance of these interactions of tau with proteins and structures other than the actin and microtubule cytoskeleton is largely unknown , especially in the context of tau - mediated neurodegeneration . still , these findings support the notion that tau is prone to a large number of heterogeneous interactions , and irregularities of some of these interactions may lead , or contribute , to protein misfolding and aggregation , and even cell death through , as yet , unknown mechanisms . as mentioned above , the progressive accumulation of nft , composed of insoluble , hyperphosphorylated tau in a filamentous form , is a common hallmark of all tauopathies , including ad . as the severity of dementia in ad was shown to correlate well with nft load , in contrast to sp load [ 4547 ] , these aggregated forms of tau were , at first , thought to be the prime toxic component . however , this concept is still under debate , as several lines of evidence indicate that the tau aggregates are not major toxic components , and may even represent a protective mechanism , by which the neuronal cell attempts to detoxify other harmful species of tau by sequestering them into relatively inert aggregates [ 10 , 48 ] . indeed , tau - mediated neuronal death , in the absence of tau filaments , is observed in drosophila and some transgenic mouse models overexpressing human tau [ 4951 ] . in addition , mouse models of tauopathies exist , in which a dissociation was found between tangle formation in some areas distinct from neuronal loss in others [ 52 , 53 ] . finally , in the transgenic mouse model rtg4510 , conditionally expressing the human tau p301l mutant , age - related nft develop , along with neuronal loss and memory impairment . yet , subsequent suppression of the mutant tau was shown to stabilize neuronal loss and improve memory function , even though nft continue to accumulate . further detailed analysis of tau pathology in this mouse model suggested that the accumulation of early - stage aggregated tau species , including hyperphosphorylated multimers of tau , and not the end - stage nft , are correlated with the development of cognitive deficits during the pathogenic progression of tauopathy . thus , these studies suggest that tau - mediated neuronal death does not require the formation of nft . rather , nonfilamentous tau , in a more soluble , hyperphosphorylated , and possibly oligomeric state , may represent the prime neurotoxic tau component . apart from the exact nature of the toxic tau species , additional controversy surrounds the question of the molecular mechanisms mediating cell death . regarding this issue , it is becoming increasingly evident that tau - mediated neurodegeneration may encompass multiple mechanisms , including both loss of normal functions and toxic gains - of - functions acquired by the aggregates and their precursors ( figure 2 ) . since the major physiological function ascribed to tau is the regulation of mt dynamics , tau malfunction has been reported to induce a loss of mt stability [ 5658 ] and hamper proper axonal transport [ 5963 ] . impairment of these cellular functions initiates synaptic damage , an early event observed in multiple tauopathies [ 6468 ] , ultimately leading to neurodegeneration . in addition , tau may also mediate neurotoxicity , at least in part , by altering the organization and dynamics of the actin cytoskeleton . through its ability to interact with the plasma membrane and to bind a variety of proteins therefore , abnormal alterations in the phosphorylation of tau , and possibly other abnormal tau modifications , may aberrantly affect its association with the plasma membrane and with various signaling molecules , possibly leading to a toxic outcome . in this respect , a model for the molecular events leading to neurodegeneration in ad was recently proposed , connecting amyloid and tau dysfunction to a fyn - dependent , nmda receptor - mediated excitotoxicity [ 6972 ] . although several aspects of this model need further confirmation , it accounts for the tau - dependency of a-induced toxicity [ 17 , 18 ] , as well as the observed requirement of activation of nmda receptors to induce cell death by tau overexpression in cultured neurons . aside from phosphorylation , proteolytic processing of tau truncation of tau may generate amyloidogenic tau fragments that initiate the aggregation of tau and/or result in tau fragments which induce neurodegeneration through unknown mechanisms , independently of tau aggregation . although caspases are known to play essential roles in apoptosis , the involvement of the latter process in the mediation of tau - induced cell death is still obscure , as both proapoptotic and antiapoptotic features of tau are described in the literature [ 7476 ] . recently , in vivo imaging techniques in the transgenic rtg4510 mouse model revealed a dissociation between caspase activation and acute neuronal death in tangle - bearing neurons . therefore , these authors suggested that neurons undergo a slow , nonapoptotic but caspase - associated form of cell death in tauopathy , in which caspase cleavage of tau seeds an aggregate that actually sequesters toxic tau species . although this process delays cell death , it results in a sick neuron that loses connections and eventually dies . aside from the well - known involvement of caspases in tau processing , the proteasome is also shown to degrade tau [ 78 , 79 ] , though hyperphosphorylated tau seems resistant to this proteasome - mediated clearance [ 13 , 80 ] . interestingly , tau aggregates have been reported to inhibit proteasome activity , correlating with the decreased activity of the proteasome in ad - affected brain [ 82 , 83 ] . as illustrated by all the studies mentioned above , tau dysfunction likely contributes to neurodegeneration via multiple mechanisms , acting at different stages of disease . still , the exact nature of toxic tau species remains unknown , as is the sequence of events leading to tau - mediated cell death . indeed , although correlations are quite easy to observe , it is more difficult to discern which is cause , and which is consequence . in addition , it is still unclear which aspects of aging , the greatest risk factor of all for disease development , are involved in the onset of tau pathology , and to what extent . aging is known to affect a plethora of processes , such as glucose and insulin metabolism , inflammation , oxidative stress management , and the protein quality control system . for most of them , the molecular mechanisms connecting these processes to tau pathology are still largely elusive [ 25 , 8891 ] . the general trend , however , is that there seems to be a bidirectional relation between these processes and tau pathology , because defects in these processes seem to induce tau hyperphosphorylation and aggregation and , on the other hand , tau pathology results , for instance , in increased oxidative stress and inflammation . thus , a picture emerges in which , when cellular stress surpasses a certain threshold level , tau toxicity is induced and a seemingly unstoppable self - sustaining cycle is created , propagating the disease throughout the brain . further elucidating the causes of tau malfunction may provide new insights into the initiating factors of tau pathology and first toxic tau intermediates . this information will be of great value in the development of new therapeutic strategies combating tau pathologies . we will now briefly review cellular aspects involved in tau phosphorylation and oxidative stress , two important determinants of tau pathology , since , as we will discuss further below , we recapitulated elements of these features in our humanized yeast system . the phosphorylation of tau plays a physiological role in regulating the affinity of tau for mt . in addition , tau in ad , and other tauopathies , is characterized by an abnormally hyperphosphorylated state . as the phosphorylation state of tau is controlled by the activities of various tau kinases and phosphatases , these enzymes , and their regulators , have received much attention for their role in tauopathies . two groups of kinases have been implicated in tau phosphorylation : proline - directed protein kinases ( pdpks ) and non - pdpks . the pdpks include glycogen synthase kinase 3 ( gsk-3 ) , cyclin - dependent protein kinase 5 ( cdk5 ) , mitogen - activated protein kinase , and several stress - activated protein kinases ( sapk ) . both kinases copurify with mt [ 9395 ] , and phosphorylate tau within a cellular environment [ 9698 ] . data from in vitro studies indicate that phosphorylation of tau by gsk-3 inhibits its ability to promote mt assembly [ 99 , 100 ] . intriguingly , gsk-3 pseudophosphorylated mutants of tau not only displayed a decreased affinity for mt , but also reduced inducer - initiated rates of nucleation and polymerization in vitro , indicating that phosphorylation of tau by gsk-3 might not per se lead to increased tau aggregation . multiple lines of evidence indicate that gsk-3 is a key player affecting tau toxicity in vivo . for instance , the cotransfection of tau with gsk-3 in a cell culture model results in more cell death compared to the expression of tau and mutant ( inactive ) gsk-3 , suggesting that tau phosphorylation by gsk-3 is toxic . in addition , inhibition of gsk3 by lithium not only reduced tau phosphorylation in vivo , but also lowered the level of aggregated tau , compared with controls . like gsk-3 , cdk5 is intensively investigated for its role in the development of tau pathology . tau is a proven cdk5 target in vivo and in vitro , it was shown that phosphorylation by cdk5 promotes tau dimerization . activation of cdk5 , by overexpressing its activator p25 , accelerates tau phosphorylation and aggregation in mice overexpressing mutant p301l tau , and has even been shown to contribute to tau pathology in mice expressing only endogenous tau [ 106 , 107 ] . of interest , cdk5 activity is elevated in the prefrontal cortex of ad brain , where nft are found , but not in the cerebellar cortex , suggesting a relationship between deregulated cdk5 activity and tau pathology in humans [ 108 , 109 ] . although cdk5 is shown to phosphorylate tau directly , there are reports that cdk5 activity also affects the tau phosphorylation status indirectly [ 110 , 111 ] , which , under certain conditions , may occur via a cdk5-mediated inhibition of gsk-3 activity . among the tau non - pdpks are cyclic amp - dependent protein kinase ( pka ) , calcium- and calmodulin - dependent protein kinase ii ( camkii ) , and microtubule affinity regulating kinase ( mark ) . mark phosphorylates tau at kxgs motifs within the mtbd of tau , thereby inducing the release of tau from mt [ 113 , 114 ] . interestingly , in an in vitro study , it was shown that phosphorylation of tau by mark and pka led to a strongly reduced affinity of tau for mt , along with a decrease of tau 's ability to assemble into paired helical filaments . although at first glance this may contradict the correlation of hyperphosphorylated tau with the occurrence of nft in tauopathies , this result is in agreement with the hypothesis that not the nft , but soluble hyperphosphorylated forms of tau represent the toxic species as discussed above . unbound tau , generated by mark and/or pka phosphorylation , may subsequently be phosphorylated by other kinases , generating the notorious hyperphosphorylated tau . in fact , the phosphorylation of tau by mark may be a prerequisite for the action of downstream kinases , including gsk-3 and cdk5 . obviously , the phosphorylation state of tau is dictated not only by kinase activity , but also by the activities of tau phosphatases . tau is dephosphorylated by protein phosphatases 2a ( pp2a ) and , to a lesser extent , by pp1 , pp2b , and pp5 [ 37 , 116118 ] . in ad brain , it is found that the mrna and protein expression levels of some of these phosphatases , as well as their activities , are decreased [ 118124 ] . therefore , downregulation of phosphatase activity , especially that of pp2a , can contribute to increased levels of hyperphosphorylated tau . in addition , pin1 , a member of the peptidyl - prolyl cis - trans isomerases , is involved in the regulation of the phosphorylation state of tau , as pin1 binds tau when it is phosphorylated at thr231 and facilitates its dephosphorylation by pp2a [ 39 , 125127 ] . notably , pin1 is significantly downregulated and oxidized in the ad hippocampus . furthermore , in ad neurons , pin1 binds hyperphosphorylated tau in filaments , potentially depleting soluble pin1 levels [ 39 , 129 ] . as hyperphosphorylation of tau constitutes an early modification , inducing further conformational changes and aggregation of tau , modulation of the activities of tau kinases and phosphatases represents an appealing strategy to combat tau pathologies . major focus has been on modulation of important tau kinases , especially gsk-3 [ 103 , 130 ] , though no successful outcome has yet been reported for clinical trials using gsk-3 inhibitors , such as lithium and valproic acid . accumulating evidence suggests that , besides the accumulation of protein aggregates , oxidative stress and mitochondrial dysfunction , which are intimately linked , also play an important role in the etiology of neurodegenerative diseases , including ad [ 89 , 90 ] . as mitochondria are a major source of reactive oxygen species ( ros ) , malfunctions of these organelles are thought to be a prime contributor to cellular oxidative stress . additionally , mitochondrial dysfunction will lead to decreased energy production , which puts an extra burden on neurons , which are heavily dependent on high atp levels to sustain many biochemical processes , especially involving neurotransmission at their synapses . still , a proteomic and functional analysis showed a mitochondrial dysfunction in p301l mice , together with reduced nadh - ubiquinone oxidoreductase activity , and , with age , impaired mitochondrial respiration and atp synthesis . in the aged transgenic mice , mitochondrial dysfunction was associated with higher levels of ros , and increased tau pathology revealed modified lipid peroxidation levels and the upregulation of antioxidant enzymes in response to oxidative stress . interestingly , as the p301l mice displayed an increased vulnerability towards a insult , this suggests that a and tau pathology work synergistically on mitochondria , through distinct mechanisms . although a direct impact of tau on some mitochondrial proteins / enzymes is hypothesized , this remains to be confirmed . in another study , the addition of annonacin , a natural mitochondrial complex i inhibitor , caused a redistribution of tau from the axons to the cell body , along with a retrograde transport of mitochondria and cell death . retrograde transport of dysfunctional mitochondria is a general feature , priming them for elimination by autophagy [ 134136 ] . interestingly , although annonacin addition caused an increase in oxidative stress , atp depletion was shown to be the primary trigger for tau relocalization , mitochondrial retrograde transport , and cell death . thus , although tau pathology seems to induce mitochondrial dysfunction , leading to increased ros , the concurrent decreased atp levels must not be overlooked , as both these signals can play vital roles in neuronal survival . oxidative stress in ad and other neurodegenerative diseases has also been linked to increased brain levels of certain metals , especially iron ( fe ) , copper ( cu ) , and zinc ( zn ) [ 137139 ] . both fe and cu are capable of inducing oxidative stress by stimulating free radical formation ( e.g. , hydroxyl radicals via fenton reaction ) . in ad , fe- and cu - induced oxidative reactions are accelerated by a , and a oligomerization is stimulated in the presence of these metals . interestingly , it was recently shown that the app protein functions as an iron - export ferroxidase , whose activity is inhibited by zn . as zn is locally concentrated in a-plaques , this will lead to excessive fe retention in app expressing neurons , creating a self - sustaining cycle , wherein increased fe retention induces further a-oligomerization and plaque formation , further inhibiting fe export . in regards to tau biology , metals may influence the self - assembly of tau , as low micromolar concentrations of zn have been shown to accelerate the fibrillization of human tau via the bridging of two cysteine residues under physiological reducing conditions . under oxidizing conditions , however , intermolecular disulfide cross - linking of tau can occur , facilitating its oligomerization . furthermore , similar to a plaques , nft were found to be capable of adventitious binding of cu and fe in a redox - competent manner , indicating that nft may exert prooxidant or antioxidant activities , depending on the balance among cellular reductants and oxidants in the local environment [ 144 , 145 ] . finally , oxidative stress not only results in directly damaging modifications of cell constituents , ros also function as signalling molecules , affecting the activity of several kinases and phosphatases . surprisingly , oxidative stress by addition of hydrogen peroxide resulted in a dephosphorylation of tau . subsequent studies indicated that , on one hand , oxidative stress induces a pin1-mediated activation of protein phosphatase 2a . on the other hand , an increased activity of protein phosphatase 1 ( pp1 ) was observed , due to a cdk5-mediated inhibition of inhibitor-2 , a negative regulator of pp1 . thus , although cdk5 is generally considered as a bona fide in vivo tau kinase , these and other results indicate that this kinase can influence the phosphorylation state of tau in multiple , antagonizing ways [ 92 , 110112 ] . intriguingly , in ad brain , oxidative stress is observed , though tau is found in a hyperphosphorylated state , in contrast to the observed oxidative stress - induced dephosphorylation of tau described above . in this respect , it was found that oxidative stress combined with okadaic acid , which inhibits both pp1 and pp2a , results in a hyperphosphorylated tau species which was significantly resistant to degradation . these data suggest that , in tauopathies , a combination of oxidative stress and hyperphosphorylation may be directly responsible for the accumulation of tau aggregates . considering the fact that oxidative stress constitutes a hallmark of numerous neurodegenerative diseases , strategies that ameliorate this stress are studied intensively as possible therapeutic treatments . these include the supplementation with antioxidants or a mixture of antioxidants ( e.g. , vitamin c and e ) , the use of specific mitochondria - targeted antioxidants , and the modulation of metal bioavailability [ 139 , 147 ] . promising initial results of some of these strategies have been described , and outcomes of clinical trials are heavily anticipated . the basic cellular machinery and molecular processes between the budding yeast saccharomyces cerevisiae and other eukaryotic species , including humans , appears to be highly conserved . consequently , as many yeast genes have functional orthologues in mammalian organisms , yeast has been an effective model system for the study of diverse cellular processes , including mechanisms involved in glucose response , apoptosis , and cancer . yeast systems have been constructed to study disease - related proteins that have no , or no apparent , functional yeast orthologue , such as the human tau protein . these humanized yeast systems have also proven to be valuable tools to unravel disease - related molecular processes and to identify novel medicinal compounds . examples of this type of studies in the field of neurodegenerative disorders include protein - misfolding disorders such as alzheimer 's , parkinson 's , and huntington 's disease [ 152 , 153 ] . in our laboratory , we expressed different isoforms and clinical ftdp-17 mutant forms of tau in yeast , and found that tau exhibits many of the same features as it does in neurons of patients with ad , that is , hyperphosphorylation , conformational changes , and partial accumulation into aggregates [ 8486 ] . we will now go over our most important findings on the impact of phosphorylation as well as oxidative stress on tau properties in our yeast model . phosphorylation of tau in mammalian cells controls its interactions with mt while its hyperphosphorylation is thought to cause , or contribute to , the aggregation and toxicity of this protein . in order to assess tau phosphorylation in yeast , we employed a series of phosphospecific tau antibodies to scan phosphorylated residues on tau . we found that tau , when expressed in yeast , became reactive to a multitude of these antibodies , proving the existence of yeast kinases and/or phosphatases able to recognize and ( de)phosphorylate human protein tau ( figure 3 ) . in addition , we could detect tau with the conformation - dependent antibody mc1 , a marker for pathological tau filaments and their precursors [ 154156 ] , and demonstrate a reproducible amount of tau present in the sarkosyl - insoluble fraction ( sint , sarkosyl - insoluble tau ) , pointing to tau aggregation in yeast . to study the role of yeast kinases in the phosphorylation and aggregation of tau , we set out to test the involvement in tau phosphorylation of mds1 and pho85 , functional yeast orthologues of mammalian gsk-3 and cdk5 , respectively . deletion of mds1 led to a significant decrease in tau 's immunoreactivity to both ad2 and pg5 . for the ad2 epitope ( p - s396/p - s404 ) , this was expected , as it constitutes a typical gsk-3 target [ 50 , 157 , 158 ] . phosphorylation of tau at s409 ( pg5 epitope ) , however , is not a typical substrate of gsk-3 , but of pka , indicating that mds1 might affect phosphorylation at this site indirectly [ 159 , 160 ] . interestingly , deletion of pho85 resulted in a hyperphosphorylation of tau , mainly at the ad2 and pg5 epitopes . this hyperphosphorylation was accompanied by an increase in mc1-reactive tau species and increased sint levels , compared to control levels . this may not be so strange , as , also in mammalian cells , evidence exists that cdk5 has indirect effects on the phosphorylation status in tau , exemplified by the study of hallows et al . , also demonstrating an increase in tau phosphorylation upon cdk5 inactivation , by knockout of its activating partner p35 . since deletion of mds1 and pho85 both affect the ad2 and pg5 epitopes in opposite ways , it is tempting to speculate that pho85 exerts its effects on tau by inhibiting mds1 . although this has not yet been proven in yeast , we demonstrated , by means of yeast epistasis analysis combined with complementation studies using the human gsk-3 and cdk5 kinases , that mds1/gsk-3 genetically operates downstream of pho85/cdk5 in the phosphorylation of tau in our yeast system . intriguingly , studies in a mouse model also imply that cdk5 might inhibit gsk-3 activity under certain conditions . additional data on the physiological consequences of tau phosphorylation in a yeast environment were obtained via two separate in vitro tests , for which tau was purified from wild - type ( wt ) , mds1 , and pho85 yeast strains using an anion exchange chromatography method [ 84 , 85 ] . we could confirm that , after purification , tau retained its phosphorylation status , characteristic for each strain . in a first assay , we observed that the in vitro tau filament formation was much faster when using tau isolated from the pho85 strain , compared to tau extracted from either a wt and mds1 yeast strain , consistent with the hyperphosphorylated state of tau in a pho85 strain . in addition , further fractionation of tau extracts yielded a hyperphosphorylated , mc1-reactive subfraction , and we demonstrated that this species could vastly accelerate the in vitro aggregation of tau extracted from a wt strain , implicating a seeding capacity of this hyperphosphorylated tau species . in a second series experiments , we investigated the in vitro mt binding capacity of purified tau , using taxol - stabilized mt formed with pig tubulin . in this assay , we could demonstrate an inverse relation between mt binding and tau phosphorylation status , as hyperphosphorylated tau , isolated from pho85 cells , showed the poorest mt binding , followed by tau extracted from a wt strain , and finally tau extracted from the mds1 strain , which showed impaired phosphorylation . to gain further insight into the relation between tau phosphorylation and aggregation in our yeast system , several clinical ftdp-17 mutants were expressed in wt , mds1 , and pho85 yeast strains , and their phosphorylation patterns and sint levels were analyzed . most notably , compared to wild - type tau , both the p301l and r406w mutants displayed a clear reduction in the phosphorylation of s409 ( pg5 epitope ) and decreased sint levels , particularly in a pho85 strain . these findings suggested that phosphorylation of tau at s409 might be an important determinant for tau aggregation . to confirm this hypothesis , we mutagenized the pg5 epitope and expressed the synthetic tau - s409a mutant and its pseudophosphorylated counterpart tau - s409e in wt and pho85 strains . analysis of these synthetic mutants indeed revealed a marked decrease of tau - s409a aggregation while the tau - s409e mutant displayed sint levels higher than , or comparable to , wild - type tau ( figure 4 ) . interestingly , we demonstrated that phosphorylation of s409 is also detrimental for tau - mt interaction , revealing a close , antagonistic link between the ability of tau to bind mt and its ability to aggregate . intriguingly , the tau - s409a mutant was characterized with a lower ad2 reactivity while the pseudophosphorylated tau - s409e exhibited an increased immunodetection with the ad2 antibody , especially in the wt strain . thus , phosphorylation of tau at the pg5 and ad2 epitopes seems interdependent , and phosphorylation of tau at s409 might prime subsequent phosphorylation of s396/s404 . these observations are in line with data from the brain of ad patients , demonstrating that the formation of the pg5 epitope on tau is an early event in the pretangle stage , and precedes the phosphorylation at s396 , which is characteristic for nft . in conclusion of this part , we can say that we have demonstrated that , when expressed in yeast , tau is phosphorylated at multiple pathologically relevant sites . interestingly , mds1 and pho85 , the yeast orthologues of human gsk-3 and cdk5 , respectively , play crucial roles in the phosphorylation of several of these epitopes . furthermore , our data substantiate the notion that hyperphosphorylation of tau leads to a loss of mt - binding capacity , along with the induction of conformational changes , detectable by the mc1 antibody , which ultimately lead to tau aggregation [ 155 , 156 ] . finally , we show that phosphorylation of s409 is a crucial mediator in both tau aggregation and mt binding . it is important to emphasize that the tau - mt binding assays described here were performed in vitro using mt built from pig tubulin . we are , as yet , unable to demonstrate binding of tau to yeast mt , likely due to differences in yeast and mammalian tubulins [ 162 , 163 ] . as discussed previously , evidence exists suggesting a link between oxidative stress and tau pathology . we investigated the effects of oxidative stress and mitochondrial dysfunction on tau aggregation by adding fe , a known inducer of oxidative stress , to yeast cells , or by examining sint levels in specific mitochondrial mutants , respectively . interestingly , in both conditions , markedly increased tau insolubility was observed through mechanisms that are not strictly dependent on phosphorylation of tau , but rather act mainly in parallel . close examination of western blot profiles indicated that oxidative stress led to a reduction in the level of tau dimers , concomitant with an increase in higher - order oligomers . furthermore , a 35 kda degradation product appeared after the addition of fe , indicative of altered processing and/or diminished clearance of tau fragments under this condition . strikingly , application of oxidative stress led to decreased phosphorylation of tau at specific epitopes , especially ad2 and pg5 ( figure 5 ) . this is consistent with several studies in neuronal cells showing a dephosphorylation of tau upon exposure to oxidative stress [ 111 , 125 , 146 ] . two mechanisms accounting for this oxidative stress - induced tau dephosphorylation have been described , both of which seem to be conserved in yeast . in the first , increased activity of protein phosphatase 1 ( pp1 ) was observed , due to a cdk5-mediated inhibition of inhibitor-2 , a negative regulator of pp1 . interestingly , the cdk5/p35 complex is functionally equivalent to the yeast pho85/pcl6,7 complex , which phosphorylates glc8 , the orthologue of mammalian inhibitor-2 , thereby controlling the activity of the glc7 phosphatase , the orthologue of mammalian pp1 . in the second mechanism , pin1 function is represented by the yeast orthologue ess1 , and our unpublished results indicate that disruption of ess1 activity leads to increased hyperphosphorylation of tau . hence , it appears that similar mechanisms may govern oxidative stress - induced tau dephosphorylation in yeast and mammalian cells , highlighting the value of studying tau biology in yeast . one important aspect of our research in tau - expressing yeast cells , is that we did not observe strong tau - related growth phenotypes in any of the strains , even under conditions which displayed a strong induction of tau aggregation . this implies that tau aggregation is not per se correlated with toxicity , a conclusion that correlates directly with findings in mammalian systems , in which nft were found not to be essential for tau - induced toxicity and may even play a protective function [ 48 , 55 ] . in addition , all the results discussed here concerning tau phosphorylation and aggregation , were obtained from exponentially growing yeast cells . since , for instance , mitochondrial activity , a factor believed to be involved in the etiology of tauopathies , is more important during the yeast 's stationary phase than during fermentation , investigating the impact of tau on cellular fitness during yeast aging may present us with new clues on tau toxicity . it is needless to say that we have just started to scratch the surface on studying possible mediators of tau biology and pathology in yeast ( see table 1 ) . in our studies , we focused mainly on the role of mds1 and pho85 , functional yeast orthologues of mammalian gsk-3 and cdk5 , respectively , in the phosphorylation of tau in yeast . obvious candidates for further research include the yeast orthologues of protein phosphatases involved in tau dephosphorylation , such as pp2a and pp1 , and of pin1 . as mentioned , our unpublished results already indicate a hyperphosphorylated tau status upon decreased activity of ess1 , the yeast orthologue of pin1 . interestingly , in contrast to a pho85 strain , we did observe a toxic effect of tau on growth of yeast cells with impaired ess1 activity . this implies that the toxicity of hyperphosphorylated tau is not a general feature , but likely applies to a specific combination of phosphorylated epitopes . detailed investigation of ess1-dependent tau modifications is currently in progress . in this regard , it would be of great interest to extend this type of analysis by employing a genome - wide screen for yeast mutants displaying tau - dependent toxicity . analysis of the phosphorylation pattern and sint levels in such mutants can provide crucial new information regarding toxic tau species and molecular players involved in their generation . as a final note , we mention that , although we did not see a tau - induced toxicity in yeast , we observed synthetic toxicity upon the coexpression of tau with -synuclein . thus , it seems that tau can exert hazardous effects in yeast , but in the absence of other stressors , this does not reveal itself by a growth defect . increase of cellular stress , by coexpression of -synuclein , however , apparently results in the exceeding of a certain threshold , thereby revealing a tau - dependent toxicity . detailed analysis of impaired cellular processes upon coexpression of tau with -synuclein , in conjunction with the above - mentioned genome - wide screening , may thus reveal which cellular machineries are affected by tau overexpression in yeast . as the group of seniors in the world 's population continues to grow , age - related neurodegenerative disorders , including tauopathies such as alzheimer 's disease , are becoming more prevalent , and pose a serious threat to an already overwhelmed health care system . despite the vast amount of research already performed , several aspects of tauopathies still await molecular elucidation . among these are the nature of toxic tau species and the way they influence cell viability . we developed a yeast model expressing human protein tau variants , and demonstrated that this model recapitulates many important aspects implicated in tau pathology , including hyperphosphorylation , conformation , and aggregation . combined with the ease of genetic manipulations , rapid genome - wide screening methods , and other advantages characteristic of yeast systems , this model may prove its value in the clarification of fundamental cellular processes involved in tau biology and pathology .

hyperphosphorylated and aggregated human protein tau constitutes a hallmark of a multitude of neurodegenerative diseases called tauopathies , exemplified by alzheimer 's disease . in spite of an enormous amount of research performed on tau biology , several crucial questions concerning the mechanisms of tau toxicity remain unanswered . in this paper we will highlight some of the processes involved in tau biology and pathology , focusing on tau phosphorylation and the interplay with oxidative stress . in addition , we will introduce the development of a human tau - expressing yeast model , and discuss some crucial results obtained in this model , highlighting its potential in the elucidation of cellular processes leading to tau toxicity .

1. Introduction 2. Tau: Physiological Functions 3. Tau Pathology 4. A Humanized Yeast Model to Study Tau Biology 5. Concluding Remarks

alzheimer 's disease ( ad ) is the most common age - related neurodegenerative disorder , clinically manifested by a progression from episodic memory problems to a slow global decline of cognitive function that leaves patients with end - stage ad bedridden and dependent on custodial care , with death occurring on average 9 years after diagnosis . although numerous studies have been performed on both a and tau biology , the exact molecular mechanisms behind the pathology are still not completely elucidated . early studies mainly focused on the a biology , in part because several genetic mutations leading to early onset familial ad were identified in the genes encoding the -amyloid precursor protein ( app ) and presenilins , components of the -secretase complex , which are involved in the formation of a peptides . in addition , tau is implicated in neurodegeneration in various other diseases , such as pick 's disease and progressive supranuclear palsy ( psp ) , which , similar to ad and ftdp-17 , are all characterized by the appearance of intraneuronal inclusions of aggregated tau proteins [ 911 ] . in this respect , many studies concerning ad have indicated an intimate link between a and tau pathology , whereby a accumulation would constitute an upstream event , triggering tau pathology and neurodegeneration [ 12 , 13 ] . indeed , amyloid deposition was demonstrated to precede tau tangle formation in a triple transgenic mouse model of alzheimer 's disease . furthermore , administration of a42 , the most fibrillogenic form of a peptide , has been shown to induce the formation of tau - containing filaments , both in tissue culture , as in p301l transgenic mice . in cultured hippocampal neurons , tau knockout resulted in the loss of neurodegeneration in the presence of a . in addition , reducing endogenous tau was shown to ameliorate a-induced behavioral deficits in an ad mouse model , without altering their high a levels . key questions that remain to be solved involve the molecular nature of the toxic tau species as well as the molecular mechanisms leading to neurodegeneration . in the central nervous system , alternative splicing of exons 2 , 3 , and 10 yields six tau isoforms ( figure 1 ) . the isoforms differ by the presence or absence of one or two acidic inserts at the n - terminal domain , and whether they contain three or four repeats of a conserved tubulin binding motif at the c - terminal . as mt - binding of tau is largely controlled by its phosphorylation status , cellular morphology and axonal transport are critically dependent on the balance of the activities of tau kinases and phosphatases . the importance of these interactions of tau with proteins and structures other than the actin and microtubule cytoskeleton is largely unknown , especially in the context of tau - mediated neurodegeneration . still , these findings support the notion that tau is prone to a large number of heterogeneous interactions , and irregularities of some of these interactions may lead , or contribute , to protein misfolding and aggregation , and even cell death through , as yet , unknown mechanisms . indeed , tau - mediated neuronal death , in the absence of tau filaments , is observed in drosophila and some transgenic mouse models overexpressing human tau [ 4951 ] . in addition , mouse models of tauopathies exist , in which a dissociation was found between tangle formation in some areas distinct from neuronal loss in others [ 52 , 53 ] . further detailed analysis of tau pathology in this mouse model suggested that the accumulation of early - stage aggregated tau species , including hyperphosphorylated multimers of tau , and not the end - stage nft , are correlated with the development of cognitive deficits during the pathogenic progression of tauopathy . in addition , tau may also mediate neurotoxicity , at least in part , by altering the organization and dynamics of the actin cytoskeleton . through its ability to interact with the plasma membrane and to bind a variety of proteins therefore , abnormal alterations in the phosphorylation of tau , and possibly other abnormal tau modifications , may aberrantly affect its association with the plasma membrane and with various signaling molecules , possibly leading to a toxic outcome . in this respect , a model for the molecular events leading to neurodegeneration in ad was recently proposed , connecting amyloid and tau dysfunction to a fyn - dependent , nmda receptor - mediated excitotoxicity [ 6972 ] . although several aspects of this model need further confirmation , it accounts for the tau - dependency of a-induced toxicity [ 17 , 18 ] , as well as the observed requirement of activation of nmda receptors to induce cell death by tau overexpression in cultured neurons . although caspases are known to play essential roles in apoptosis , the involvement of the latter process in the mediation of tau - induced cell death is still obscure , as both proapoptotic and antiapoptotic features of tau are described in the literature [ 7476 ] . still , the exact nature of toxic tau species remains unknown , as is the sequence of events leading to tau - mediated cell death . in addition , it is still unclear which aspects of aging , the greatest risk factor of all for disease development , are involved in the onset of tau pathology , and to what extent . aging is known to affect a plethora of processes , such as glucose and insulin metabolism , inflammation , oxidative stress management , and the protein quality control system . the general trend , however , is that there seems to be a bidirectional relation between these processes and tau pathology , because defects in these processes seem to induce tau hyperphosphorylation and aggregation and , on the other hand , tau pathology results , for instance , in increased oxidative stress and inflammation . this information will be of great value in the development of new therapeutic strategies combating tau pathologies . we will now briefly review cellular aspects involved in tau phosphorylation and oxidative stress , two important determinants of tau pathology , since , as we will discuss further below , we recapitulated elements of these features in our humanized yeast system . in addition , tau in ad , and other tauopathies , is characterized by an abnormally hyperphosphorylated state . two groups of kinases have been implicated in tau phosphorylation : proline - directed protein kinases ( pdpks ) and non - pdpks . for instance , the cotransfection of tau with gsk-3 in a cell culture model results in more cell death compared to the expression of tau and mutant ( inactive ) gsk-3 , suggesting that tau phosphorylation by gsk-3 is toxic . in addition , inhibition of gsk3 by lithium not only reduced tau phosphorylation in vivo , but also lowered the level of aggregated tau , compared with controls . like gsk-3 , cdk5 is intensively investigated for its role in the development of tau pathology . activation of cdk5 , by overexpressing its activator p25 , accelerates tau phosphorylation and aggregation in mice overexpressing mutant p301l tau , and has even been shown to contribute to tau pathology in mice expressing only endogenous tau [ 106 , 107 ] . although at first glance this may contradict the correlation of hyperphosphorylated tau with the occurrence of nft in tauopathies , this result is in agreement with the hypothesis that not the nft , but soluble hyperphosphorylated forms of tau represent the toxic species as discussed above . in addition , pin1 , a member of the peptidyl - prolyl cis - trans isomerases , is involved in the regulation of the phosphorylation state of tau , as pin1 binds tau when it is phosphorylated at thr231 and facilitates its dephosphorylation by pp2a [ 39 , 125127 ] . as hyperphosphorylation of tau constitutes an early modification , inducing further conformational changes and aggregation of tau , modulation of the activities of tau kinases and phosphatases represents an appealing strategy to combat tau pathologies . accumulating evidence suggests that , besides the accumulation of protein aggregates , oxidative stress and mitochondrial dysfunction , which are intimately linked , also play an important role in the etiology of neurodegenerative diseases , including ad [ 89 , 90 ] . in the aged transgenic mice , mitochondrial dysfunction was associated with higher levels of ros , and increased tau pathology revealed modified lipid peroxidation levels and the upregulation of antioxidant enzymes in response to oxidative stress . interestingly , although annonacin addition caused an increase in oxidative stress , atp depletion was shown to be the primary trigger for tau relocalization , mitochondrial retrograde transport , and cell death . oxidative stress in ad and other neurodegenerative diseases has also been linked to increased brain levels of certain metals , especially iron ( fe ) , copper ( cu ) , and zinc ( zn ) [ 137139 ] . in ad , fe- and cu - induced oxidative reactions are accelerated by a , and a oligomerization is stimulated in the presence of these metals . in regards to tau biology , metals may influence the self - assembly of tau , as low micromolar concentrations of zn have been shown to accelerate the fibrillization of human tau via the bridging of two cysteine residues under physiological reducing conditions . surprisingly , oxidative stress by addition of hydrogen peroxide resulted in a dephosphorylation of tau . intriguingly , in ad brain , oxidative stress is observed , though tau is found in a hyperphosphorylated state , in contrast to the observed oxidative stress - induced dephosphorylation of tau described above . in this respect , it was found that oxidative stress combined with okadaic acid , which inhibits both pp1 and pp2a , results in a hyperphosphorylated tau species which was significantly resistant to degradation . these data suggest that , in tauopathies , a combination of oxidative stress and hyperphosphorylation may be directly responsible for the accumulation of tau aggregates . considering the fact that oxidative stress constitutes a hallmark of numerous neurodegenerative diseases , strategies that ameliorate this stress are studied intensively as possible therapeutic treatments . consequently , as many yeast genes have functional orthologues in mammalian organisms , yeast has been an effective model system for the study of diverse cellular processes , including mechanisms involved in glucose response , apoptosis , and cancer . examples of this type of studies in the field of neurodegenerative disorders include protein - misfolding disorders such as alzheimer 's , parkinson 's , and huntington 's disease [ 152 , 153 ] . in our laboratory , we expressed different isoforms and clinical ftdp-17 mutant forms of tau in yeast , and found that tau exhibits many of the same features as it does in neurons of patients with ad , that is , hyperphosphorylation , conformational changes , and partial accumulation into aggregates [ 8486 ] . we will now go over our most important findings on the impact of phosphorylation as well as oxidative stress on tau properties in our yeast model . in order to assess tau phosphorylation in yeast , we employed a series of phosphospecific tau antibodies to scan phosphorylated residues on tau . we found that tau , when expressed in yeast , became reactive to a multitude of these antibodies , proving the existence of yeast kinases and/or phosphatases able to recognize and ( de)phosphorylate human protein tau ( figure 3 ) . in addition , we could detect tau with the conformation - dependent antibody mc1 , a marker for pathological tau filaments and their precursors [ 154156 ] , and demonstrate a reproducible amount of tau present in the sarkosyl - insoluble fraction ( sint , sarkosyl - insoluble tau ) , pointing to tau aggregation in yeast . to study the role of yeast kinases in the phosphorylation and aggregation of tau , we set out to test the involvement in tau phosphorylation of mds1 and pho85 , functional yeast orthologues of mammalian gsk-3 and cdk5 , respectively . this may not be so strange , as , also in mammalian cells , evidence exists that cdk5 has indirect effects on the phosphorylation status in tau , exemplified by the study of hallows et al . although this has not yet been proven in yeast , we demonstrated , by means of yeast epistasis analysis combined with complementation studies using the human gsk-3 and cdk5 kinases , that mds1/gsk-3 genetically operates downstream of pho85/cdk5 in the phosphorylation of tau in our yeast system . additional data on the physiological consequences of tau phosphorylation in a yeast environment were obtained via two separate in vitro tests , for which tau was purified from wild - type ( wt ) , mds1 , and pho85 yeast strains using an anion exchange chromatography method [ 84 , 85 ] . in a first assay , we observed that the in vitro tau filament formation was much faster when using tau isolated from the pho85 strain , compared to tau extracted from either a wt and mds1 yeast strain , consistent with the hyperphosphorylated state of tau in a pho85 strain . in addition , further fractionation of tau extracts yielded a hyperphosphorylated , mc1-reactive subfraction , and we demonstrated that this species could vastly accelerate the in vitro aggregation of tau extracted from a wt strain , implicating a seeding capacity of this hyperphosphorylated tau species . in this assay , we could demonstrate an inverse relation between mt binding and tau phosphorylation status , as hyperphosphorylated tau , isolated from pho85 cells , showed the poorest mt binding , followed by tau extracted from a wt strain , and finally tau extracted from the mds1 strain , which showed impaired phosphorylation . to gain further insight into the relation between tau phosphorylation and aggregation in our yeast system , several clinical ftdp-17 mutants were expressed in wt , mds1 , and pho85 yeast strains , and their phosphorylation patterns and sint levels were analyzed . to confirm this hypothesis , we mutagenized the pg5 epitope and expressed the synthetic tau - s409a mutant and its pseudophosphorylated counterpart tau - s409e in wt and pho85 strains . analysis of these synthetic mutants indeed revealed a marked decrease of tau - s409a aggregation while the tau - s409e mutant displayed sint levels higher than , or comparable to , wild - type tau ( figure 4 ) . interestingly , we demonstrated that phosphorylation of s409 is also detrimental for tau - mt interaction , revealing a close , antagonistic link between the ability of tau to bind mt and its ability to aggregate . intriguingly , the tau - s409a mutant was characterized with a lower ad2 reactivity while the pseudophosphorylated tau - s409e exhibited an increased immunodetection with the ad2 antibody , especially in the wt strain . thus , phosphorylation of tau at the pg5 and ad2 epitopes seems interdependent , and phosphorylation of tau at s409 might prime subsequent phosphorylation of s396/s404 . these observations are in line with data from the brain of ad patients , demonstrating that the formation of the pg5 epitope on tau is an early event in the pretangle stage , and precedes the phosphorylation at s396 , which is characteristic for nft . we investigated the effects of oxidative stress and mitochondrial dysfunction on tau aggregation by adding fe , a known inducer of oxidative stress , to yeast cells , or by examining sint levels in specific mitochondrial mutants , respectively . close examination of western blot profiles indicated that oxidative stress led to a reduction in the level of tau dimers , concomitant with an increase in higher - order oligomers . strikingly , application of oxidative stress led to decreased phosphorylation of tau at specific epitopes , especially ad2 and pg5 ( figure 5 ) . this is consistent with several studies in neuronal cells showing a dephosphorylation of tau upon exposure to oxidative stress [ 111 , 125 , 146 ] . in the second mechanism , pin1 function is represented by the yeast orthologue ess1 , and our unpublished results indicate that disruption of ess1 activity leads to increased hyperphosphorylation of tau . hence , it appears that similar mechanisms may govern oxidative stress - induced tau dephosphorylation in yeast and mammalian cells , highlighting the value of studying tau biology in yeast . one important aspect of our research in tau - expressing yeast cells , is that we did not observe strong tau - related growth phenotypes in any of the strains , even under conditions which displayed a strong induction of tau aggregation . in addition , all the results discussed here concerning tau phosphorylation and aggregation , were obtained from exponentially growing yeast cells . since , for instance , mitochondrial activity , a factor believed to be involved in the etiology of tauopathies , is more important during the yeast 's stationary phase than during fermentation , investigating the impact of tau on cellular fitness during yeast aging may present us with new clues on tau toxicity . it is needless to say that we have just started to scratch the surface on studying possible mediators of tau biology and pathology in yeast ( see table 1 ) . in our studies , we focused mainly on the role of mds1 and pho85 , functional yeast orthologues of mammalian gsk-3 and cdk5 , respectively , in the phosphorylation of tau in yeast . obvious candidates for further research include the yeast orthologues of protein phosphatases involved in tau dephosphorylation , such as pp2a and pp1 , and of pin1 . analysis of the phosphorylation pattern and sint levels in such mutants can provide crucial new information regarding toxic tau species and molecular players involved in their generation . as a final note , we mention that , although we did not see a tau - induced toxicity in yeast , we observed synthetic toxicity upon the coexpression of tau with -synuclein . increase of cellular stress , by coexpression of -synuclein , however , apparently results in the exceeding of a certain threshold , thereby revealing a tau - dependent toxicity . as the group of seniors in the world 's population continues to grow , age - related neurodegenerative disorders , including tauopathies such as alzheimer 's disease , are becoming more prevalent , and pose a serious threat to an already overwhelmed health care system . despite the vast amount of research already performed , several aspects of tauopathies still await molecular elucidation . we developed a yeast model expressing human protein tau variants , and demonstrated that this model recapitulates many important aspects implicated in tau pathology , including hyperphosphorylation , conformation , and aggregation . combined with the ease of genetic manipulations , rapid genome - wide screening methods , and other advantages characteristic of yeast systems , this model may prove its value in the clarification of fundamental cellular processes involved in tau biology and pathology .

the shift in therapeutic concepts from resection to regeneration has significantly impacted the practice of periodontology in last quarter of the century . the key to tissue regeneration is to stimulate a cascade of healing events which , if coordinated , can result in completion of integrated tissue formation . although the achievement of the goal of complete regeneration of the periodontal tissues may not be possible for many years , recent developments in nanomaterials and nanotechnology have provided a promising insight into the commercial applications of nanomaterials in the management of periodontal diseases e.g. , nanocrystalline hydroxyapatite ( nha ) paste ( ostim , heraeus kulzer , hanau , germany ) containing 65% water and 35% nanostructured apatite particles has widely been used for augmentation procedures in osseous defects . nanomaterials have significant surface effects , size effects , quantum effects , and exhibit much better performance properties than traditional materials . another form of synthetic nha is obtained from chemical precipitation using aqueous solution of calcium nitrate tetrahydrate and ammonium dihydrogen phosphate by hydrothermal treatment . advantages of this material are the close contact with surrounding tissues , quick resorption characteristics and large number of molecules on the surface . nanophase ha also can promote proliferation and osteogenic differentiation of periodontal ligament cells and further it may be used as a bioresorbable agent in osseous restoration . therefore , in view of various positive effects of nha in bone regeneration the current study was designed to clinically and radiographically evaluate the efficacy of nha and synthetic resorbable ha graft in the treatment of intrabony defects . a clinical and radiographic study was carried out to assess the efficacy of treatment of human intrabony defects with nha ( particle size : ~20 nm ) ( group a ) and with conventional ha ( particle size : 15 - 35 10 nm ) ( group b ) in the treatment of human intrabony defects . ten patients ( 8 males and 2 females ) aged between 20 and 50 years with moderate to advanced chronic periodontitis with bilateral clinical and radiographic evidence of angular defects were recruited for the study . moderate to severe periodontitis , diagnosed on the basis of bleeding on probing , probing depth , and clinical attachment losspatients having pocket depth of 6 - 9 mmpatients with vertical intrabony component of 4 mm . moderate to severe periodontitis , diagnosed on the basis of bleeding on probing , probing depth , and clinical attachment loss patients having pocket depth of 6 - 9 mm patients with vertical intrabony component of 4 mm . any systemic disease that might affect the periodontiumany recent periodontal surgery within 6 monthssmokinguncooperative patients . any systemic disease that might affect the periodontium any recent periodontal surgery within 6 months uncooperative patients . the patients selected on the above criteria were then explained about the treatment procedure and the associated risks and benefits and their written consent was obtained . four weeks following phase i therapy a periodontal re - evaluation was performed to confirm the suitability of the sites for the study . the following recordings were made at baseline demographic data , medical history , dental history , and personal historyclinical examination of the dentitionparameters : bleeding on probing ( ainamo and bay)gingival index ( loe and sillness)plaque index ( sillness and loe)oral hygiene index s ( greene and vermillion)probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . ( hu - friedy)}. a wire was placed on the bucco - occlusal line angle of teeth during fabrication of stent . this wire was then exposed by making grooves in the inter - dental regions , such that the probe when inserted in the inter - proximal area was in contact with the wire ; hence , the probe position and angulation remained the same for all pre and post operative measurements . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recordeddigital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . the size of the defect was calculated using the rvg software x - ray vision apetryx . the infrabony component was assessed by identifying the parameters and calculated as stated by eickholz . demographic data , medical history , dental history , and personal history clinical examination of the dentition bleeding on probing ( ainamo and bay)gingival index ( loe and sillness)plaque index ( sillness and loe)oral hygiene index s ( greene and vermillion)probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . ( hu - friedy)}. a wire was placed on the bucco - occlusal line angle of teeth during fabrication of stent . this wire was then exposed by making grooves in the inter - dental regions , such that the probe when inserted in the inter - proximal area was in contact with the wire ; hence , the probe position and angulation remained the same for all pre and post operative measurements . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recordeddigital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . the size of the defect was calculated using the rvg software x - ray vision apetryx . the infrabony component was assessed by identifying the parameters and calculated as stated by eickholz . bleeding on probing ( ainamo and bay ) gingival index ( loe and sillness ) plaque index ( sillness and loe ) oral hygiene index s ( greene and vermillion ) probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . ( hu - friedy)}. a wire was placed on the bucco - occlusal line angle of teeth during fabrication of stent . this wire was then exposed by making grooves in the inter - dental regions , such that the probe when inserted in the inter - proximal area was in contact with the wire ; hence , the probe position and angulation remained the same for all pre and post operative measurements . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recorded digital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . the size of the defect was calculated using the rvg software x - ray vision apetryx . the infrabony component was assessed by identifying the parameters and calculated as stated by eickholz . the experimental material was synthesized and characterized at smart materials research laboratory , department of physics , iit roorkee , india . 0.5 m ca ( no3).24h2o ( merck ) in ethanol with a ph 10.5 was added to 0.5 m ammonium phosphate [ ( nh4)2 po4](merck ) slowly at a rate of 8ml / min and at constant temperature ( 80c ) with vigorous stirring . the resultant sol - gel was continuously stirred at a constant ph of 10 was kept constant by adding ca ( oh)2 solution and a constant temperature of 70c for 5 hours . after allowing the product to cool , it was kept inside the oven at 50c overnight . the formation and quality of the synthesized compound as prepared and heat treated at 350c for 2.5 was studied using an x - ray diffractometer ( xrd ) ( pw1140/90 ) , using cu k(=0.15418 nm ) in a wide range of bragg angles ( 10-2-70 ) at room temperature . scanning electron micrographs were obtained with scanning electron microscope ( sem ) leo vp stereoscan . elemental analysis of the synthesized powder was determined by energy dispersive x - ray analysis ( edax ) attached with the sem . the stochiometric ( ca / p ) ratio of the as synthesized sample was determined by edax . it was found that ca / p ratios nearly coincide with the theoretical value ( 1.67 ) of ha . x - ray crystallography is a method of determining the arrangement of atoms within a crystal , in which a beam of x - rays strikes a crystal and diffracts into many specific directions . x - ray diffraction techniques play an important role in the analysis of crystallite sizes . the patterns due to the as - prepared material bear with it the characteristic patterns of ha but not with much resolution and intensity . the broad patterns around at 30 indicate that the crystallites are very tiny ( ~20 nm ) in nature with much atomic oscillations . here the mother liquor is expected to permit selective growth of multitudes of ha crystallites with high rate thus avoiding orderly growth of large crystals . the xrd patterns of the heat treated material at 350c show an increase in intensity of the diffraction peaks . there are many spherical agglomerations and crystallites of submicrometric in size with a tendency to agglomerate leaving submicrometric pores in between . local anesthesia ( 2% lidocaine , epinephrine 1 : 100,000 ) was injected in the site of surgery . crevicular incision was given in the sextent of the defect and a mucoperiosteal flap was raised . the area was degranulated , curetted , and irrigation was done with diluted betadine solution . defect was isolated and the graft ( nha or ha ) was wetted in patient 's own blood and placed in small increments in the defect using an amalgam carrier and condensed until the defect was filled . the sutures were then tightened over the defect site and also placed in the adjacent sites so as to ensure complete approximation of the flaps . following this a periodontal pack was applied over the site [ figures 1 and 2 ] . ( c ) clinical view of the intrabony defect after debridement ( a ) nha powder in place prior to suturing . ( c ) healing at 6 month postoperatively the data regarding the clinical and radiographic parameters was tabulated and subjected to statistical analysis . student 's paired t - test was used to compare data from baseline to that of 3 months and from baseline to 6 months for each treatment group . comparison between the test and the control group ( intergroup ) at baseline 3 months and 6 months were accomplished using student 's unpaired t - test . moderate to severe periodontitis , diagnosed on the basis of bleeding on probing , probing depth , and clinical attachment losspatients having pocket depth of 6 - 9 mmpatients with vertical intrabony component of 4 mm . moderate to severe periodontitis , diagnosed on the basis of bleeding on probing , probing depth , and clinical attachment loss patients having pocket depth of 6 - 9 mm patients with vertical intrabony component of 4 mm . any systemic disease that might affect the periodontiumany recent periodontal surgery within 6 monthssmokinguncooperative patients . any systemic disease that might affect the periodontium any recent periodontal surgery within 6 months uncooperative patients . the patients selected on the above criteria were then explained about the treatment procedure and the associated risks and benefits and their written consent was obtained . four weeks following phase i therapy a periodontal re - evaluation was performed to confirm the suitability of the sites for the study . the following recordings were made at baseline demographic data , medical history , dental history , and personal historyclinical examination of the dentitionparameters : bleeding on probing ( ainamo and bay)gingival index ( loe and sillness)plaque index ( sillness and loe)oral hygiene index s ( greene and vermillion)probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . ( hu - friedy)}. a wire was placed on the bucco - occlusal line angle of teeth during fabrication of stent . this wire was then exposed by making grooves in the inter - dental regions , such that the probe when inserted in the inter - proximal area was in contact with the wire ; hence , the probe position and angulation remained the same for all pre and post operative measurements . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recordeddigital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . the size of the defect was calculated using the rvg software x - ray vision apetryx . the infrabony component was assessed by identifying the parameters and calculated as stated by eickholz . demographic data , medical history , dental history , and personal history clinical examination of the dentition bleeding on probing ( ainamo and bay)gingival index ( loe and sillness)plaque index ( sillness and loe)oral hygiene index s ( greene and vermillion)probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . ( hu - friedy)}. a wire was placed on the bucco - occlusal line angle of teeth during fabrication of stent . this wire was then exposed by making grooves in the inter - dental regions , such that the probe when inserted in the inter - proximal area was in contact with the wire ; hence , the probe position and angulation remained the same for all pre and post operative measurements . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recordeddigital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . the size of the defect was calculated using the rvg software x - ray vision apetryx . the infrabony component was assessed by identifying the parameters and calculated as stated by eickholz . bleeding on probing ( ainamo and bay ) gingival index ( loe and sillness ) plaque index ( sillness and loe ) oral hygiene index s ( greene and vermillion ) probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . ( hu - friedy)}. a wire was placed on the bucco - occlusal line angle of teeth during fabrication of stent . this wire was then exposed by making grooves in the inter - dental regions , such that the probe when inserted in the inter - proximal area was in contact with the wire ; hence , the probe position and angulation remained the same for all pre and post operative measurements . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recorded digital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . the size of the defect was calculated using the rvg software x - ray vision apetryx . the infrabony component was assessed by identifying the parameters and calculated as stated by eickholz . the experimental material was synthesized and characterized at smart materials research laboratory , department of physics , iit roorkee , india . 0.5 m ca ( no3).24h2o ( merck ) in ethanol with a ph 10.5 was added to 0.5 m ammonium phosphate [ ( nh4)2 po4](merck ) slowly at a rate of 8ml / min and at constant temperature ( 80c ) with vigorous stirring . the resultant sol - gel was continuously stirred at a constant ph of 10 was kept constant by adding ca ( oh)2 solution and a constant temperature of 70c for 5 hours . after allowing the product to cool , it was kept inside the oven at 50c overnight . the formation and quality of the synthesized compound as prepared and heat treated at 350c for 2.5 was studied using an x - ray diffractometer ( xrd ) ( pw1140/90 ) , using cu k(=0.15418 nm ) in a wide range of bragg angles ( 10-2-70 ) at room temperature . scanning electron micrographs elemental analysis of the synthesized powder was determined by energy dispersive x - ray analysis ( edax ) attached with the sem . the stochiometric ( ca / p ) ratio of the as synthesized sample was determined by edax . it was found that ca / p ratios nearly coincide with the theoretical value ( 1.67 ) of ha . x - ray crystallography is a method of determining the arrangement of atoms within a crystal , in which a beam of x - rays strikes a crystal and diffracts into many specific directions . x - ray diffraction techniques play an important role in the analysis of crystallite sizes . the patterns due to the as - prepared material bear with it the characteristic patterns of ha but not with much resolution and intensity . the broad patterns around at 30 indicate that the crystallites are very tiny ( ~20 nm ) in nature with much atomic oscillations . here the mother liquor is expected to permit selective growth of multitudes of ha crystallites with high rate thus avoiding orderly growth of large crystals . the xrd patterns of the heat treated material at 350c show an increase in intensity of the diffraction peaks . there are many spherical agglomerations and crystallites of submicrometric in size with a tendency to agglomerate leaving submicrometric pores in between . local anesthesia ( 2% lidocaine , epinephrine 1 : 100,000 ) was injected in the site of surgery . crevicular incision was given in the sextent of the defect and a mucoperiosteal flap was raised . the area was degranulated , curetted , and irrigation was done with diluted betadine solution . defect was isolated and the graft ( nha or ha ) was wetted in patient 's own blood and placed in small increments in the defect using an amalgam carrier and condensed until the defect was filled . the sutures were then tightened over the defect site and also placed in the adjacent sites so as to ensure complete approximation of the flaps . following this a periodontal pack was applied over the site [ figures 1 and 2 ] . ( c ) clinical view of the intrabony defect after debridement ( a ) nha powder in place prior to suturing . the data regarding the clinical and radiographic parameters was tabulated and subjected to statistical analysis . student 's paired t - test was used to compare data from baseline to that of 3 months and from baseline to 6 months for each treatment group . comparison between the test and the control group ( intergroup ) at baseline 3 months and 6 months were accomplished using student 's unpaired t - test . twenty subjects ( n = 10 in each group ) completed the 6-month follow - up period . in all treated sites , radiographs and clinical photographs for nha and ha are shown in figures 1 and 2 . the mean and standard deviation scores at baseline , 3 months , and 6 months for test and control groups are summarized in table 1 . no statistically significant differences were found between the groups for any of the investigated parameters at baseline . intra group comparison by means of paired t test from baseline to 3 months and from baseline to 6 months and from 3 to 6 months for each treatment group is summarized in table 2 . meansd scores at baseline , 3 months , and 6 months ( n=10 subjects in each group ) for test and control groups paired t test data from baseline to that of 3 months and from baseline to 6 months and from 3 to 6 months for each treatment group following treatment , pi values remained low throughout the study period in both groups . on comparison of the mean difference in pd between the two groups at baseline , 3 months and 6 months , scores were found to be statistically insignificant ( p > 0.01 ) , as the calculated t values at baseline , 3 months , and 6 months were 1.9384 , 0.88465 , and 1.38054 ( ttab 2.88 ) [ table 3 ] . unpaired t test data from baseline to that of 3 months and from baseline to 6 months and from 3 to 6 months for each treatment group on comparison of the mean difference in relative attachment level between the two groups after the unpaired t test was applied at baseline , 3 months , and 6 months , scores were found to be statistically non - significant ( p > 0.01 ) , as the calculated t values at baseline , 3 months , and 6 months were 2.031 , 1.878 , and 1.897 ( ttab 2.88 ) [ table 3 ] . on comparison of the mean difference in radiographic measurements between the two groups after the unpaired t test was applied at baseline , 3 months , and 6 months , scores were found to be statistically non - significant ( p > 0.01 ) , as the calculated t values at baseline , 3 months , and 6 months were 0.17 , 0.46 , and 0.2410 ( ttab 2.88 ) [ table 3 ] . conventional ha graft ( periobone g ) , a bioceramic resorbable alloplast , with a particle size of 150 - 250 used in the current study served as a control group . the results of the present study demonstrated an improvement in clinical and radiographic parameters with use of ha graft . the difference in the oral hygiene status from baseline to 3 months and 6 months respectively and from 3 months to 6 months were statistically insignificant . also reported no effect on the plaque index when ha graft was used in treatment of intrabony defects . the above studies , like the present followed strict plaque control and maintenance of oral hygiene throughout the study period . these observations suggest that ha was well tolerated in the hard and soft tissues and does not seem to evoke any inflammatory response significantly . the similar results have been confirmed clinically by kenny et al . who reported uneventful post - operative healing . the ha was well tolerated by the gingival tissues during initial healing and thereafter including 6-month evaluation period . the present study demonstrates a decrease in pd from baseline to 3 months and 6 months with difference of 3.25 1.2304 mm and 3.75 1.6874 mm respectively which was found to be statistically significant . these findings signify the use of ha graft in the clinical resolution of intrabony defects . also , kenny et al . reported in the re - entry data that there was a measurable improvement in the gain of attachment level which was duplicated with an equivalent improvement in the depth of the defect seen at re - entry . in accordance with the observations of the current study , yukna et al . also reported a decrease in pocket depth of 2.8 1.4 mm and gain in clinical attachment level of 1 0.1 mm . was of 5 years during which some recurrence might have occurred due to the lack of oral hygiene maintenance by the patients . the improvement in the clinical probing and relative attachment level was well supported by the decrease in the radiographic area of the intrabony defect in the control group of the present study , which was determined in a manner similar to that as described by eickholz et al . the observations of the present study show a decrease in the size of the defect from baseline to 3 and 6 months respectively to be statistically significant . also , the difference from 3 months to 6 months was statistically significant . the increase in the radiodensity in the defect , and hence a decrease in the defect size , signifies that use of ha graft results in resolution of the intrabony defect . however , the nature of the restoration of the defect that whether the graft acted as a filler material or allowed for ingrowth of the bone can not be inferred from the clinical and radiographic observations of the present study . evaluation of the true nature of attachment requires histological investigation . in accordance with the radiological observations of the current study , okuda et al . and scabbia et al . evaluated the depth of the intrabony defect radiographically using the same landmarks used in the current study after a period of 12 months and reported significant gain in the bone height and reduction of the depth of the defect . osteoconductivity , solubility , sinterability , and mechanical reliability of ha can be enhanced by controlling its particle size and structural morphology in the order of a nanoscale . nha which has been used as a test material in the present study posseses exceptional biocompatibility and bioactivity properties with respect to bone cells and tissues , probably due to its similarity with the hard tissues of the body . beside a good biocompatibility , a synthetic bone substitute should also ensure the formation of new bone after their implantation . it would seem rational to suggest that particles too large in size will resorb at a slower rate and offer a overall reduced surface area , while particles too small in size may induce inflammation , be readily resorbed or phagocytosed and result in an interparticulate space of a reduced dimension that would not be conducive to cellular migration and ingrowth . in fact , an optimal synthetic bone substitute is resorbed by hydrolytic and cellular degradation process involving the action of macrophages and further is replaced by vital bone over time . a major prerequisite for this process is angiogenesis because newly formed blood vessels transport oxygen and nutrients into the implanted bone substitute , a physiological milieu is created , which supports the ingrowth of bone cells and also differentiation of pluripotent stem cells from the surrounding tissue to an osteoblastic phenotype . however , 2 weeks is too early a rate of resorption and clinical results following and any gain in clinical attachment level or decrease in an intrabony defect is highly unexpected . in contrast , our study showed a significant decrease in pocket pd and decrease in radiographic area of defect . furthur laschke also reported that within these areas , vascularized granulation tissue could directly invade the biomaterial . this guided vascularization may accelerate formation of new bone in bone defects , because osteoblasts are facilitated to migrate into these vascularized areas where the remaining nondegraded fragments serve as scaffold for invading cells . in this process , the newly developing blood vessels play an important role , because it has previously been reported that endothelial cells stimulate the differentiation of preosteoblasts to osteoblasts by expression of osteotropic growth factors , such as endothelin-1 and insulin like growth factor ( igf -1 ) . vice - versa , the expression of vascular endothelial growth factor ( vegf ) by osteoblasts further sustains the proliferation of endothelial cells and thus the formation of new blood vessels . vitro study demonstrated that nha can promote periodontal ligament fibroblast proliferation and osteogenic differentiation in comparison with dense ha . furthermore , they reported that the increased proliferation capability of periodontal ligament fibroblasts under the influence of nanometer order ha indicated that latter had better compatibility and resorbability than dense ha . nanostructured ha promotes up - regulation of fibroblast growth factor ( fgf)-2 and primes endothelial cells to vegf action . fgf-2 plays a biological pleiotropic role , including cell migration , angiogenesis , bone development and repair . also , a synthetic bone substitute must not only support the growth and foster the phenotype of the cell type for the tissue it is to replace ( i.e. , osteoblasts ) , but also support the cells that are responsible for maintaining the bone cells ( i.e. , fibroblasts ) the upregulation of fgf-2 through gene transcription is an important event which explains the role of ha nanocrystals in inducing a positive and controlled angiogenic phenotype in endothelium . the robust increase of fgf-2 m rna ( 3 - 6 fold ) translates into a significant production of the soluble fgf-2 isoform . in the current study it was decided to use powder form of nha which served as the test group of the study . the results of the test group demonstrate statistically insignificant difference in plaque scores from 3 to 6 months . a similar trend was seen in the simplified oral hygiene index scores and gingival index scores . on comparison of the test group with the control no significant difference was observed , which showed that nha did not have any adjunctive effect in improving the plaque scores or the oral hygiene of the patient . these factors were more influenced by the fact that bilateral defects were chosen and patient 's own maintenance which was reinforced timely during the recall periods . in the present study there was statistically significant difference in the pd and relative attachment level between baseline and 3 months and baseline and 6 months in the test group . but there was no significant difference between 3 months and 6 months in the test group . similar to the other parameters , no significant improvement of the test group was seen over the control group . these observations parallel that of the study of kasaj et al . who reported significant improvement in pd and clinical attachment level ( cal ) at 6 months after surgery compared to baseline . in a previous study evaluating the healing of intrabony peri - implantitis defects following an application of an nha paste as a graft or a bovine derived xenograft in combination with a collagen membrane , both treatment procedures produced clinically significant pd reductions and cal gains . the use of nha in the treatment of periodontal defects was evaluated in the pioneer study of zuev et al . who found ostim to be not inferior to bone transplant and devoid of its shortcomings in the treatment of 395 patients with periodontal defects including mainly periodontal abcesses . later kasaj et al . stated that at 6 months after surgery , the treatment of intrabony periodontal defects with an nha paste produced clinically and statistically significant pd reductions and cal gains compared to open flap debridement alone . on compared clinical outcomes of papilla preservation flap surgery with or without the application of a novel nha bone graft substitute . they observed that after 6 months statistically significant reduction in probing pocket depths and gain in probing bone levels in the test group . schnettler et al . found that nha binds to the bone and stimulates bone healing by stimulation of osteoblast activity . the current study also demonstrates a statistically significant reduction in area of defect from baseline to 6 months and from 3 to 6 months . however , the decrease in area from baseline to 3 months was statistically insignificant . this might be because of the very small particle size and early resorption within 12 weeks as reported by thorwarth et al . who used microradiography to assess the mineralization content and degradation of the test material . they stated that the accelerated initial ceramolysis of the material did not hinder the bone healing process which follows the principles of a primary angiogenic reossification . they stated that newly formed bone was discriminated from local bone by lower degree of mineralization , the different architecture of spongiosa and lack of alignment of the trabecular trajectory . the present study coincides with the above study and the clinical findings in the present study also reveal evidence of regeneration after 12 weeks ( 3 months ) . also , no additional effect of the test group was noted over the control group . similar findings were reported by klawitter et al . who stated that trabeculae of bone vary in size from 20 to over 100 m . when a trabecula reaches about 100 m , it carries its own blood vessels , much in the same way an osteon does via haversian canal . thus , to support trabecular bone ingrowth , the pores would need to be at least 40 - 100 m , and to support osteonal bone ingrowth , pores of at least 100 m would appear necessary . later hirschorn reported that particle size of about 380 m in diameter would yield this minimal dimension and particles yielding under 100 m space dimensions may possess less mineralization potential . therefore in the present study the tested material i.e. , nha which had a particle size of ~20 nm shows statistically insignificant results over the control group . in view of above , fucini et al . found that there was no difference in defect fill between demineralized freeze dried bone allograft particles of 250 - 500 m size compared to those of 850 - 1000 m . in vitro analysis of the interparticulate space among bone replacement grafts condensed under a uniform standard force showed that autogenous bone harvested low and high speed rotary instruments , freeze dried bone allograft ( 250 - 710 m ) . bio - oss ( cancellous and cortical ) , osteograf ld , perioglas and osteogen yielded a 40 - 100 m interparticulate space . autogenous bone harvested by back action chisels , demineralized freeze dried bone allograft ( 350 - 500 m ) . osteograf n ( 300 - 700 m ) , biocoral , biogran , interpore 200 and calcitite ( 40 - 60 m ) ha granules yielded an interparticulate space that was equal to or greater than 100 m . another important property of nanomaterials which might have played a major role in regeneration in the current study is development of self assembly which helps to integrate different functions into synthetic extracellular matrices . these synthetic extracellular matrices will need to perform functions such as to sustain cell viability and proliferation , allow the establishment of a blood vessel network formation and provide sufficient support to prevent tissue collapse . in recent times , developments in this field have seen the use of ph - induced self - assembly of a peptide - amphiphile to artificially construct a nanostructured fibrous scaffold with the structural features of extracellular matrices . after cross linking , the newly produced fibers are able to direct mineralization of ha to form a composite material in which the crystallographic axes of ha are aligned with the long axes of the fibers . this alignment is similar as that observed in vivo between collagen fibrils and ha crystals in bone . despite good clinical results the current study was not subjected to histological analysis . also , differences in the physiochemical and structural characteristics between nha and ha used in the past and in the present study may lead to differences in the regenerative / osteoconductive properties . material properties including porosity , surface geometry , and surface chemistry play a role in determining the osteoconductive capacities of a graft which must be analyzed histologically . further studies using more subjects and histologic analysis could clarify the benefits of the new synthetic bone grafting material . in addition , a longer post treatment observation interval may be needed to confirm the stability of clinical outcomes .

background : the aim of this study is to compare , clinically and radiographically , the effectiveness of nanocrystalline hydroxyapatite ( nha ) and synthetic resorbable hydroxyapatite ( ha ) in the treatment of intrabony defects.materials and methods : ten subjects with bilateral defects , with probing depth ( pd ) 6 - 9 mm and radiographic evidence of an intraosseous component 4 mm participated in the present study . subjects were allocated randomly to treatment with nha ( test group ) or ha ( control group ) . at baseline , 3 and 6 months after surgery , the following clinical parameters were recorded : plaque index , gingival index , pd , relative attachment level ( ral ) , and radiographic reduction in intrabony defect.results:at 6 months following therapy , the test group showed a reduction in mean pd from 6.4 0.843 to 3.3 0.8232 mm and a change in mean ral from 12.9 1.197 to 10.1 0.7378 mm , whereas in the control group the mean pd decreased from 7.65 1.8566 to 3.9 1.1005 mm , and mean ral decreased from 13.9 0.9944 to 10.7 0.6749 mm . on comparison of the mean difference in probing depth between the two groups after the unpaired t - test was applied at baseline , 3 months and 6 months , scores were found to be statistically non - significant ( p > 0.01).conclusion : the results of the present study indicate that both nha and conventional ha led to the improvement of clinical and radiographic parameters over the course of the study . however , the test group did not show any significant improvement over the control group .

INTRODUCTION MATERIALS AND METHODS Inclusion criteria Exclusion criteria Preparation of nanocrystalline hydroxyapatite Determination of Ca/P ratio X-ray diffraction patterns Scanning electron microscopy Surgical procedure Statistical analysis RESULTS DISCUSSION CONCLUSION

although the achievement of the goal of complete regeneration of the periodontal tissues may not be possible for many years , recent developments in nanomaterials and nanotechnology have provided a promising insight into the commercial applications of nanomaterials in the management of periodontal diseases e.g. , nanocrystalline hydroxyapatite ( nha ) paste ( ostim , heraeus kulzer , hanau , germany ) containing 65% water and 35% nanostructured apatite particles has widely been used for augmentation procedures in osseous defects . therefore , in view of various positive effects of nha in bone regeneration the current study was designed to clinically and radiographically evaluate the efficacy of nha and synthetic resorbable ha graft in the treatment of intrabony defects . a clinical and radiographic study was carried out to assess the efficacy of treatment of human intrabony defects with nha ( particle size : ~20 nm ) ( group a ) and with conventional ha ( particle size : 15 - 35 10 nm ) ( group b ) in the treatment of human intrabony defects . ten patients ( 8 males and 2 females ) aged between 20 and 50 years with moderate to advanced chronic periodontitis with bilateral clinical and radiographic evidence of angular defects were recruited for the study . moderate to severe periodontitis , diagnosed on the basis of bleeding on probing , probing depth , and clinical attachment losspatients having pocket depth of 6 - 9 mmpatients with vertical intrabony component of 4 mm . moderate to severe periodontitis , diagnosed on the basis of bleeding on probing , probing depth , and clinical attachment loss patients having pocket depth of 6 - 9 mm patients with vertical intrabony component of 4 mm . four weeks following phase i therapy a periodontal re - evaluation was performed to confirm the suitability of the sites for the study . the following recordings were made at baseline demographic data , medical history , dental history , and personal historyclinical examination of the dentitionparameters : bleeding on probing ( ainamo and bay)gingival index ( loe and sillness)plaque index ( sillness and loe)oral hygiene index s ( greene and vermillion)probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recordeddigital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . demographic data , medical history , dental history , and personal history clinical examination of the dentition bleeding on probing ( ainamo and bay)gingival index ( loe and sillness)plaque index ( sillness and loe)oral hygiene index s ( greene and vermillion)probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . this wire was then exposed by making grooves in the inter - dental regions , such that the probe when inserted in the inter - proximal area was in contact with the wire ; hence , the probe position and angulation remained the same for all pre and post operative measurements . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recordeddigital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . bleeding on probing ( ainamo and bay ) gingival index ( loe and sillness ) plaque index ( sillness and loe ) oral hygiene index s ( greene and vermillion ) probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recorded digital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . the formation and quality of the synthesized compound as prepared and heat treated at 350c for 2.5 was studied using an x - ray diffractometer ( xrd ) ( pw1140/90 ) , using cu k(=0.15418 nm ) in a wide range of bragg angles ( 10-2-70 ) at room temperature . the broad patterns around at 30 indicate that the crystallites are very tiny ( ~20 nm ) in nature with much atomic oscillations . crevicular incision was given in the sextent of the defect and a mucoperiosteal flap was raised . defect was isolated and the graft ( nha or ha ) was wetted in patient 's own blood and placed in small increments in the defect using an amalgam carrier and condensed until the defect was filled . the sutures were then tightened over the defect site and also placed in the adjacent sites so as to ensure complete approximation of the flaps . ( c ) clinical view of the intrabony defect after debridement ( a ) nha powder in place prior to suturing . ( c ) healing at 6 month postoperatively the data regarding the clinical and radiographic parameters was tabulated and subjected to statistical analysis . student 's paired t - test was used to compare data from baseline to that of 3 months and from baseline to 6 months for each treatment group . comparison between the test and the control group ( intergroup ) at baseline 3 months and 6 months were accomplished using student 's unpaired t - test . moderate to severe periodontitis , diagnosed on the basis of bleeding on probing , probing depth , and clinical attachment losspatients having pocket depth of 6 - 9 mmpatients with vertical intrabony component of 4 mm . moderate to severe periodontitis , diagnosed on the basis of bleeding on probing , probing depth , and clinical attachment loss patients having pocket depth of 6 - 9 mm patients with vertical intrabony component of 4 mm . four weeks following phase i therapy a periodontal re - evaluation was performed to confirm the suitability of the sites for the study . the following recordings were made at baseline demographic data , medical history , dental history , and personal historyclinical examination of the dentitionparameters : bleeding on probing ( ainamo and bay)gingival index ( loe and sillness)plaque index ( sillness and loe)oral hygiene index s ( greene and vermillion)probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . this wire was then exposed by making grooves in the inter - dental regions , such that the probe when inserted in the inter - proximal area was in contact with the wire ; hence , the probe position and angulation remained the same for all pre and post operative measurements . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recordeddigital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . demographic data , medical history , dental history , and personal history clinical examination of the dentition bleeding on probing ( ainamo and bay)gingival index ( loe and sillness)plaque index ( sillness and loe)oral hygiene index s ( greene and vermillion)probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . this wire was then exposed by making grooves in the inter - dental regions , such that the probe when inserted in the inter - proximal area was in contact with the wire ; hence , the probe position and angulation remained the same for all pre and post operative measurements . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recordeddigital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . bleeding on probing ( ainamo and bay ) gingival index ( loe and sillness ) plaque index ( sillness and loe ) oral hygiene index s ( greene and vermillion ) probing depth ( pd ) and relative attachment level : an occlusal stent was fabricated for positioning of the periodontal probe { university of north carolina no . using the groove and wire as a guide , the periodontal probe was inserted into the gingival sulcus and pd ( using the gingival margin as reference ) , and relative attachment level ( using the stent wire as reference ) was recorded digital radiography : radiographs were taken using the rinn xcp system ( dentsply , usa ) by the standardized paralleling technique with the digital radiovisiography ( rvg ) ( suni ray suni imaging micro system inc . ) at baseline , 3 months and 6 months post - operatively . the formation and quality of the synthesized compound as prepared and heat treated at 350c for 2.5 was studied using an x - ray diffractometer ( xrd ) ( pw1140/90 ) , using cu k(=0.15418 nm ) in a wide range of bragg angles ( 10-2-70 ) at room temperature . the broad patterns around at 30 indicate that the crystallites are very tiny ( ~20 nm ) in nature with much atomic oscillations . crevicular incision was given in the sextent of the defect and a mucoperiosteal flap was raised . defect was isolated and the graft ( nha or ha ) was wetted in patient 's own blood and placed in small increments in the defect using an amalgam carrier and condensed until the defect was filled . the sutures were then tightened over the defect site and also placed in the adjacent sites so as to ensure complete approximation of the flaps . the data regarding the clinical and radiographic parameters was tabulated and subjected to statistical analysis . student 's paired t - test was used to compare data from baseline to that of 3 months and from baseline to 6 months for each treatment group . comparison between the test and the control group ( intergroup ) at baseline 3 months and 6 months were accomplished using student 's unpaired t - test . the mean and standard deviation scores at baseline , 3 months , and 6 months for test and control groups are summarized in table 1 . no statistically significant differences were found between the groups for any of the investigated parameters at baseline . intra group comparison by means of paired t test from baseline to 3 months and from baseline to 6 months and from 3 to 6 months for each treatment group is summarized in table 2 . meansd scores at baseline , 3 months , and 6 months ( n=10 subjects in each group ) for test and control groups paired t test data from baseline to that of 3 months and from baseline to 6 months and from 3 to 6 months for each treatment group following treatment , pi values remained low throughout the study period in both groups . on comparison of the mean difference in pd between the two groups at baseline , 3 months and 6 months , scores were found to be statistically insignificant ( p > 0.01 ) , as the calculated t values at baseline , 3 months , and 6 months were 1.9384 , 0.88465 , and 1.38054 ( ttab 2.88 ) [ table 3 ] . unpaired t test data from baseline to that of 3 months and from baseline to 6 months and from 3 to 6 months for each treatment group on comparison of the mean difference in relative attachment level between the two groups after the unpaired t test was applied at baseline , 3 months , and 6 months , scores were found to be statistically non - significant ( p > 0.01 ) , as the calculated t values at baseline , 3 months , and 6 months were 2.031 , 1.878 , and 1.897 ( ttab 2.88 ) [ table 3 ] . on comparison of the mean difference in radiographic measurements between the two groups after the unpaired t test was applied at baseline , 3 months , and 6 months , scores were found to be statistically non - significant ( p > 0.01 ) , as the calculated t values at baseline , 3 months , and 6 months were 0.17 , 0.46 , and 0.2410 ( ttab 2.88 ) [ table 3 ] . conventional ha graft ( periobone g ) , a bioceramic resorbable alloplast , with a particle size of 150 - 250 used in the current study served as a control group . the results of the present study demonstrated an improvement in clinical and radiographic parameters with use of ha graft . the difference in the oral hygiene status from baseline to 3 months and 6 months respectively and from 3 months to 6 months were statistically insignificant . also reported no effect on the plaque index when ha graft was used in treatment of intrabony defects . the above studies , like the present followed strict plaque control and maintenance of oral hygiene throughout the study period . the present study demonstrates a decrease in pd from baseline to 3 months and 6 months with difference of 3.25 1.2304 mm and 3.75 1.6874 mm respectively which was found to be statistically significant . these findings signify the use of ha graft in the clinical resolution of intrabony defects . reported in the re - entry data that there was a measurable improvement in the gain of attachment level which was duplicated with an equivalent improvement in the depth of the defect seen at re - entry . also reported a decrease in pocket depth of 2.8 1.4 mm and gain in clinical attachment level of 1 0.1 mm . the improvement in the clinical probing and relative attachment level was well supported by the decrease in the radiographic area of the intrabony defect in the control group of the present study , which was determined in a manner similar to that as described by eickholz et al . the observations of the present study show a decrease in the size of the defect from baseline to 3 and 6 months respectively to be statistically significant . also , the difference from 3 months to 6 months was statistically significant . the increase in the radiodensity in the defect , and hence a decrease in the defect size , signifies that use of ha graft results in resolution of the intrabony defect . however , the nature of the restoration of the defect that whether the graft acted as a filler material or allowed for ingrowth of the bone can not be inferred from the clinical and radiographic observations of the present study . evaluated the depth of the intrabony defect radiographically using the same landmarks used in the current study after a period of 12 months and reported significant gain in the bone height and reduction of the depth of the defect . osteoconductivity , solubility , sinterability , and mechanical reliability of ha can be enhanced by controlling its particle size and structural morphology in the order of a nanoscale . nha which has been used as a test material in the present study posseses exceptional biocompatibility and bioactivity properties with respect to bone cells and tissues , probably due to its similarity with the hard tissues of the body . however , 2 weeks is too early a rate of resorption and clinical results following and any gain in clinical attachment level or decrease in an intrabony defect is highly unexpected . in the current study it was decided to use powder form of nha which served as the test group of the study . the results of the test group demonstrate statistically insignificant difference in plaque scores from 3 to 6 months . on comparison of the test group with the control no significant difference was observed , which showed that nha did not have any adjunctive effect in improving the plaque scores or the oral hygiene of the patient . in the present study there was statistically significant difference in the pd and relative attachment level between baseline and 3 months and baseline and 6 months in the test group . but there was no significant difference between 3 months and 6 months in the test group . similar to the other parameters , no significant improvement of the test group was seen over the control group . who reported significant improvement in pd and clinical attachment level ( cal ) at 6 months after surgery compared to baseline . the use of nha in the treatment of periodontal defects was evaluated in the pioneer study of zuev et al . who found ostim to be not inferior to bone transplant and devoid of its shortcomings in the treatment of 395 patients with periodontal defects including mainly periodontal abcesses . stated that at 6 months after surgery , the treatment of intrabony periodontal defects with an nha paste produced clinically and statistically significant pd reductions and cal gains compared to open flap debridement alone . they observed that after 6 months statistically significant reduction in probing pocket depths and gain in probing bone levels in the test group . the current study also demonstrates a statistically significant reduction in area of defect from baseline to 6 months and from 3 to 6 months . however , the decrease in area from baseline to 3 months was statistically insignificant . the present study coincides with the above study and the clinical findings in the present study also reveal evidence of regeneration after 12 weeks ( 3 months ) . also , no additional effect of the test group was noted over the control group . thus , to support trabecular bone ingrowth , the pores would need to be at least 40 - 100 m , and to support osteonal bone ingrowth , pores of at least 100 m would appear necessary . therefore in the present study the tested material i.e. , nha which had a particle size of ~20 nm shows statistically insignificant results over the control group . also , differences in the physiochemical and structural characteristics between nha and ha used in the past and in the present study may lead to differences in the regenerative / osteoconductive properties .

the study was a prospective , observational study designed to follow eyes / patients with mild npdr ( early treatment diabetic retinopathy study [ etdrs ] grades 20 and 35 ) for a period of 2 years or at the time of development of a vtdr complication , csme needing laser photocoagulation . ( 10 ) for a statistical power of 90% , an level of 0.05 , and a dropout rate of 10% ( i.e. , 60% of the patients with mild npdr and high ma turnover will develop csme , and patients with high ma turnover represent only 20% of the patients with mild npdr ) . four hundred ten patients , men and women , were included with the following inclusion criteria : diagnosed adult - onset type 2 diabetes , age 4075 years , mild npdr ( grades 20 and 35 of etdrs classification ) , without clinical evidence of macular edema , best corrected visual acuity ( bcva ) 95 , etdrs letters ( 20/25 ) , and refraction with a spherical equivalent less than 5d . exclusion criteria included the presence of cataract or other eye disease that may interfere with fundus examination , glaucoma , other retinal disease , previous intraocular surgery , dilatation of the pupil < 5 mm , and previous laser therapy or intravitreal injections . tenets of the declaration of helsinki were followed , and approval from the institutional review board was obtained ( clinical trial registration number : nct00763802 ) . at the baseline visit ( v0 ) , patient s body weight , height , blood pressure , and concomitant medications were recorded . a physical examination by a diabetologist was also performed . laboratory analyses were performed at baseline ( v0 ) , at 6-month ( v6 ) and at 24-month ( v24 ) visits , or at the pretreatment visit . laboratory analyses included glucose and hba1c concentration , red blood cell count , white blood cell count , platelet amount , hemoglobin concentration , and packed cell volume . metabolic control was also assessed by measuring in the plasma concentrations of hba1c and lipid fractionation identifying total cholesterol , hdl , ldl , and triglycerides . one eye per patient was selected as the study eye by the physician based on the inclusion / exclusion criteria . when both eyes fulfill the same criteria , one of the eyes was selected by choosing sequentially the right or the left eye . at the three study visits , v0 , v6 , and v24 ( or pretreatment visit ) , the study eyes underwent a complete eye examination , which included bcva , as tested in the etdrs , slit - lamp examination , intraocular pressure measurements , fundus photography , and optical coherence tomography ( oct ) . csme was identified on clinical examination by retinal thickening within 500 m of the center of the fovea , the presence of exudates within 500 m of the center of the fovea , or adjacent thickening or thickening of at least one disc area of any part within 1 disc diameter of the center of the fovea ( 11 ) . the 7-field photographs were obtained at 30 using a zeiss ff450 camera ( carl zeiss meditec , dublin , ca ) for dr classification according to etdrs grading . the field-2 color fundus images ( macula ) the automated computer - aided diagnostic system , retmarkerdr ( critical health sa , coimbra , portugal ) , was used to automatically detect ma on the field-2 color fundus images . this automated computer - aided diagnostic system consists of software earmarking mas and vascular lesions ; it includes a coregistration algorithm that allows comparison within the same retinal location between different visits for the same eye . the algorithm detects the presence of mas and red dot like lesions . for this purpose , for each of these candidates , features such as area , shape , intensity distribution , and gradient magnitude distribution are extracted using a region covariance descriptor . next , a state of the art classifier , based on support vector machines , is used to classify the candidates as true or false ( classifier s training was done using a dataset of color fundus images in which graders were asked to earmark only small lesions that appeared as a round or ovoid red spot of 20200 m in diameter with regular borders and located within the superior and inferior arcades ) . the images from field-2 are coregistered ( 12 ) to indicate disease activity in the central 3,000-m circle of the macula . image coregistration is achieved by extracting a retinal vascular tree , which is used for landmarks during the registration process . this rigid transformation is then adjusted to obtain exact pairings of selected landmarks ( 13 ) . the retmarkerdr ( critical health sa ) therefore computes for each eye / patient the number of ma in each visit and the number of ma that appears and/or disappears from one visit to the other , allowing calculation of the number of ma appearing and/or disappearing per time interval ( i.e. , the ma formation rate and the ma disappearance rate , respectively ) . the ma turnover is computed as the sum of the ma formation and disappearance rates . for example , a patient who has three ma on the first visit , and those same three ma remain unchanged on the second visit , will have a formation rate of zero , indicating no disease activity . in contrast , a patient can have the total of three ma on the first and second visits , but if those ma are all registered in different retinal locations , an ma formation rate of 3 and a ma disappearance rate of 3 will be calculated , indicating an ma turnover of 6 per time interval . previous work from our group ( 10,14 ) showed a good intergrader agreement for the total number of mas earmarked and the ma turnover for three independent human graders . the retmarkerdr ( critical health sa ) shows a similar intergrader agreement for the total number of mas and the ma turnover ( when compared with a human grader , intraclass correlation coefficients were 0.857 and 0.806 , respectively ) while showing no intragrader variability as opposed to human graders , being , therefore , a reliable tool for ma assessment . oct scans were performed using the stratus oct ( carl zeiss meditec ) . to obtain a more detailed retinal thickness map in the central macular region , proprietary software for increased - resolution oct maps was used ( 15 ) . to compute the new increased - resolution oct maps ( composed by 124 equal areas ) , two acquisition protocols were performed in each eye , the fast macular acquisition protocol , acquiring six radial scans 30 apart , 6 mm long , and the fast rnfl acquisition protocol , acquiring five circular scans within the central 6 mm in diameter area . the following parameters were collected at baseline , computed , and compared between eyes / patients that developed csme needing photocoagulation treatment ( csme group ) and the eyes / patients that did not develop csme ( non - csme group ) : age , diabetes duration , hba1c , blood pressure , cholesterol , hdl , ldl , and triglyceride levels , macular retinal thickness ( in the central 500 and 1,500 m in diameter area ) , number of ma , ma formation and disappearance rates , and ma turnover . changes in the number of ma in field-2 , including ma formation rate , disappearance rate , and turnover , were computed for the first 6-month period of follow - up , v0 , and v6 . a univariate analysis was performed to test for statistically significant differences between csme and non - csme eyes . due to the skewed distribution for the parameters under analysis , for the csme and the non - csme groups , the nonparametric mann whitney test was used . to analyze the predictiveness of the different ma parameters , a receiver operating curve ( roc ) cutoff values for csme and non - csme eyes were identified for the parameter that showed simultaneously the higher sensitivity and specificity ( i.e. , the higher roc area ) . a multivariate analysis , considering the entire set of parameters at baseline ( i.e. , sex , age , diabetes duration , bcva , diabetes treatment , systolic and diastolic blood pressure , glycose , cholesterol , hdl and ldl , triglycerides , central retinal thickness [ in the 500 and 1,500 m in diameter area ] , number of ma , and ma turnover [ from baseline to month 6 ] ) , was also performed using a poisson regression analysis to identify predictive factors for csme development considering patient s time of follow - up . all statistical analyses were performed using the stata software version 12.0 ( statacorp lp , college station , texas ) , and p values 0.05 were considered as statistically significant results . csme was identified on clinical examination by retinal thickening within 500 m of the center of the fovea , the presence of exudates within 500 m of the center of the fovea , or adjacent thickening or thickening of at least one disc area of any part within 1 disc diameter of the center of the fovea ( 11 ) . the 7-field photographs were obtained at 30 using a zeiss ff450 camera ( carl zeiss meditec , dublin , ca ) for dr classification according to etdrs grading . the field-2 color fundus images ( macula ) were subjected to automated ma analysis . the automated computer - aided diagnostic system , retmarkerdr ( critical health sa , coimbra , portugal ) , was used to automatically detect ma on the field-2 color fundus images . this automated computer - aided diagnostic system consists of software earmarking mas and vascular lesions ; it includes a coregistration algorithm that allows comparison within the same retinal location between different visits for the same eye . the algorithm detects the presence of mas and red dot like lesions . for this purpose , then , dark objects of a given size are detected and used as candidates . for each of these candidates , features such as area , shape , intensity distribution , and gradient magnitude distribution next , a state of the art classifier , based on support vector machines , is used to classify the candidates as true or false ( classifier s training was done using a dataset of color fundus images in which graders were asked to earmark only small lesions that appeared as a round or ovoid red spot of 20200 m in diameter with regular borders and located within the superior and inferior arcades ) . the images from field-2 are coregistered ( 12 ) to indicate disease activity in the central 3,000-m circle of the macula . image coregistration is achieved by extracting a retinal vascular tree , which is used for landmarks during the registration process . this rigid transformation is then adjusted to obtain exact pairings of selected landmarks ( 13 ) . the retmarkerdr ( critical health sa ) therefore computes for each eye / patient the number of ma in each visit and the number of ma that appears and/or disappears from one visit to the other , allowing calculation of the number of ma appearing and/or disappearing per time interval ( i.e. , the ma formation rate and the ma disappearance rate , respectively ) . the ma turnover is computed as the sum of the ma formation and disappearance rates . for example , a patient who has three ma on the first visit , and those same three ma remain unchanged on the second visit , will have a formation rate of zero , indicating no disease activity . in contrast , a patient can have the total of three ma on the first and second visits , but if those ma are all registered in different retinal locations , an ma formation rate of 3 and a ma disappearance rate of 3 will be calculated , indicating an ma turnover of 6 per time interval . previous work from our group ( 10,14 ) showed a good intergrader agreement for the total number of mas earmarked and the ma turnover for three independent human graders . the retmarkerdr ( critical health sa ) shows a similar intergrader agreement for the total number of mas and the ma turnover ( when compared with a human grader , intraclass correlation coefficients were 0.857 and 0.806 , respectively ) while showing no intragrader variability as opposed to human graders , being , therefore , a reliable tool for ma assessment . oct scans were performed using the stratus oct ( carl zeiss meditec ) . to obtain a more detailed retinal thickness map in the central macular region , proprietary software for increased - resolution oct maps was used ( 15 ) . to compute the new increased - resolution oct maps ( composed by 124 equal areas ) , two acquisition protocols were performed in each eye , the fast macular acquisition protocol , acquiring six radial scans 30 apart , 6 mm long , and the fast rnfl acquisition protocol , acquiring five circular scans within the central 6 mm in diameter area . the following parameters were collected at baseline , computed , and compared between eyes / patients that developed csme needing photocoagulation treatment ( csme group ) and the eyes / patients that did not develop csme ( non - csme group ) : age , diabetes duration , hba1c , blood pressure , cholesterol , hdl , ldl , and triglyceride levels , macular retinal thickness ( in the central 500 and 1,500 m in diameter area ) , number of ma , ma formation and disappearance rates , and ma turnover . changes in the number of ma in field-2 , including ma formation rate , disappearance rate , and turnover , were computed for the first 6-month period of follow - up , v0 , and v6 . a univariate analysis was performed to test for statistically significant differences between csme and non - csme eyes . due to the skewed distribution for the parameters under analysis , for the csme and the non - csme groups , the nonparametric mann whitney test was used . to analyze the predictiveness of the different ma parameters , a receiver operating curve ( roc ) cutoff values for csme and non - csme eyes were identified for the parameter that showed simultaneously the higher sensitivity and specificity ( i.e. , the higher roc area ) . a multivariate analysis , considering the entire set of parameters at baseline ( i.e. , sex , age , diabetes duration , bcva , diabetes treatment , systolic and diastolic blood pressure , glycose , cholesterol , hdl and ldl , triglycerides , central retinal thickness [ in the 500 and 1,500 m in diameter area ] , number of ma , and ma turnover [ from baseline to month 6 ] ) , was also performed using a poisson regression analysis to identify predictive factors for csme development considering patient s time of follow - up . all statistical analyses were performed using the stata software version 12.0 ( statacorp lp , college station , texas ) , and p values 0.05 were considered as statistically significant results . three hundred forty - eight eyes / patients were considered for analysis because they reached the study end point , csme needing laser photocoagulation , or performed the last study visit ( v24 ) ( fig . baseline characteristics for the 410 patients included are shown in table 1 ( no statistically significant differences were found between excluded and included eyes / patients except for the cholesterol and ldl levels ) . baseline characteristics of the patients in the study of these 348 eyes / patients , 26 were diagnosed during the 2-year period of follow - up as having csme and treated with laser photocoagulation . the other 322 eyes / patients completed the last study visit of follow - up without developing csme ( v24 ) . eyes / patients characteristics by csme and non - csme are shown in table 2 . patient characteristics at baseline comparing the two patient groups , the ones that did not develop csme and the ones that developed csme fifteen eyes / patients progressed to more advanced etdrs levels . fourteen eyes progressed to moderate npdr ( 11 with 43a and 3 with 43b ) , and 1 eye progressed to moderate proliferative dr ( 65b ) . the average number of ma was 6.2 5.4 in the 26 eyes / patients that developed csme and 3.3 3.7 in the remaining 322 eyes / patients ( p < 0.001 ) . the ma turnover was 11.2 11.2 in the 26 eyes / patients that developed csme and 5.0 5.2 in the remaining 322 eyes / patients ( p < 0.001 ) . the ma turnover shows a higher predictiveness for csme than the remaining ma parameters ( the roc area was for the ma turnover , 0.695 ; for the number of ma , 0.676 ; for the ma formation rate , 0.658 ; and for the ma disappearance rate , 0.656 ) . for an ma turnover cutoff value of 9 , a sensitivity of 57.7% and a specificity of 81.2% was achieved ( i.e. , 79.4% of the eyes are correctly classified ) . eyes with an ma turnover > 9 during the initial 6-month period showed a higher risk for csme development than eyes with a lower ma turnover ( odds ratio 5.886 [ 95% ci 2.50313.844 ] ) . the ma turnover predictive values for csme development were : for the positive predictive value , 20.0% ; and for the negative predictive value , 95.9% , showing that a low ma turnover value is associated with less likelihood for csme development in a 2-year period . furthermore , considering only the eyes with an ma turnover 9 , eyes that developed csme before the 24-month visit presented higher ma turnover values ( 26.6 15.9 ) when compared with the eyes in which csme was detected only at month 24 ( 12.8 3.6 ) ( p = 0.018 ) , indicating again that there is a correlation between high turnover values and risk for the development of csme for eyes with the same etdrs retinopathy level . considering the central retinal thickness values at baseline , a statistically significant difference was found for the central 500 m in diameter area ( p = 0.025 ) but not for the central 1,500-m area ( p = 0.052 ) . for bcva , no statistically significant differences were found between csme and non - csme eyes at baseline ( p = 0.499 ) or in the last study visit ( p = 0.593 ) . however , there was an overall decrease in bcva in both csme and non - csme ( p < 0.018 ) . when considering the systemic parameters examined , the eyes that developed csme during the 2-year study period had higher hba1c level at baseline ( p = 0.022 ) . the other parameters examined such as blood pressure ( systolic and diastolic ) and other blood lipids ( triglycerides , cholesterol , hdl , and ldl ) did not show any correlation with occurrence of csme ( table 2 ) . no statistically significant changes in the systemic parameter were found between the baseline and 6-month examinations . the multivariate analysis ( poisson regression analysis ) shows that ma turnover values and hba1c levels are independently predictive for csme development in the early stages of npdr ( table 3 ) . multivariate poisson regression analysis combining the two factors ma turnover and hba1c to identify eyes at risk for csme development , using < 9 and 9 for the ma turnover ( sensitivity and specificity of 57.7 and 81.2% , respectively ) and < 9 and 9 for the hba1c levels ( sensitivity and specificity of 32.6 and 77.0% , respectively ) , the sensitivity for csme development when considering ma turnover < 9 and hba1c levels < 9 increased to 76.9% , decreasing the specificity to 64.9% . this 2-year prospective , longitudinal study of patients with diabetes type 2 and mild npdr ( etdrs levels 20 and 35 at baseline ) shows that ma turnover in field-2 is a good indicator of retinopathy worsening and development of csme needing photocoagulation . on ophthalmoscopic examination and color fundus photography , red - dot lesions , . they may be counted , and retinal ma counting has been proposed as an appropriate marker of retinopathy worsening ( 16 ) . in a previous study by our group ( 10 ) , determination of ma formation rates taking into account the exact location of new ma in successive color fundus photographs showed higher sensitivity in predicting worsening of retinopathy for a 10-year period of follow - up than the simple counting of ma at one time . moreover , we found that ma formation rates obtained during an initial 2-year follow - up period gave more accurate information on the activity of the retinopathy and that there was much better agreement between graders when determining ma formation rates than ma counts ( 14 ) . ma turnover values in this study show a high sd relatively to the mean , indicating that ma turnover values vary widely between eyes having similar etdrs grading level . ( 17 ) and may indicate that ma turnover is an indicator of different vascular disease activity in different eyes . of particular interest in this study is the observation that ma turnover values , including both the ma formation rate and ma disappearance rate , determined over a period of only 6 months predicts with a high degree of confidence the eyes that do not go on to develop csme for at least a period of 2 years . this observation is interpreted as indicating that eyes with low ma turnover have less disease activity and therefore less risk of worsening . it should be realized that retinal thickness was only measured in the central 6,000 m in diameter area , within the vascular arcades , corresponding to etdrs field-2 . therefore , any retinal edema outside of this central area was not considered . the observation that , in this group of patients with diabetes type 2 , the level of metabolic control , given by hba1c values , correlates with retinopathy worsening confirms a previous report ( 10 ) . it is also of major interest that at these early retinopathy stages , other variables , such as blood pressure and blood lipid levels , do not appear to be associated with the development of csme . it is also considered of major interest that ma turnover can be determined in diabetic retinas using only noninvasive color fundus photographs , without the need for fluorescein angiography ( 18 ) . the identification of the eyes that show very slow worsening of retinopathy and low disease activity with low risk for development of vision - threatening complications , based on computer - assisted detection of ma turnover using digital color fundus images , is considered of potential relevance for management of diabetic retinal disease . the validation of digital color fundus cameras as a tool for following dr worsening to vtdr is considered a valuable development , taking into account their cost and availability . quality assurance of digital color fundus imaging is straightforward and will allow more efficient utilization of widespread screening and prevention programs . automated analysis techniques offer advantages of repeatability and consistency , and , although not better than trained graders , particularly in identifying all ma , they avoid the variability inherent to human grading , which has its own subjective reference standards . it is also considered relevant that ma turnover calculated by the retmarkerdr ( critical health sa ) is much less time consuming than ma counting by an expert grader . the observations reported in this article are also expected to have impact on clinical trial design . excluding from the trials eyes / patients that show little disease activity and are not expected to progress during the trial period would offer a better chance for development of vtdr in the placebo control eyes , thus creating the conditions to identify efficacy of the drug being tested . limitations of this study include the relatively short duration of the study ( 2 years ) and the lack of more detailed information on the systemic parameters such as lipid stratification . the results of this study confirm that in a prospective study of a relatively large number of patients , ma turnover values obtained from noninvasive color fundus photography based solely on field-2 images may help to identify the eyes / patients at risk for developing csme and potential vision loss .

objectiveto examine the relationship between microaneurysm ( ma ) turnover using automated analysis of fundus photographs ( retmarkerdr ; critical health sa ) and development of clinically significant macular edema ( csme ) in nonproliferative diabetic retinopathy ( npdr).research design and methodsa prospective , monocenter , observational study was designed to follow eyes / patients with type 2 diabetes and npdr ( early treatment diabetic retinopathy study levels 20 and 35 ) with no prior laser treatment for 2 years or until development of csme . a total of 410 patients , one eye per patient , fulfilled the inclusion / exclusion criteria and were included in the study . ophthalmologic examinations including best corrected visual acuity , fundus photography , and optical coherence tomography were performed at baseline , 6 months , and at the last study visit ( 24 months or before laser treatment).resultsa total of 348 eyes / patients performed the 24-month visit or developed csme . of these 348 eyes / patients , 26 developed csme . hba1c levels at baseline and ma turnover ( i.e. , the sum of the ma formation and disappearance rates ) computed during the first 6 months of follow - up were found to be independently predictive factors for development of csme . ma turnover was 11.2 11.2 in the 26 eyes / patients that developed csme and 5.0 5.2 in the remaining 322 ( p < 0.001 ) . higher ma turnover values correlated with earlier development of csme . ma turnover predictive values for csme development were , for the positive predictive value , 20% and for the negative predictive value , 96%.conclusionsma turnover calculated with the retmarkerdr predicts development of csme in eyes with npdr . low ma turnover values identify well the eyes that are less likely to develop csme in a 2-year period .

RESEARCH DESIGN AND METHODS CSME Color fundus photography OCT Data analysis RESULTS CONCLUSIONS

the study was a prospective , observational study designed to follow eyes / patients with mild npdr ( early treatment diabetic retinopathy study [ etdrs ] grades 20 and 35 ) for a period of 2 years or at the time of development of a vtdr complication , csme needing laser photocoagulation . ( 10 ) for a statistical power of 90% , an level of 0.05 , and a dropout rate of 10% ( i.e. , 60% of the patients with mild npdr and high ma turnover will develop csme , and patients with high ma turnover represent only 20% of the patients with mild npdr ) . four hundred ten patients , men and women , were included with the following inclusion criteria : diagnosed adult - onset type 2 diabetes , age 4075 years , mild npdr ( grades 20 and 35 of etdrs classification ) , without clinical evidence of macular edema , best corrected visual acuity ( bcva ) 95 , etdrs letters ( 20/25 ) , and refraction with a spherical equivalent less than 5d . exclusion criteria included the presence of cataract or other eye disease that may interfere with fundus examination , glaucoma , other retinal disease , previous intraocular surgery , dilatation of the pupil < 5 mm , and previous laser therapy or intravitreal injections . tenets of the declaration of helsinki were followed , and approval from the institutional review board was obtained ( clinical trial registration number : nct00763802 ) . at the baseline visit ( v0 ) , patient s body weight , height , blood pressure , and concomitant medications were recorded . laboratory analyses were performed at baseline ( v0 ) , at 6-month ( v6 ) and at 24-month ( v24 ) visits , or at the pretreatment visit . one eye per patient was selected as the study eye by the physician based on the inclusion / exclusion criteria . when both eyes fulfill the same criteria , one of the eyes was selected by choosing sequentially the right or the left eye . at the three study visits , v0 , v6 , and v24 ( or pretreatment visit ) , the study eyes underwent a complete eye examination , which included bcva , as tested in the etdrs , slit - lamp examination , intraocular pressure measurements , fundus photography , and optical coherence tomography ( oct ) . csme was identified on clinical examination by retinal thickening within 500 m of the center of the fovea , the presence of exudates within 500 m of the center of the fovea , or adjacent thickening or thickening of at least one disc area of any part within 1 disc diameter of the center of the fovea ( 11 ) . the field-2 color fundus images ( macula ) the automated computer - aided diagnostic system , retmarkerdr ( critical health sa , coimbra , portugal ) , was used to automatically detect ma on the field-2 color fundus images . for this purpose , for each of these candidates , features such as area , shape , intensity distribution , and gradient magnitude distribution are extracted using a region covariance descriptor . the images from field-2 are coregistered ( 12 ) to indicate disease activity in the central 3,000-m circle of the macula . the retmarkerdr ( critical health sa ) therefore computes for each eye / patient the number of ma in each visit and the number of ma that appears and/or disappears from one visit to the other , allowing calculation of the number of ma appearing and/or disappearing per time interval ( i.e. , the ma formation rate and the ma disappearance rate , respectively ) . the ma turnover is computed as the sum of the ma formation and disappearance rates . for example , a patient who has three ma on the first visit , and those same three ma remain unchanged on the second visit , will have a formation rate of zero , indicating no disease activity . in contrast , a patient can have the total of three ma on the first and second visits , but if those ma are all registered in different retinal locations , an ma formation rate of 3 and a ma disappearance rate of 3 will be calculated , indicating an ma turnover of 6 per time interval . previous work from our group ( 10,14 ) showed a good intergrader agreement for the total number of mas earmarked and the ma turnover for three independent human graders . the retmarkerdr ( critical health sa ) shows a similar intergrader agreement for the total number of mas and the ma turnover ( when compared with a human grader , intraclass correlation coefficients were 0.857 and 0.806 , respectively ) while showing no intragrader variability as opposed to human graders , being , therefore , a reliable tool for ma assessment . to compute the new increased - resolution oct maps ( composed by 124 equal areas ) , two acquisition protocols were performed in each eye , the fast macular acquisition protocol , acquiring six radial scans 30 apart , 6 mm long , and the fast rnfl acquisition protocol , acquiring five circular scans within the central 6 mm in diameter area . the following parameters were collected at baseline , computed , and compared between eyes / patients that developed csme needing photocoagulation treatment ( csme group ) and the eyes / patients that did not develop csme ( non - csme group ) : age , diabetes duration , hba1c , blood pressure , cholesterol , hdl , ldl , and triglyceride levels , macular retinal thickness ( in the central 500 and 1,500 m in diameter area ) , number of ma , ma formation and disappearance rates , and ma turnover . changes in the number of ma in field-2 , including ma formation rate , disappearance rate , and turnover , were computed for the first 6-month period of follow - up , v0 , and v6 . due to the skewed distribution for the parameters under analysis , for the csme and the non - csme groups , the nonparametric mann whitney test was used . to analyze the predictiveness of the different ma parameters , a receiver operating curve ( roc ) cutoff values for csme and non - csme eyes were identified for the parameter that showed simultaneously the higher sensitivity and specificity ( i.e. a multivariate analysis , considering the entire set of parameters at baseline ( i.e. , sex , age , diabetes duration , bcva , diabetes treatment , systolic and diastolic blood pressure , glycose , cholesterol , hdl and ldl , triglycerides , central retinal thickness [ in the 500 and 1,500 m in diameter area ] , number of ma , and ma turnover [ from baseline to month 6 ] ) , was also performed using a poisson regression analysis to identify predictive factors for csme development considering patient s time of follow - up . all statistical analyses were performed using the stata software version 12.0 ( statacorp lp , college station , texas ) , and p values 0.05 were considered as statistically significant results . csme was identified on clinical examination by retinal thickening within 500 m of the center of the fovea , the presence of exudates within 500 m of the center of the fovea , or adjacent thickening or thickening of at least one disc area of any part within 1 disc diameter of the center of the fovea ( 11 ) . the automated computer - aided diagnostic system , retmarkerdr ( critical health sa , coimbra , portugal ) , was used to automatically detect ma on the field-2 color fundus images . for each of these candidates , features such as area , shape , intensity distribution , and gradient magnitude distribution next , a state of the art classifier , based on support vector machines , is used to classify the candidates as true or false ( classifier s training was done using a dataset of color fundus images in which graders were asked to earmark only small lesions that appeared as a round or ovoid red spot of 20200 m in diameter with regular borders and located within the superior and inferior arcades ) . the images from field-2 are coregistered ( 12 ) to indicate disease activity in the central 3,000-m circle of the macula . the retmarkerdr ( critical health sa ) therefore computes for each eye / patient the number of ma in each visit and the number of ma that appears and/or disappears from one visit to the other , allowing calculation of the number of ma appearing and/or disappearing per time interval ( i.e. , the ma formation rate and the ma disappearance rate , respectively ) . the ma turnover is computed as the sum of the ma formation and disappearance rates . in contrast , a patient can have the total of three ma on the first and second visits , but if those ma are all registered in different retinal locations , an ma formation rate of 3 and a ma disappearance rate of 3 will be calculated , indicating an ma turnover of 6 per time interval . previous work from our group ( 10,14 ) showed a good intergrader agreement for the total number of mas earmarked and the ma turnover for three independent human graders . the retmarkerdr ( critical health sa ) shows a similar intergrader agreement for the total number of mas and the ma turnover ( when compared with a human grader , intraclass correlation coefficients were 0.857 and 0.806 , respectively ) while showing no intragrader variability as opposed to human graders , being , therefore , a reliable tool for ma assessment . to compute the new increased - resolution oct maps ( composed by 124 equal areas ) , two acquisition protocols were performed in each eye , the fast macular acquisition protocol , acquiring six radial scans 30 apart , 6 mm long , and the fast rnfl acquisition protocol , acquiring five circular scans within the central 6 mm in diameter area . the following parameters were collected at baseline , computed , and compared between eyes / patients that developed csme needing photocoagulation treatment ( csme group ) and the eyes / patients that did not develop csme ( non - csme group ) : age , diabetes duration , hba1c , blood pressure , cholesterol , hdl , ldl , and triglyceride levels , macular retinal thickness ( in the central 500 and 1,500 m in diameter area ) , number of ma , ma formation and disappearance rates , and ma turnover . changes in the number of ma in field-2 , including ma formation rate , disappearance rate , and turnover , were computed for the first 6-month period of follow - up , v0 , and v6 . due to the skewed distribution for the parameters under analysis , for the csme and the non - csme groups , the nonparametric mann whitney test was used . to analyze the predictiveness of the different ma parameters , a receiver operating curve ( roc ) cutoff values for csme and non - csme eyes were identified for the parameter that showed simultaneously the higher sensitivity and specificity ( i.e. a multivariate analysis , considering the entire set of parameters at baseline ( i.e. , sex , age , diabetes duration , bcva , diabetes treatment , systolic and diastolic blood pressure , glycose , cholesterol , hdl and ldl , triglycerides , central retinal thickness [ in the 500 and 1,500 m in diameter area ] , number of ma , and ma turnover [ from baseline to month 6 ] ) , was also performed using a poisson regression analysis to identify predictive factors for csme development considering patient s time of follow - up . three hundred forty - eight eyes / patients were considered for analysis because they reached the study end point , csme needing laser photocoagulation , or performed the last study visit ( v24 ) ( fig . baseline characteristics for the 410 patients included are shown in table 1 ( no statistically significant differences were found between excluded and included eyes / patients except for the cholesterol and ldl levels ) . baseline characteristics of the patients in the study of these 348 eyes / patients , 26 were diagnosed during the 2-year period of follow - up as having csme and treated with laser photocoagulation . the other 322 eyes / patients completed the last study visit of follow - up without developing csme ( v24 ) . eyes / patients characteristics by csme and non - csme are shown in table 2 . patient characteristics at baseline comparing the two patient groups , the ones that did not develop csme and the ones that developed csme fifteen eyes / patients progressed to more advanced etdrs levels . fourteen eyes progressed to moderate npdr ( 11 with 43a and 3 with 43b ) , and 1 eye progressed to moderate proliferative dr ( 65b ) . the average number of ma was 6.2 5.4 in the 26 eyes / patients that developed csme and 3.3 3.7 in the remaining 322 eyes / patients ( p < 0.001 ) . the ma turnover was 11.2 11.2 in the 26 eyes / patients that developed csme and 5.0 5.2 in the remaining 322 eyes / patients ( p < 0.001 ) . the ma turnover shows a higher predictiveness for csme than the remaining ma parameters ( the roc area was for the ma turnover , 0.695 ; for the number of ma , 0.676 ; for the ma formation rate , 0.658 ; and for the ma disappearance rate , 0.656 ) . for an ma turnover cutoff value of 9 , a sensitivity of 57.7% and a specificity of 81.2% was achieved ( i.e. , 79.4% of the eyes are correctly classified ) . eyes with an ma turnover > 9 during the initial 6-month period showed a higher risk for csme development than eyes with a lower ma turnover ( odds ratio 5.886 [ 95% ci 2.50313.844 ] ) . the ma turnover predictive values for csme development were : for the positive predictive value , 20.0% ; and for the negative predictive value , 95.9% , showing that a low ma turnover value is associated with less likelihood for csme development in a 2-year period . furthermore , considering only the eyes with an ma turnover 9 , eyes that developed csme before the 24-month visit presented higher ma turnover values ( 26.6 15.9 ) when compared with the eyes in which csme was detected only at month 24 ( 12.8 3.6 ) ( p = 0.018 ) , indicating again that there is a correlation between high turnover values and risk for the development of csme for eyes with the same etdrs retinopathy level . considering the central retinal thickness values at baseline , a statistically significant difference was found for the central 500 m in diameter area ( p = 0.025 ) but not for the central 1,500-m area ( p = 0.052 ) . for bcva , no statistically significant differences were found between csme and non - csme eyes at baseline ( p = 0.499 ) or in the last study visit ( p = 0.593 ) . however , there was an overall decrease in bcva in both csme and non - csme ( p < 0.018 ) . when considering the systemic parameters examined , the eyes that developed csme during the 2-year study period had higher hba1c level at baseline ( p = 0.022 ) . the other parameters examined such as blood pressure ( systolic and diastolic ) and other blood lipids ( triglycerides , cholesterol , hdl , and ldl ) did not show any correlation with occurrence of csme ( table 2 ) . no statistically significant changes in the systemic parameter were found between the baseline and 6-month examinations . the multivariate analysis ( poisson regression analysis ) shows that ma turnover values and hba1c levels are independently predictive for csme development in the early stages of npdr ( table 3 ) . multivariate poisson regression analysis combining the two factors ma turnover and hba1c to identify eyes at risk for csme development , using < 9 and 9 for the ma turnover ( sensitivity and specificity of 57.7 and 81.2% , respectively ) and < 9 and 9 for the hba1c levels ( sensitivity and specificity of 32.6 and 77.0% , respectively ) , the sensitivity for csme development when considering ma turnover < 9 and hba1c levels < 9 increased to 76.9% , decreasing the specificity to 64.9% . this 2-year prospective , longitudinal study of patients with diabetes type 2 and mild npdr ( etdrs levels 20 and 35 at baseline ) shows that ma turnover in field-2 is a good indicator of retinopathy worsening and development of csme needing photocoagulation . on ophthalmoscopic examination and color fundus photography , red - dot lesions , . in a previous study by our group ( 10 ) , determination of ma formation rates taking into account the exact location of new ma in successive color fundus photographs showed higher sensitivity in predicting worsening of retinopathy for a 10-year period of follow - up than the simple counting of ma at one time . moreover , we found that ma formation rates obtained during an initial 2-year follow - up period gave more accurate information on the activity of the retinopathy and that there was much better agreement between graders when determining ma formation rates than ma counts ( 14 ) . ma turnover values in this study show a high sd relatively to the mean , indicating that ma turnover values vary widely between eyes having similar etdrs grading level . ( 17 ) and may indicate that ma turnover is an indicator of different vascular disease activity in different eyes . of particular interest in this study is the observation that ma turnover values , including both the ma formation rate and ma disappearance rate , determined over a period of only 6 months predicts with a high degree of confidence the eyes that do not go on to develop csme for at least a period of 2 years . this observation is interpreted as indicating that eyes with low ma turnover have less disease activity and therefore less risk of worsening . the observation that , in this group of patients with diabetes type 2 , the level of metabolic control , given by hba1c values , correlates with retinopathy worsening confirms a previous report ( 10 ) . it is also of major interest that at these early retinopathy stages , other variables , such as blood pressure and blood lipid levels , do not appear to be associated with the development of csme . the identification of the eyes that show very slow worsening of retinopathy and low disease activity with low risk for development of vision - threatening complications , based on computer - assisted detection of ma turnover using digital color fundus images , is considered of potential relevance for management of diabetic retinal disease . automated analysis techniques offer advantages of repeatability and consistency , and , although not better than trained graders , particularly in identifying all ma , they avoid the variability inherent to human grading , which has its own subjective reference standards . it is also considered relevant that ma turnover calculated by the retmarkerdr ( critical health sa ) is much less time consuming than ma counting by an expert grader . excluding from the trials eyes / patients that show little disease activity and are not expected to progress during the trial period would offer a better chance for development of vtdr in the placebo control eyes , thus creating the conditions to identify efficacy of the drug being tested . limitations of this study include the relatively short duration of the study ( 2 years ) and the lack of more detailed information on the systemic parameters such as lipid stratification . the results of this study confirm that in a prospective study of a relatively large number of patients , ma turnover values obtained from noninvasive color fundus photography based solely on field-2 images may help to identify the eyes / patients at risk for developing csme and potential vision loss .

all patients with md1 confirmed by genetic and electromyographic study and treated by our university institution 's multidisciplinary team for the care of patients with neuromuscular disease were recruited . some of the patients included in the present study have already been described in previous studies.13 15 new patients with md1 who were presented to the multidisciplinary team due to recent disease diagnosis , or new symptoms or new resting ecg abnormalities were also include in this study . the annual check - up consisted of a physical examination by a cardiologist , a neurologist and a chest specialist . functional status , supplemented by the recording of resting ecg , sa - ecg and pulmonary capacity , was monitored once a year . the md patients without pacemakers and who had previously undergone an electrophysiological study were selected for our study . a second electrophysiological study was proposed to the patients in the event of new symptoms ( unexplained syncope or presyncope , palpitations ) , new atrioventricular conduction disturbances on annual resting ecg , significant modifications of sa - ecg with respect to the previous study,15 and atrial arrhythmias justifying anti - arrhythmic drugs , or in asymptomatic patients with a follow - up of at least 60 months . this time interval between the first and second electrophysiological tests in asymptomatic patients was chosen from the study of prystowsky et al,14 who reported a 5 ms prolongation of the hv interval over a 3-year period in several patients , and from the study of laurent et al,13 who reported a complete avb in one patient with a normal hv interval 5 years earlier . therefore , 60 months after the first electrophysiological test the hv interval would be prolonged by 10 ms and could exceed 70 ms . once identified , md patients with these criteria underwent electrophysiological testing after informed consent was obtained and signed . patients who were initially studied less than 60 months without new symptoms , or without significant changes in resting ecg or sa - ecg were not considered for a new electrophysiological study . the electrophysiological testing was performed using standard techniques with two quadripolar catheters introduced into the femoral vein and worked up into the bundle of his region of the right atrium . several variables were measured during the electrophysiological study : atrial - his ( ah ) interval ( ms ) , hv interval ( ms ) , atrioventricular wenckebach points ( beats / min ) during right atrial stimulation at increasing rate , sinus node recovery time ( ms ) . the criterion for proposing a prophylactic pacemaker implantation was a hv interval of 70 ms or greater . recordings were performed using simson 's method with an art recorder ( model 120 ; arrhythmia research technology , austin , texas , usa ) . signals were amplified , averaged and filtered with bidirectional filtered frequencies of 40250 hz . as previously reported,15 the association of qrsd ( total qrs duration ) of 100 ms or greater and las40 ( duration of low amplitude signal<40v ) of 36 ms or greater on sa - ecg contributes to the identification of md patients with a prolonged hv interval . five patients reported in the previous study in 1999 were also included in the present study . these criteria are not those commonly used to identify patients at risk of sustained ventricular tachycardia post - myocardial infarction , but in md they are linked to infrahisian conduction alteration and not to ventricular arrhythmia substrate.16 the primary endpoint was to evaluate the evolution over time of the infrahissian conduction time in adult md patients . the second endpoint was to define the predictive clinical and electrocardiographic parameters of a severe alteration in infrahissian conduction time that would justify a repetition of electrophysiological testing in md patients with a view to proposing the prophylactic implantation of a pacemaker . baseline characteristics in patients with and without prolonged hv interval were compared using univariate analysis and the fisher 's exact test for qualitative parameters and the mann the correlation between the variation of hv interval ( hv2hv1 ) and the size of the mutation was analysed using the linear regression method . all patients with md1 confirmed by genetic and electromyographic study and treated by our university institution 's multidisciplinary team for the care of patients with neuromuscular disease were recruited . some of the patients included in the present study have already been described in previous studies.13 15 new patients with md1 who were presented to the multidisciplinary team due to recent disease diagnosis , or new symptoms or new resting ecg abnormalities were also include in this study . the annual check - up consisted of a physical examination by a cardiologist , a neurologist and a chest specialist . functional status , supplemented by the recording of resting ecg , sa - ecg and pulmonary capacity , was monitored once a year . the md patients without pacemakers and who had previously undergone an electrophysiological study were selected for our study . a second electrophysiological study was proposed to the patients in the event of new symptoms ( unexplained syncope or presyncope , palpitations ) , new atrioventricular conduction disturbances on annual resting ecg , significant modifications of sa - ecg with respect to the previous study,15 and atrial arrhythmias justifying anti - arrhythmic drugs , or in asymptomatic patients with a follow - up of at least 60 months . this time interval between the first and second electrophysiological tests in asymptomatic patients was chosen from the study of prystowsky et al,14 who reported a 5 ms prolongation of the hv interval over a 3-year period in several patients , and from the study of laurent et al,13 who reported a complete avb in one patient with a normal hv interval 5 years earlier . therefore , 60 months after the first electrophysiological test the hv interval would be prolonged by 10 ms and could exceed 70 ms . once identified , md patients with these criteria underwent electrophysiological testing after informed consent was obtained and signed . patients who were initially studied less than 60 months without new symptoms , or without significant changes in resting ecg or sa - ecg were not considered for a new electrophysiological study . the electrophysiological testing was performed using standard techniques with two quadripolar catheters introduced into the femoral vein and worked up into the bundle of his region of the right atrium . several variables were measured during the electrophysiological study : atrial - his ( ah ) interval ( ms ) , hv interval ( ms ) , atrioventricular wenckebach points ( beats / min ) during right atrial stimulation at increasing rate , sinus node recovery time ( ms ) . the criterion for proposing a prophylactic pacemaker implantation was a hv interval of 70 ms or greater . recordings were performed using simson 's method with an art recorder ( model 120 ; arrhythmia research technology , austin , texas , usa ) . signals were amplified , averaged and filtered with bidirectional filtered frequencies of 40250 hz . as previously reported,15 the association of qrsd ( total qrs duration ) of 100 ms or greater and las40 ( duration of low amplitude signal<40v ) of 36 ms or greater on sa - ecg contributes to the identification of md patients with a prolonged hv interval . five patients reported in the previous study in 1999 were also included in the present study . these criteria are not those commonly used to identify patients at risk of sustained ventricular tachycardia post - myocardial infarction , but in md they are linked to infrahisian conduction alteration and not to ventricular arrhythmia substrate.16 the primary endpoint was to evaluate the evolution over time of the infrahissian conduction time in adult md patients . the second endpoint was to define the predictive clinical and electrocardiographic parameters of a severe alteration in infrahissian conduction time that would justify a repetition of electrophysiological testing in md patients with a view to proposing the prophylactic implantation of a pacemaker . baseline characteristics in patients with and without prolonged hv interval were compared using univariate analysis and the fisher 's exact test for qualitative parameters and the mann the correlation between the variation of hv interval ( hv2hv1 ) and the size of the mutation was analysed using the linear regression method . starting in 1994 , 70 of 127 consecutively screened patients with md1 were not initially implanted with a pacemaker . twenty - five of these 70 patients were enrolled and underwent a second electrophysiological testing . no significant differences were observed between the initial clinical characteristics of the 25 patients enrolled in the present study ( age , sex , brooke score , epworth score , resting ecg , right and left ventricular ejection fraction , ctg amplification ) and the 45 patients who did not initially receive a pacemaker ( table 1 ) and who were not enrolled in the present study for different reasons set out in figure 1 . the upgraded clinical characteristics of the 25 patients undergoing a second electrophysiological study are summarised in table 2 . initial mean characteristics of the md patients without a pacemaker group 1 all patients without pacemaker in our global myotonic dystrophy ( md ) population ; group 2 patients excluded from the present study for different reasons explained in figure 1 ; group 3 patients enrolled in the study who underwent two electrophysiological studies . af , atrial fibrillation ; ctg , cytosine - thymine - guanine ; lvedd , left ventricular end - diastolic dimension ; lvef , left ventricular ejection fraction ; nsvt , non sustained ventricular tachycardia ; rvef , right ventricular ejection fraction . avb , atrioventricular block ; ep , electrophysiological test ; hv , his - ventricle ; sa - ecg , signal - averaged ecg . upgraded clinical characteristics of the patients who underwent a second electrophysiological test ctg , cytosine - thymine - guanine ; levd ; lvedd , left ventricular end - diastolic dimension ; lvef , left ventricular ejection fraction ; nsvt , non sustained ventricular tachycardia ; rvef , right ventricular ejection fraction . neurological functional status was evaluated by the brooke score.17 the severity of muscular weakness was scored at the level of arms ( range from 1 to 6 ) and legs ( range from 1 to 10 ) . no patient was under permanent mechanical respirator at the time of the second electrophysiological testing . the excessive daytime sleepiness was evaluated by the epworth sleepiness scale with eight items.18 each item accounts for 03 points . this sleep apnoea syndrome was treated by nocturnal non - invasive positive pressure ventilation in eight patients . the mean value of creatine phosphokinase was 205165 ui ( from 64 to 712 , normal value < 145 ui ) and five patients had significant vesicular lithiasis . left ventricular ejection fraction was normal ( > 50% ) in 21 patients ( 84% ) and slightly altered in four patients ( ranging from 45% to 50% ) . right ventricular radionuclide function was available for 13 patients and was normal in four of them ( > 45% ) and slightly altered in nine others ( ranging from 30% to 45% ) . initially , 17 patients ( 68% ) showed normal conduction intervals on their first ecg . among the remaining patients , four had left anterior block , two had isolated 1 avb , one had 1 avb associated with right bundle branch block , and one had both right bundle branch block and left anterior block . at the time of the second electrophysiological test electrophysiological characteristics of patients who underwent a second electrophysiological study signal - averaged ( sa)-ecg was regarded as the presence of qrsd of 100 ms or greater and las40 of 36 ms or greater.15 pacemaker implanted for brady - tachycardia syndrome . avb 1 , atrioventricular block 1 ; cp , chest pain ; ep , electrophysiological testing ; hv , his - ventricle ; lab , left anterior block ; lbbbg , left bundle branch block , rbbb , right bundle branch block . atrial fibrillation was treated by amiodarone ( n=1 ) which does not severely depress the infrahisian conduction.19 atrial flutter was treated by cavo - tricuspid isthmus ablation ( n=1 ) . three patients were not treated by anti - arrhythmic drugs because the atrial arrhythmias were not symptomatic . on 24 h ecg , paroxysmal avb of second and third degree was not observed . modifications of sa - ecg reaching the significant cut - off defined by qrsd of 100 ms or greater and las of 36 ms15 or greater were observed in six patients . in one patient , sa - ecg was invalid because of the level of noise , and in another it could not be performed due to a technical problem . the mean values of sa - ecg parameters were : qrsd 10510.8 ms , under 40 36.512 ms , hfrmsa ( root mean square voltage of signals in the last 40 ms of the qrs complexes ) 30.324.7 v . the mean age at the second electrophysiological test was 44.413.6 years . the mean interval between the first and the second electrophysiological test was 90.836 months ( minimum 24 months , maximum 144 months ) . four patients were given the second electrophysiological test because they developed new symptoms , four others on account of modifications in resting ecg , one other for a bradytachycardia syndrome , only three for abnormal sa - ecg , and 13 patients merely because the follow - up exceeded 60 months . in asymptomatic patients with no ecg or sa - ecg modification , the mean interval between the first and the second electrophysiological test tended to be longer than in other patients ( 10134 months versus 8129 months , ns ) . at the second electrophysiological test the hv interval was 70 ms or greater in five cases ( 20% ) . the mean value of the hv interval increased significantly between the two electrophysiological tests ( figure 2 and table 3 ) . five patients with hv interval of 70 ms or greater were implanted with a prophylactic dual - chamber pacemaker . the patient with bradytachycardia syndrome was given an oral anti - arrhythmic agent , amiodarone 200 mg / day and a pacemaker . evolution of his - ventricle ( hv ) interval ( ms ) in myotonic dystrophy patients . full black square : individual hv interval at the first electrophysiological study ( hv1 ) and at the second electrophysiological study ( hv2 ) full black circles : mean value of hv intervales ( p<0.001 ) . the five patients with a hv interval of 70 ms or greater at the second electrophysiological test showed a modification in their resting ecg over time ( n=4 ) or significant modifications in the sa - ecg ( n=3 ) ( table 3 and figure 2 ) . one of the md patients with stable resting ecg , despite a prolonged hv interval , showed significant lengthening of sa - ecg parameters ( patient 24 in table 3 ) . qrsd and under 40 tended to be longer in the prolonged hv interval group than in normal hv interval group ( qrds 109.86 vs 103.611 ms , under 40 42.418 vs 349.6 ms).the mean duration of the follow - up was not significantly different between the group of patients with a hv interval of 70 ms or greater at the second electrophysiological test and those without a hv interval of 70 ms or greater at the second electrophysiological test ( 8046 vs 93.534 months , ns ) . none of the clinical characteristics were significantly different between the two groups in terms of age , severity of muscular weakness and frequency of sleep apnoea syndrome ; nor was the size of the mutation significantly different in those patients ( 2.51.1 vs 2.21.9 kb , ns ) . no correlation was established between the variation in the hv interval and the size of the mutation ( r=0.03 , ns ) . resting ecg is very useful for the identification of patients with an alteration of infrahissian conduction . of the five patients with a new prolongation of the hv interval , four showed significant modification in their resting ecg . only one patient without modification of ecg had a prolonged hv interval ( sensitivity 80% , specificity 99% ) . sa - ecg was significantly modified in six patients but was associated with prolonged interval in only three patients ( sensitivity 100% , specificity 50% ) . the combination of modifications of resting ecg and sa - ecg identified all patients with prolonged hv interval ( sensitivity 100% and specificity 75% ) . initially , 17 patients ( 68% ) showed normal conduction intervals on their first ecg . among the remaining patients , four had left anterior block , two had isolated 1 avb , one had 1 avb associated with right bundle branch block , and one had both right bundle branch block and left anterior block . at the time of the second electrophysiological test electrophysiological characteristics of patients who underwent a second electrophysiological study signal - averaged ( sa)-ecg was regarded as the presence of qrsd of 100 ms or greater and las40 of 36 ms or greater.15 pacemaker implanted for brady - tachycardia syndrome . avb 1 , atrioventricular block 1 ; cp , chest pain ; ep , electrophysiological testing ; hv , his - ventricle ; lab , left anterior block ; lbbbg , left bundle branch block , rbbb , right bundle branch block . atrial fibrillation was treated by amiodarone ( n=1 ) which does not severely depress the infrahisian conduction.19 atrial flutter was treated by cavo - tricuspid isthmus ablation ( n=1 ) . three patients were not treated by anti - arrhythmic drugs because the atrial arrhythmias were not symptomatic . on 24 h ecg , paroxysmal avb of second and third degree was not observed . modifications of sa - ecg reaching the significant cut - off defined by qrsd of 100 ms or greater and las of 36 ms15 or greater were observed in six patients . in one patient , sa - ecg was invalid because of the level of noise , and in another it could not be performed due to a technical problem . the mean values of sa - ecg parameters were : qrsd 10510.8 ms , under 40 36.512 ms , hfrmsa ( root mean square voltage of signals in the last 40 ms of the qrs complexes ) 30.324.7 v . the mean age at the second electrophysiological test was 44.413.6 years . the mean interval between the first and the second electrophysiological test was 90.836 months ( minimum 24 months , maximum 144 months ) . four patients were given the second electrophysiological test because they developed new symptoms , four others on account of modifications in resting ecg , one other for a bradytachycardia syndrome , only three for abnormal sa - ecg , and 13 patients merely because the follow - up exceeded 60 months . in asymptomatic patients with no ecg or sa - ecg modification , the mean interval between the first and the second electrophysiological test tended to be longer than in other patients ( 10134 months versus 8129 months , ns ) . at the second electrophysiological test the hv interval was 70 ms or greater in five cases ( 20% ) . the mean value of the hv interval increased significantly between the two electrophysiological tests ( figure 2 and table 3 ) . five patients with hv interval of 70 ms or greater were implanted with a prophylactic dual - chamber pacemaker . the patient with bradytachycardia syndrome was given an oral anti - arrhythmic agent , amiodarone 200 mg / day and a pacemaker . evolution of his - ventricle ( hv ) interval ( ms ) in myotonic dystrophy patients . full black square : individual hv interval at the first electrophysiological study ( hv1 ) and at the second electrophysiological study ( hv2 ) full black circles : mean value of hv intervales ( p<0.001 ) . the five patients with a hv interval of 70 ms or greater at the second electrophysiological test showed a modification in their resting ecg over time ( n=4 ) or significant modifications in the sa - ecg ( n=3 ) ( table 3 and figure 2 ) . one of the md patients with stable resting ecg , despite a prolonged hv interval , showed significant lengthening of sa - ecg parameters ( patient 24 in table 3 ) . qrsd and under 40 tended to be longer in the prolonged hv interval group than in normal hv interval group ( qrds 109.86 vs 103.611 ms , under 40 42.418 vs 349.6 ms).the mean duration of the follow - up was not significantly different between the group of patients with a hv interval of 70 ms or greater at the second electrophysiological test and those without a hv interval of 70 ms or greater at the second electrophysiological test ( 8046 vs 93.534 months , ns ) . none of the clinical characteristics were significantly different between the two groups in terms of age , severity of muscular weakness and frequency of sleep apnoea syndrome ; nor was the size of the mutation significantly different in those patients ( 2.51.1 vs 2.21.9 kb , ns ) . no correlation was established between the variation in the hv interval and the size of the mutation ( r=0.03 , ns ) . resting ecg is very useful for the identification of patients with an alteration of infrahissian conduction . of the five patients with a new prolongation of the hv interval , four showed significant modification in their resting ecg . only one patient without modification of ecg had a prolonged hv interval ( sensitivity 80% , specificity 99% ) . sa - ecg was significantly modified in six patients but was associated with prolonged interval in only three patients ( sensitivity 100% , specificity 50% ) . the combination of modifications of resting ecg and sa - ecg identified all patients with prolonged hv interval ( sensitivity 100% and specificity 75% ) . this study , for the first time , shows the evolution of the resting ecg and sa - ecg characteristics and of the hv interval in non - selected adult md patients monitored every year in the same reference centre . some significant modifications of resting ecg were observed in four patients and abnormal sa - ecg in six patients , and were strongly associated with an hv interval prolongation . the hv interval increase was not linked to any new symptoms such as syncope or palpitations , or to the duration of the follow - up . nguyen et al20 reported that abnormalities of the cardiac conduction system were commonly observed in md due to frequent fatty infiltration and fibrosis of the his bundle . the electrophysiological consequences of these histopathological lesions were frequent infrahissian conduction disturbances on electrophysiological testing ( prolonged hv interval ) and a high risk of complete avb in md patients.12 13 21 in animal models for dystrophy myotonic protein kinase ( dmpk ) reduction the same histopathological lesions and his purkinje system alterations were reported.22 a time - dependent degenerative process has been suggested by groh et al,23 who observed a correlation between age and ecg abnormalities , and by prystowsky et al,14 who in serial electrophysiological studies demonstrated the progression of infrahissian conduction time in a small number ( n=9 ) of non - consecutive md patients . however , in the large study by laurent et al,13 including 51 md patients with a normal hv interval of less than 70 ms followed up during 5736 months , only one patient developed a complete avb , 52 months after the initial electrophysiological testing , whereas 19 of 49 patients with hv interval of 70 ms or greater developed 3 avb . these results suggest two categories of adult md patients : one characterised by early infrahissian conduction disturbances ( hv interval 70 ms ) frequently risking complete avb , and a second category of adult md patients who may exhibit a very slow progression of infrahissian conduction time . this hypothesis is consistent with our results , which through serial electrophysiological testing demonstrated a progression of hv interval in only five of 25 patients during a prolonged period of 90 months as opposed to prystowsky et al,14 who reported an aggravation of hv interval in seven of nine selected patients during a short 3-year period . this difference can be explained by the high rate of abnormal resting ecg ( 7/9 ) and prolonged hv interval ( 3/9 ) at the initial evaluation in the prystowsky study.14 second , the high prevalence of complete avb documented in memory pacemaker in md patients with an early hv interval of 70 ms or greater points to a severe evolution of the infrahissian conduction time in this subgroup of patients.12 13 this heterogeneity of md patients in terms of infrahissian characteristics is compatible with the heterogeneity of the size of the mutation in the different organs ( somatic mosaicism ) , which is not exactly the same for leucocytes . no correlation has been established between respiratory insufficiency , weakness , or cardiac disease , probably because the effect of the non - translated ctg repeat expansion is not unique but multiple24 and is not uniform from one patient to another . a second major finding of our study is the strong relationship between the evolution of cardiac conduction disturbances on resting ecg and the increase in the hv interval in a serial study . no correlation between resting ecg and hv interval or histopathological lesions12 14 15 20 has been reported in the literature , but the previous studies did not integrate the dynamism of the atrioventricular conduction parameters in their analysis . in our study patients with a significant increase in the hv interval corresponded approximately to those with a significant progression of cardiac conduction abnormalities on resting ecg . mrner et al25 pointed out the importance of analysing the progression of the atrioventricular conduction disturbances on ecg . a progressive increase in pr interval and qrs duration on resting ecg has been demonstrated as being statistically associated with higher mortality in md patients.10 25 groh et al5 showed in a large population of md patients that the resting ecg was a crucial key in defining those patients at high risk of sudden death . in the subgroup of patients at high risk of sudden death in accordance with the groh criteria , laurent et al13 showed that the implantation of a prophylactic pacemaker in the presence of a prolonged hv interval decreased the incidence of sudden death . therefore , the evaluation of the risk of sudden death should be re - checked by ecg every year . ventricular arrhythmias are the second mechanism evoked to explain sudden death but the method of identifying md patients at risk is unknown . ventricular arrhythmias induced during electrophysiological study do not predict spontaneous ventricular arrhythmias.16 we can only suggest discussing the implantation of an implantable cardioverter defibrillator instead of a pacemaker in md patients with prolonged hv interval and depressed left ventricular ejection fraction below 30%.26 breton and mathieu11 proposed integrating the analysis of qt interval on resting ecg to identify md patients at risk of sudden death or pacemaker implantation . in presence of a qt interval longer than 450 ms the age - adjusted rr was 3 . however , rigorous analysis of the qt interval remains a major problem.27 an automatic analysis and a dynamic analysis on 24 h ecg with the help of new software could be interesting to limit the variability of the qt interval measurement . sa - ecg is a useful tool in evaluating the infrahissian conduction characteristics of md patients.15 babuty et al15 demonstrated that an association of qrsd of 100 ms or greater and las of 36 ms or greater selects patients with a prolonged hv interval of 70 ms or greater with high sensitivity ( 80% ) and specificity ( 83.3% ) . in the present study significant changes in the sa - ecg correlate with prolonged hv interval in three of six patients . the combination of resting ecg and sa - ecg appears to be the best method of identifying patients showing an alteration over time of the infrahissian conduction time , with excellent sensitivity ( 100% ) and acceptable specificity ( 75% ) . however , the usefulness of sa - ecg to identify md patients with prolonged infrahisian conduction has not been validated by an another group . the fact that not all the patients without a pacemaker were systematically re - investigated could introduce a bias in this study . however , we have verified that the clinical characteristics of the 25 patients included in the study did not differ statistically from the 45 patients not re - investigated for different reasons ( table 1 and figure 1 ) . some patients could have a normal ecg and prolonged hv interval due to a short ah interval.12 these patients , however , can be identified by the sa - ecg , which , as previously mentioned in the discussion , accurately reveals infrahissian conduction abnormalities . for the moment there is no randomised , controlled study in the literature clearly indicating that preventive pacemaker implantation in patients with a hv interval greater than 70 ms decreases mortality and morbidity in md . only observational and non - controlled studies suggest a real benefit from the prophylactic implantation of a pacemaker in the presence of a prolonged hv interval of 70 ms or greater.12 13 the fact that not all the patients without a pacemaker were systematically re - investigated could introduce a bias in this study . however , we have verified that the clinical characteristics of the 25 patients included in the study did not differ statistically from the 45 patients not re - investigated for different reasons ( table 1 and figure 1 ) . some patients could have a normal ecg and prolonged hv interval due to a short ah interval.12 these patients , however , can be identified by the sa - ecg , which , as previously mentioned in the discussion , accurately reveals infrahissian conduction abnormalities . for the moment there is no randomised , controlled study in the literature clearly indicating that preventive pacemaker implantation in patients with a hv interval greater than 70 ms decreases mortality and morbidity in md . only observational and non - controlled studies suggest a real benefit from the prophylactic implantation of a pacemaker in the presence of a prolonged hv interval of 70 ms or greater.12 13 the infrahissian conduction disturbances frequently observed in adult md patients justify the early identification of patients with a prolonged hv interval , who are at high risk of complete avb . this can be done by carrying out an early electrophysiological study at the time of md diagnosis . prophylactic implantation of a pacemaker should be proposed to patients with a hv interval of 70 ms or greater . in patients with an initially normal infrahissian conduction time on the electrophysiological study , the risk of complete avb is very low , and a second electrophysiological study should be performed only in the event of modifications in atrioventricular conduction ecg parameters or sa - ecg in annual check - ups . in the future , it will be important to conduct a controlled and randomised study in order to demonstrate clearly the benefits of prophylactic pacemaker implantation in md patients with a hv interval of 70 ms or greater .

backgroundmyotonic dystrophy ( md1 ) is a hereditary autosomal dominant disease with variable penetrance . cardiac conduction disturbances are frequent and may be responsible for sudden death , but its progression was heretofore unknown.aimsthe aim of the study was to analyse the natural history of infrahissian conduction time in patients with a normal first electrophysiological test , and to identify the predictive value of the clinical and ecg factors accompanying an alteration of infrahissian conduction.methodsamong 127 consecutive screened md patients , 25 were enrolled and underwent a second electrophysiological testing . the second electrophysiological test was carried out on patients showing new symptoms , new atrioventricular conduction disturbances on ecg , or significant modifications of signal - averaged ( sa)-ecg , and on asymptomatic patients with a follow - up of at least 60 months since the first electrophysiological test.resultsamong the 25 patients , four had new clinical symptoms , four others developed new atrioventricular conduction abnormalities on ecg and six had significant modifications of the sa - ecg . the mean his - ventricle ( hv ) interval increased significantly between the two electrophysiological studies ( initial hv interval 52.1 ms1.6 ms , final hv interval 61.4 ms2.2 ms , p<0.005 ) , with a mean increase of 1.2 ms / year . the five patients with hv interval of 70 ms or greater were implanted with a prophylactic dual - chamber pacemaker . modifications of resting ecg and sa - ecg were strongly associated with hv interval prolongation.conclusionin patients with a normal initial electrophysiological study , modifications on the resting ecg and/or sa - ecg , on annual check - up , were associated with an alteration of infrahissian conduction .

Methods Population Electrophysiological testing Signal-averaged ECG Study endpoints Statistical analysis Results ECG patterns Signal-averaged ECG Electrophysiological testing Characteristics of patients with abnormal HV interval Predictive value of resting ECG and SA-ECG Discussion Limitations of the study Conclusion on clinical implications

some of the patients included in the present study have already been described in previous studies.13 15 new patients with md1 who were presented to the multidisciplinary team due to recent disease diagnosis , or new symptoms or new resting ecg abnormalities were also include in this study . functional status , supplemented by the recording of resting ecg , sa - ecg and pulmonary capacity , was monitored once a year . a second electrophysiological study was proposed to the patients in the event of new symptoms ( unexplained syncope or presyncope , palpitations ) , new atrioventricular conduction disturbances on annual resting ecg , significant modifications of sa - ecg with respect to the previous study,15 and atrial arrhythmias justifying anti - arrhythmic drugs , or in asymptomatic patients with a follow - up of at least 60 months . this time interval between the first and second electrophysiological tests in asymptomatic patients was chosen from the study of prystowsky et al,14 who reported a 5 ms prolongation of the hv interval over a 3-year period in several patients , and from the study of laurent et al,13 who reported a complete avb in one patient with a normal hv interval 5 years earlier . therefore , 60 months after the first electrophysiological test the hv interval would be prolonged by 10 ms and could exceed 70 ms . patients who were initially studied less than 60 months without new symptoms , or without significant changes in resting ecg or sa - ecg were not considered for a new electrophysiological study . the criterion for proposing a prophylactic pacemaker implantation was a hv interval of 70 ms or greater . as previously reported,15 the association of qrsd ( total qrs duration ) of 100 ms or greater and las40 ( duration of low amplitude signal<40v ) of 36 ms or greater on sa - ecg contributes to the identification of md patients with a prolonged hv interval . these criteria are not those commonly used to identify patients at risk of sustained ventricular tachycardia post - myocardial infarction , but in md they are linked to infrahisian conduction alteration and not to ventricular arrhythmia substrate.16 the primary endpoint was to evaluate the evolution over time of the infrahissian conduction time in adult md patients . the second endpoint was to define the predictive clinical and electrocardiographic parameters of a severe alteration in infrahissian conduction time that would justify a repetition of electrophysiological testing in md patients with a view to proposing the prophylactic implantation of a pacemaker . functional status , supplemented by the recording of resting ecg , sa - ecg and pulmonary capacity , was monitored once a year . a second electrophysiological study was proposed to the patients in the event of new symptoms ( unexplained syncope or presyncope , palpitations ) , new atrioventricular conduction disturbances on annual resting ecg , significant modifications of sa - ecg with respect to the previous study,15 and atrial arrhythmias justifying anti - arrhythmic drugs , or in asymptomatic patients with a follow - up of at least 60 months . this time interval between the first and second electrophysiological tests in asymptomatic patients was chosen from the study of prystowsky et al,14 who reported a 5 ms prolongation of the hv interval over a 3-year period in several patients , and from the study of laurent et al,13 who reported a complete avb in one patient with a normal hv interval 5 years earlier . therefore , 60 months after the first electrophysiological test the hv interval would be prolonged by 10 ms and could exceed 70 ms . patients who were initially studied less than 60 months without new symptoms , or without significant changes in resting ecg or sa - ecg were not considered for a new electrophysiological study . the criterion for proposing a prophylactic pacemaker implantation was a hv interval of 70 ms or greater . as previously reported,15 the association of qrsd ( total qrs duration ) of 100 ms or greater and las40 ( duration of low amplitude signal<40v ) of 36 ms or greater on sa - ecg contributes to the identification of md patients with a prolonged hv interval . these criteria are not those commonly used to identify patients at risk of sustained ventricular tachycardia post - myocardial infarction , but in md they are linked to infrahisian conduction alteration and not to ventricular arrhythmia substrate.16 the primary endpoint was to evaluate the evolution over time of the infrahissian conduction time in adult md patients . the second endpoint was to define the predictive clinical and electrocardiographic parameters of a severe alteration in infrahissian conduction time that would justify a repetition of electrophysiological testing in md patients with a view to proposing the prophylactic implantation of a pacemaker . twenty - five of these 70 patients were enrolled and underwent a second electrophysiological testing . no significant differences were observed between the initial clinical characteristics of the 25 patients enrolled in the present study ( age , sex , brooke score , epworth score , resting ecg , right and left ventricular ejection fraction , ctg amplification ) and the 45 patients who did not initially receive a pacemaker ( table 1 ) and who were not enrolled in the present study for different reasons set out in figure 1 . the upgraded clinical characteristics of the 25 patients undergoing a second electrophysiological study are summarised in table 2 . initial mean characteristics of the md patients without a pacemaker group 1 all patients without pacemaker in our global myotonic dystrophy ( md ) population ; group 2 patients excluded from the present study for different reasons explained in figure 1 ; group 3 patients enrolled in the study who underwent two electrophysiological studies . avb , atrioventricular block ; ep , electrophysiological test ; hv , his - ventricle ; sa - ecg , signal - averaged ecg . at the time of the second electrophysiological test electrophysiological characteristics of patients who underwent a second electrophysiological study signal - averaged ( sa)-ecg was regarded as the presence of qrsd of 100 ms or greater and las40 of 36 ms or greater.15 pacemaker implanted for brady - tachycardia syndrome . modifications of sa - ecg reaching the significant cut - off defined by qrsd of 100 ms or greater and las of 36 ms15 or greater were observed in six patients . the mean values of sa - ecg parameters were : qrsd 10510.8 ms , under 40 36.512 ms , hfrmsa ( root mean square voltage of signals in the last 40 ms of the qrs complexes ) 30.324.7 v . the mean age at the second electrophysiological test was 44.413.6 years . the mean interval between the first and the second electrophysiological test was 90.836 months ( minimum 24 months , maximum 144 months ) . four patients were given the second electrophysiological test because they developed new symptoms , four others on account of modifications in resting ecg , one other for a bradytachycardia syndrome , only three for abnormal sa - ecg , and 13 patients merely because the follow - up exceeded 60 months . in asymptomatic patients with no ecg or sa - ecg modification , the mean interval between the first and the second electrophysiological test tended to be longer than in other patients ( 10134 months versus 8129 months , ns ) . at the second electrophysiological test the hv interval was 70 ms or greater in five cases ( 20% ) . the mean value of the hv interval increased significantly between the two electrophysiological tests ( figure 2 and table 3 ) . five patients with hv interval of 70 ms or greater were implanted with a prophylactic dual - chamber pacemaker . evolution of his - ventricle ( hv ) interval ( ms ) in myotonic dystrophy patients . full black square : individual hv interval at the first electrophysiological study ( hv1 ) and at the second electrophysiological study ( hv2 ) full black circles : mean value of hv intervales ( p<0.001 ) . the five patients with a hv interval of 70 ms or greater at the second electrophysiological test showed a modification in their resting ecg over time ( n=4 ) or significant modifications in the sa - ecg ( n=3 ) ( table 3 and figure 2 ) . one of the md patients with stable resting ecg , despite a prolonged hv interval , showed significant lengthening of sa - ecg parameters ( patient 24 in table 3 ) . qrsd and under 40 tended to be longer in the prolonged hv interval group than in normal hv interval group ( qrds 109.86 vs 103.611 ms , under 40 42.418 vs 349.6 ms).the mean duration of the follow - up was not significantly different between the group of patients with a hv interval of 70 ms or greater at the second electrophysiological test and those without a hv interval of 70 ms or greater at the second electrophysiological test ( 8046 vs 93.534 months , ns ) . resting ecg is very useful for the identification of patients with an alteration of infrahissian conduction . of the five patients with a new prolongation of the hv interval , four showed significant modification in their resting ecg . the combination of modifications of resting ecg and sa - ecg identified all patients with prolonged hv interval ( sensitivity 100% and specificity 75% ) . at the time of the second electrophysiological test electrophysiological characteristics of patients who underwent a second electrophysiological study signal - averaged ( sa)-ecg was regarded as the presence of qrsd of 100 ms or greater and las40 of 36 ms or greater.15 pacemaker implanted for brady - tachycardia syndrome . modifications of sa - ecg reaching the significant cut - off defined by qrsd of 100 ms or greater and las of 36 ms15 or greater were observed in six patients . the mean age at the second electrophysiological test was 44.413.6 years . the mean interval between the first and the second electrophysiological test was 90.836 months ( minimum 24 months , maximum 144 months ) . four patients were given the second electrophysiological test because they developed new symptoms , four others on account of modifications in resting ecg , one other for a bradytachycardia syndrome , only three for abnormal sa - ecg , and 13 patients merely because the follow - up exceeded 60 months . in asymptomatic patients with no ecg or sa - ecg modification , the mean interval between the first and the second electrophysiological test tended to be longer than in other patients ( 10134 months versus 8129 months , ns ) . at the second electrophysiological test the hv interval was 70 ms or greater in five cases ( 20% ) . the mean value of the hv interval increased significantly between the two electrophysiological tests ( figure 2 and table 3 ) . five patients with hv interval of 70 ms or greater were implanted with a prophylactic dual - chamber pacemaker . evolution of his - ventricle ( hv ) interval ( ms ) in myotonic dystrophy patients . full black square : individual hv interval at the first electrophysiological study ( hv1 ) and at the second electrophysiological study ( hv2 ) full black circles : mean value of hv intervales ( p<0.001 ) . the five patients with a hv interval of 70 ms or greater at the second electrophysiological test showed a modification in their resting ecg over time ( n=4 ) or significant modifications in the sa - ecg ( n=3 ) ( table 3 and figure 2 ) . one of the md patients with stable resting ecg , despite a prolonged hv interval , showed significant lengthening of sa - ecg parameters ( patient 24 in table 3 ) . qrsd and under 40 tended to be longer in the prolonged hv interval group than in normal hv interval group ( qrds 109.86 vs 103.611 ms , under 40 42.418 vs 349.6 ms).the mean duration of the follow - up was not significantly different between the group of patients with a hv interval of 70 ms or greater at the second electrophysiological test and those without a hv interval of 70 ms or greater at the second electrophysiological test ( 8046 vs 93.534 months , ns ) . resting ecg is very useful for the identification of patients with an alteration of infrahissian conduction . of the five patients with a new prolongation of the hv interval , four showed significant modification in their resting ecg . the combination of modifications of resting ecg and sa - ecg identified all patients with prolonged hv interval ( sensitivity 100% and specificity 75% ) . this study , for the first time , shows the evolution of the resting ecg and sa - ecg characteristics and of the hv interval in non - selected adult md patients monitored every year in the same reference centre . some significant modifications of resting ecg were observed in four patients and abnormal sa - ecg in six patients , and were strongly associated with an hv interval prolongation . the hv interval increase was not linked to any new symptoms such as syncope or palpitations , or to the duration of the follow - up . the electrophysiological consequences of these histopathological lesions were frequent infrahissian conduction disturbances on electrophysiological testing ( prolonged hv interval ) and a high risk of complete avb in md patients.12 13 21 in animal models for dystrophy myotonic protein kinase ( dmpk ) reduction the same histopathological lesions and his purkinje system alterations were reported.22 a time - dependent degenerative process has been suggested by groh et al,23 who observed a correlation between age and ecg abnormalities , and by prystowsky et al,14 who in serial electrophysiological studies demonstrated the progression of infrahissian conduction time in a small number ( n=9 ) of non - consecutive md patients . however , in the large study by laurent et al,13 including 51 md patients with a normal hv interval of less than 70 ms followed up during 5736 months , only one patient developed a complete avb , 52 months after the initial electrophysiological testing , whereas 19 of 49 patients with hv interval of 70 ms or greater developed 3 avb . these results suggest two categories of adult md patients : one characterised by early infrahissian conduction disturbances ( hv interval 70 ms ) frequently risking complete avb , and a second category of adult md patients who may exhibit a very slow progression of infrahissian conduction time . this difference can be explained by the high rate of abnormal resting ecg ( 7/9 ) and prolonged hv interval ( 3/9 ) at the initial evaluation in the prystowsky study.14 second , the high prevalence of complete avb documented in memory pacemaker in md patients with an early hv interval of 70 ms or greater points to a severe evolution of the infrahissian conduction time in this subgroup of patients.12 13 this heterogeneity of md patients in terms of infrahissian characteristics is compatible with the heterogeneity of the size of the mutation in the different organs ( somatic mosaicism ) , which is not exactly the same for leucocytes . a second major finding of our study is the strong relationship between the evolution of cardiac conduction disturbances on resting ecg and the increase in the hv interval in a serial study . no correlation between resting ecg and hv interval or histopathological lesions12 14 15 20 has been reported in the literature , but the previous studies did not integrate the dynamism of the atrioventricular conduction parameters in their analysis . in our study patients with a significant increase in the hv interval corresponded approximately to those with a significant progression of cardiac conduction abnormalities on resting ecg . ventricular arrhythmias induced during electrophysiological study do not predict spontaneous ventricular arrhythmias.16 we can only suggest discussing the implantation of an implantable cardioverter defibrillator instead of a pacemaker in md patients with prolonged hv interval and depressed left ventricular ejection fraction below 30%.26 breton and mathieu11 proposed integrating the analysis of qt interval on resting ecg to identify md patients at risk of sudden death or pacemaker implantation . sa - ecg is a useful tool in evaluating the infrahissian conduction characteristics of md patients.15 babuty et al15 demonstrated that an association of qrsd of 100 ms or greater and las of 36 ms or greater selects patients with a prolonged hv interval of 70 ms or greater with high sensitivity ( 80% ) and specificity ( 83.3% ) . the combination of resting ecg and sa - ecg appears to be the best method of identifying patients showing an alteration over time of the infrahissian conduction time , with excellent sensitivity ( 100% ) and acceptable specificity ( 75% ) . some patients could have a normal ecg and prolonged hv interval due to a short ah interval.12 these patients , however , can be identified by the sa - ecg , which , as previously mentioned in the discussion , accurately reveals infrahissian conduction abnormalities . only observational and non - controlled studies suggest a real benefit from the prophylactic implantation of a pacemaker in the presence of a prolonged hv interval of 70 ms or greater.12 13 the fact that not all the patients without a pacemaker were systematically re - investigated could introduce a bias in this study . some patients could have a normal ecg and prolonged hv interval due to a short ah interval.12 these patients , however , can be identified by the sa - ecg , which , as previously mentioned in the discussion , accurately reveals infrahissian conduction abnormalities . only observational and non - controlled studies suggest a real benefit from the prophylactic implantation of a pacemaker in the presence of a prolonged hv interval of 70 ms or greater.12 13 the infrahissian conduction disturbances frequently observed in adult md patients justify the early identification of patients with a prolonged hv interval , who are at high risk of complete avb . prophylactic implantation of a pacemaker should be proposed to patients with a hv interval of 70 ms or greater . in patients with an initially normal infrahissian conduction time on the electrophysiological study , the risk of complete avb is very low , and a second electrophysiological study should be performed only in the event of modifications in atrioventricular conduction ecg parameters or sa - ecg in annual check - ups . in the future , it will be important to conduct a controlled and randomised study in order to demonstrate clearly the benefits of prophylactic pacemaker implantation in md patients with a hv interval of 70 ms or greater .

one of the most stable and intriguing properties of autistic intelligence is that the relative level of difficulty of the different tasks used to estimate intelligence is not the same for autistic and non - autistic people . for instance , autistic individuals obtain a better score when evaluated with the raven 's progressive matrices ( rspm ) test ( raven , 1976 ) than with the wechsler iq test , whereas non - autistic individuals obtain similar scores on both tests ( dawson et al . , 2007 ; in parallel , autistic individuals tend to exhibit a relative advantage on visuospatial tasks in comparison to verbal tasks , as reflected by their ability on visual search , embedded figures , wechsler 's block design , pattern discrimination and mental imagery tasks ( soulires et al . , 2011 ; stevenson and gernsbacher , 2013 ) . the unique pattern of cognitive performance described above is accompanied by an alteration in underlying patterns of cerebral activity and connectivity ( samson et al . , 2012 ; stronger recruitment of visual perceptual brain regions can be found in a wide array of tasks using faces , objects and words as stimuli ( samson et al . , 2012 ) , including higher order tasks such as fluid reasoning ( sahyoun et al . , 2010 ; soulires et al . , 2009 ; yamada et al . , , initial investigations yielded results supporting widespread long - range underconnectivity and local overconnectivity in autism ( courchesne and pierce , 2005 ; just et al . , 2007 ) . more recent studies temper this view , demonstrating that local over - connectivity depends on the specific type of analyses conducted ( vissers et al . , 2012 ) and that perceptual long - range functional connectivity is sometimes stronger in autistic relative to non - autistic participants ( keehn et al . , 2013 ; leveille et al . , 2010 ; mcgrath et al . , white matter microstructural alterations are also found and correlate with autistic signs and symptoms ( gibbard et al . , 2013 ; ikuta et al . , 2014 ; mcfadden and minshew , 2013 ) , performance in visuospatial tasks ( mcgrath et al . , 2013 ) , and fluid reasoning abilities ( ellmore et al . , 2013 ) . fluid reasoning relates to the ability to infer logical solutions when solving novel problems ( cattell , 1987 ) . a spatially extended brain network underlies fluid reasoning , with crucial components in prefrontal and parietal regions ( jung and haier , 2007 ; perfetti et al . , 2009 ) . activity in the fronto - parietal reasoning network is modulated by reasoning complexity and is associated with individual reasoning ability levels ( gray et al . , 2003 ; lee et al . , 2006 ; perfetti et al . , 2009 ) . increasing reasoning complexity is associated with widespread increases in activity in the reasoning network , with marked increases in dorsolateral prefrontal cortex ( kalbfleisch et al . , 2007 ; kroger et al . , 2002 ; wendelken et al . , 2008 ) . higher reasoning abilities are associated with higher activity in frontal and parietal regions ( perfetti , 2009 ) , and particularly in posterior parietal cortex ( lee et al . , 2006 ) . in autistic individuals , solving fluid reasoning problems is accompanied by higher activity in occipital and temporal regions , but lower activity in some frontal ( middle frontal gyrus ) and parietal regions ( precuneus ) , compared to non - autistic individuals ( soulires et al . , 2009 ) , and lower structural connectivity between frontal language areas and temporal regions ( sahyoun et al . , 2010 ; yamada et al . , 2012 ) . in a previous fmri study ( soulires et al . , 2009 ) , we recorded brain activity while participants performed the rspm , the emblematic fluid reasoning test ( see examples in fig . autistic participants performed the self - paced rspm task with an accuracy similar to that of their comparison group , but unexpectedly exhibited a 40% shorter response time . during the resolution of the matrices , left middle occipital gyrus ( ba18 ) was disproportionally engaged in autistic participants , suggesting that autistic reasoning might rely more heavily on the involvement of occipital regions and their associated perceptual processes during fluid reasoning . however , in light of recent findings identifying increased activity in perceptual areas in autistic individuals in a wide range of tasks ( see samson et al . , 2012 for a meta - analysis ) , one can question whether the increased activity in occipital areas during a reasoning task contributes to reasoning processes per se . we inferred that observing connectivity between this region and other elements of the reasoning network that was modulated by problem complexity would provide further evidence that occipital areas genuinely contribute to autistic reasoning . the objective of this study was to characterize how regional cerebral activity and connectivity are modulated by task complexity in autistic individuals during fluid reasoning . we were specifically interested in verifying whether the increased activity previously observed in visuospatial areas in autistic individuals during matrix reasoning would be associated with activity and connectivity modulations according to problem complexity . we therefore conducted psychophysiological interaction analyses ( ppi ) on data from our previous rspm study ( soulires et al . , 2009 ) , with two sets of seed regions . the first set was based on areas of maximal activity common to both groups while solving the rspm . often in studies with clinical samples , the selection of seed regions is based on task - related activity patterns observed in the control group , with those seed regions possibly being slightly displaced from activity peaks in the clinical group . this selection approach could result in a bias towards detecting reduced functional connectivity in the clinical sample . here , we employed a more neutral approach , selecting seed regions based on the conjunction of task - related activity observed in both groups . then , using a second set of seed regions based on areas of between - group differences in task - related activity , we more specifically addressed our primary goal to examine the contribution of occipital regions to autistic reasoning . we predicted that autistic participants would exhibit higher functional connectivity involving the occipital seeds with increasing reasoning complexity , relative to non - autistic participants , as suggested by previous observations of stronger reasoning - related activity and anatomical local connectivity in occipital regions ( sahyoun et al . given numerous previous findings of lower parieto - frontal functional connectivity in autism and the lower frontal activity seen during reasoning in autistic individuals in our previous study , we also predicted lower functional connectivity between prefrontal and posterior parietal regions during the resolution of the more complex reasoning items in autistic relative to non - autistic participants . mri data were collected from 15 autistic participants ( aged 1435 , m = 22.40 , sd = 5.95 , 2f ) and 18 non - autistic participants ( aged 1436 , m = 21.72 , sd = 5.20 , 3f ) ( dataset from soulires et al . , 2009 ; see participant characteristics in table 1 ) . autistic participants received a diagnosis according to dsm - iv criteria and were evaluated with two diagnostic instruments , the autism diagnosis observation schedule ( ados - g ; lord et al . , 2000 ) and autism diagnostic interview ( adi - r ; le couteur et al . , 1989 ) , by a multidisciplinary team of expert clinicians . a comparison group of participants self - reporting no psychiatric or neurological conditions were recruited from the local population . primary exclusion criteria for both groups were uncorrected visual impairment , use of psychoactive or vasoactive medication as well as use of illegal drugs or excessive alcohol consumption . the study was approved by the institutional review boards of regroupement neuroimagerie / qubec and rivire - des - prairies hospital . a computerized version of the rspm test was used ( see fig . 1 for examples similar to the items in the rspm test ) . for each of the 60 test items , participants selected the missing entry in a matrix among 8 possible choices , with no time limit . the rspm task was slightly modified from its original paper version to suit the fmri environment . first , the two rows of answer choices were shifted horizontally to simplify the mapping between answers and response pads . participants had to answer by pressing a button with their left ( choices 14 ) or right hand ( choices 58 ) . the item remained visible until an answer was given by the participant , followed by a fixation period varying from 4 to 7 s following an exponential distribution . the second modification made to the original test was to present the 60 items in a randomized order ( instead of ascending order of complexity as in the original test ) , to avoid any presentation order / difficulty confound in the analyses . participants first had two practice sessions of a relatively easy pattern matching task sharing the same stimulus presentation specificities as the rspm task . the first practice session was done while sitting in front of a computer screen , while the second was done in a mock scanner . the rspm task took between 14 to 35 min , as the participants were instructed to take as much time as needed to solve each item . images were acquired using a siemens trio 3 t scanner with an 8-channel phased - array head coil . functional data were obtained using an echo planar imaging ( epi ) bold sequence , using a variable epoch design ( 48 contiguous slices , 3 mm cubic voxels , tr = 2850 ms , te = 30 ms , flip angle = 90 , fov = 192 mm ) , and structural data were t1 weighted ( mp - rage , 176 slices , 1 mm cubic voxels , tr = 2530 ms , te = 3.48 ms , flip angle = 7 , fov = 256 mm ) . the 60 rspm test items were divided into 3 complexity levels : figural ( 16 items ) , analytical ( 23 items ) and complex analytical ( 21 items ) . first , the items were divided into figural versus analytical types based on two previous classifications ( lynn et al . the figural items can be solved with visual strategies , such as visual pattern completion . conversely , the analytical and complex analytical items require abstraction and application of one or more rules ( carpenter et al . , 1990 ) . the analytical items were then further divided into two complexity levels based on rspm accuracy data from 26 non - autistic adults in our research database . the complex analytical items required the application of more complex rules and/or a combination of rules and resulted in lower accuracy than the easier analytical items and figural items . group ( autistic , non - autistic ) complexity ( figural , analytical , complex analytical ) analysis of variance ( anova ) were conducted on accuracy and response times using spss ( ibm corp . images were preprocessed with spm5 ( http://www.fil.ion.ucl.ac.uk/spm/ ) following the same procedure as in soulires et al . statistical modeling and visualization were done with spm8 ( http://www.fil.ion.ucl.ac.uk/spm/ ) and mricron ( rorden and brett , 2000 ) . slice timing was corrected to the middle slice of the volume , and then a two - pass realignment involving first registration to the first image and then to the mean of the realigned images , followed by reslicing with 4th degree b - spline interpolation . eight mm full - width at half maximum fwhm source image smoothing was applied and images were resampled to 2 2 2 mm . finally , normalized epi images were smoothed using an isotropic gaussian smoothing kernel with fwhm of 9 9 9 mm , to compensate for residual individual and group anatomical differences . to verify the presence of atypical oculomotor movement sometimes reported in autism we compared saccade density during the reasoning task in the two groups . we extracted the time course of the bold contrast signal activity within two 12.5 mm spherical roi ( one for each eye ) , for each participant . an estimate of the net saccade density during the rspm was obtained by computing the standard deviation of the activity time course , and compared between groups with a t test . head motion data ( head translation and rotation estimates ) was extracted during the preprocessing realignment and included as covariates in the first - level models . moreover , as to insure that there was no group difference in head movement , head motion data was directly compared between groups . mean displacement rate ( mm / s ) and rotation rate ( degrees / s ) , as well as peak - to - peak translation ( x , y , z ) and rotation ( pitch , roll , yaw ) for each axis , were computed and compared with independent samples t tests . for each participant , a first - level general linear model was generated with three task regressors ( figural , analytical and complex analytical ) , using all trials ( correct and incorrect ) , and six motion estimate regressors . individual contrasts for each of the three levels of complexity were used in the subsequent second - level model . within and between - group mixed - effect analyses on the complexity factor ( figural < analytical < complex analytical ) were computed using a threshold of p < 0.001 uncorrected with extent threshold k = 50 contiguous voxels . additional analyses were conducted in each group to verify the influence of response times on task - related activity , by entering response times as a covariate in a t - test . a first set of seed regions was derived from a conjunction analysis of the overall rspm task - related activity across all levels of complexity , allowing identification of areas of activity common to both groups . using conjunction analysis for seed selection ensured having high levels of activity in both groups in order to guide the seed selection for the connectivity analyses . coordinates of local task - related maximal activity were identified in each of the frontal , parietal and occipital lobes . these seeds were located in left inferior occipital gyrus ( ba18 ) , left superior parietal lobule ( ba7 ) and right inferior frontal gyrus ( ba9 ) . a second set of seed regions was selected using the results of the between - group contrast analysis exploring the complexity factor , in order to represent areas maximizing activity differences between the two groups . peak coordinates of clusters more active in autistic participants ( autistic > non - autistic ) , as well as in non - autistic participants ( non - autistic > autistic ) , were identified on the complexity contrast ( figural < analytical < complex analytical ) , yielding seeds in the left superior occipital gyrus , left middle frontal gyrus and left precuneus . the modulation of functional connectivity by task complexity was assessed using a ppi approach ( friston , 2011 ; friston et al . , 1997 ; gitelman et al . , 2003 ) . using a 6 mm radius sphere around each of the seeds , eigenvariates ( reflecting the mean activity of voxels showing maximum variance within the sphere ) were extracted for each participant , while adjusting for the effects of interest . each extracted time series was then entered in a ppi analysis model in order to form the interaction between the seed region activity and task complexity ( figural < analytical < complex analytical ) . this step created 3 vectors : the interaction term , the volume of interest ( voi ) eigenvariate and the relative complexity contrast vector . first - level analyses were then performed using the movement parameters and the 3 vectors . finally , 6 s - level factorial models , one per seed region , were computed to illustrate the interaction between functional connectivity involving the seed regions and task complexity . the group complexity anova on accuracy revealed a significant main effect of complexity , f(2 , 90 ) = 63.019 , p < 0.001 , but no significant effect of group , f(1 , 90 ) = 0.964 , p = 0.329 , and no significant interaction , f(2 , 90 ) = 0.133 , p = 0.876 . post - hoc comparisons confirmed a lower accuracy for complex analytical ( 58.25% ) problems than for figural ( 90.35% ) and analytical problems ( 83.50% ) , both p < 0.001 . the group complexity anova on response times revealed a significant main effect of group , f(1 , 90 ) = 14.047 , p < 0.001 , and of complexity , f(2 , 90 ) = 55.185 , p < 0.001 , with no significant interaction , f(2 , 90 ) = 2.242 , p = 0.112 . autistic participants ( 12.52 s ) were significantly faster than non - autistic participants ( 17.29 s ) by an average of 4.77 s. response times were longer for complex analytical ( 23.87 s ) than for analytical problems ( 13.03 s ) , p < 0.001 , and analytical than figural problems ( 7.82 s ) , p = 0.002 ( see table 2 ) . a linear contrast examining reasoning complexity ( figural < analytical < complex analytical ) revealed an extended network involving frontal , parietal , occipital , cerebellar and basal ganglia regions , all associated with increasing reasoning complexity ( table 3 ) . entering response times as a covariate in within - group analyses did not result in any significant changes in the findings . significant between - group differences in the effects of complexity on task - related activity were revealed in several areas . first , increased complexity - related activity was observed in the left superior occipital gyrus and the left middle occipital gyrus for autistic participants relative to non - autistic participants . second , non - autistic participants showed increased activity in the left middle frontal gyrus ( mfg ) and bilateral precuneus , relative to autistic participants ( fig . 2 ) . fig . 2c and d further illustrates that left mfg was only recruited for complex analytical problems in autistic participants , and that precuneus was not significantly recruited during the task in the autistic group . these results suggest a stronger reliance on occipital areas for autistic participants as task complexity increased . the eye movement analysis showed similar levels of net saccade density during the rspm task between the autistic participants ( 3.96 ) and non - autistic participants ( 4.44 ; t = 0.89 , p = 0.38 ) . similar mean displacement rates were observed in autistic ( 0.032 mm / s ) and non - autistic participants ( 0.038 mm / s ; t = 0.72 , p = 0.48 ) . similar mean rotation rates were observed in the two groups ( autistic group : 0.025/s ; non - autistic group : 0.036/s ; t = 1.28 , p = 0.21 ) . no significant differences were observed in the peak - to - peak translations or rotations ( all p > 0.05 ) . conjunction seeds ( right ifg ( ba9 ) , left spl ( ba7 ) and left inferior occipital gyrus ( ba18 ) ) represent the common areas of activity in both groups during the rspm task . their associated time series were used in ppi analyses in order to assess how these key fluid reasoning regions interact with other brain regions as complexity increases . within - group analyses in the autistic group revealed an interaction between the frontal seed ( right ifg ) and the left ifg . the occipital seed showed an interaction with frontal , temporal , occipital , parietal regions as well as the cerebellum . moreover , the parietal seed interacted with the left ifg , left ipl , left angular gyrus , right postcentral gyrus and left cerebellum . the within - group analyses in the non - autistic group showed interaction between the 3 seeds and multiple regions in frontal , parietal , temporal and occipital areas . a between - group contrast ( aut > non - aut ) showed that autistic participants had greater connectivity than non - autistic participants ( p < 0.001 , unc . ) between the occipital seed ( left inferior occipital gyrus ) and areas in the left superior frontal gyrus , right spl , right middle occipital gyrus and right inferior temporal gyrus . in contrast , non - autistic participants had greater connectivity than autistic participants between all 3 seeds and multiple other regions involved in reasoning ( frontal , parietal , temporal and occipital regions ) as complexity increased ( table 4 ) . overall , non - autistic participants exhibited more complexity - related connectivity than autistic participants in the selected frontal , parietal and occipital seeds ( fig . 3 ) , demonstrating that autistic individuals exhibit reduced modulation of connectivity by reasoning complexity in those areas . in order to explore group - specific patterns of connectivity in relation to complexity , maximum difference seeds were used to identify the key areas associated with reasoning complexity that are unique to each group in within - group analyses . in the autistic group , reasoning complexity modulated connectivity between the occipital seed ( left superior occipital gyrus ) and circumscribed frontal , temporal , occipital and subcortical regions ( table 5 ) . however no interaction involving the frontal and precuneus seeds were seen . in the non - autistic group , reasoning complexity modulated connectivity between each of the 3 seeds and an extended network of cortical and subcortical regions ( fig . 4 ) . this second set of ppi analyses also suggests less extended network interactions involved in reasoning in autistic individuals relative to non - autistic individuals . our objective was to investigate how task complexity modulates the level of activity and connectivity among brain regions during fluid reasoning in autism . while both groups showed similar accuracy in solving rspm problems , autistic participants performed faster than non - autistic participants at all complexity levels , as we previously reported in a study identifying occipital cortex as being more active in autistic individuals during matrix reasoning ( soulires et al . , 2009 ) . moreover , while autistic participants exhibited less modulation of fronto - parietal activity and connectivity as reasoning complexity increased , connectivity involving occipital areas was more consistently modulated by task complexity in autistic than non - autistic participants . occipital regions are not only known for their involvement in visuospatial processes , but also in the identification of correct answers with abstract rules ( skosnik et al . , 2002 ) , number subtraction and addition ( benn et al . , 2012 ) . the greater use of these regions as complexity increases in a reasoning task has an equivalent in typical individuals , albeit to a lesser extent ( goel , 2007 ; soulires et al . , 2009 ; yamada et al . , in contrast , compared to autistic participants , non - autistic participants more strongly engaged a combination of regions related to verbal and visuospatial processing as task complexity increased . the left middle frontal gyrus is known to be involved in inner speech ( geva et al . , 2011 ; jones and fernyhough , 2007 ) , working memory ( liakakis et al . , 2011 ) and other verbal processes ( prado et al . , 2013 ) . the precuneus in both hemispheres is involved in visuospatial imagery , episodic memory retrieval and self - referential processing ( cavanna and trimble , 2006 ) . in this group an occipital to frontal / parietal activation shift is seen when participants inhibit perceptual information to engage in logical reasoning , and by extension with increasing reasoning complexity ( houde et al . , 2000 ) . the present results suggest that this shift might not be as visible in autistic individuals , as perceptual processes able to support this type of reasoning are engaged more strongly . thus , we suggest that autistic individuals rely more specifically on visuospatial processes to resolve complex matrices whereas non - autistic individuals rely more prominently on a combination of verbal and visuospatial processes . our hypothesis that autistic participants would exhibit higher cortical connectivity in the posterior parts of the reasoning network as task complexity increased , compared to non - autistic participants , was partially confirmed . indeed , a stronger long - range functional connectivity was identified in autistic participants , relative to non - autistic participants , between the left inferior occipital gyrus and the left superior frontal gyrus and the right spl as task complexity increased . the possibility of enhanced connectivity between visual associative areas and other parts of the brain , even the most distant , has been raised by several prior studies ( leveille et al . , 2010 ; 2013 ) , views at odds with dominant accounts of reduced long - range connectivity ( dichter , 2012 ; just et al . , 2012 ) . in combination with the demonstration of enhanced activity associated with visual processing in many contexts in autism ( samson et al . , 2012 ) , we believe that autism is characterized , not only by enhanced perceptual performance , but also by enhanced role of perceptual processing in higher order cognitive processes ( mottron et al . , 2013 ) . by contrast , none of the analyses involving seeds in prefrontal cortex revealed increased functional connectivity in autistic participants relative to non - autistic participants . to the contrary , autistic participants generally exhibited less connectivity in the anterior elements of the network , a finding also reported by previous studies ( sahyoun et al . mcgrath and colleagues also obtained similar results in a mental rotation task , as they observed a general decrease in functional connectivity as task complexity increased in autistic individuals , the visual cortex being the only exception to this decrease ( mcgrath et al . , 2012 ) . this decreased modulation of the fronto - parietal network could suggest that autistic individuals are less influenced by task complexity than non - autistic individuals , as they showed less change in the network 's connectivity as complexity increased , while maintaining equal accuracy . considering that both groups showed similar accuracy , and autistic individuals responded more quickly during the task , we can not conclude that our results are best interpreted as altered network activity in the network as suggested by yamada et al . rather , they may reflect lower complexity - induced modulation of the network in autistic individuals , with potential positive consequences on performance . as the enhanced perceptual functioning ( epf ) model suggests , autistic cognition is , among other elements , characterized by a stronger engagement of perceptual processes and a different equation between neuronal engagement and task difficulty ( mottron et al . , 2013 ) . our results of a stronger engagement of visuospatial processes and lower modulation of the reasoning network as complexity increases during reasoning directly contribute to this model and add to the body of work describing a stronger engagement of perceptual processes in higher cognition in autistic individuals . this study focussed on the influence of visuospatial processes on fluid reasoning in autistic individuals and may not reflect all the cognitive process involved in other aspects of intelligence . also , we chose to use a liberal threshold ( p < 0.001 uncorrected ) in order to observe the full extent of between - group differences in the cerebral activity and connectivity underlying reasoning , as a more stringent threshold may smooth out some interesting differences . finally , our sample was composed of participants with measured intelligence in the normal range only ; hence , these results may or may not apply to the whole spectrum of autistic intelligence . nevertheless , these results provide us with a better understanding of the atypical , yet not dysfunctional , reasoning abilities of autistic individuals ( dawson et al . , 2007 ) . indeed , the relative advantage of autistic individuals at the rspm might be linked to a more efficient recruitment of posterior brain regions analyzing the visuospatial information provided in the problems . further studies could explore the effect of complexity in other domains of autistic cognition , as well as how the modulation of cognitive processes in relation to complexity occurs in autistic individuals with lower reasoning abilities . this could lead to the application of these findings to optimizing learning and work environments for autistic individuals .

different test types lead to different intelligence estimates in autism , as illustrated by the fact that autistic individuals obtain higher scores on the raven 's progressive matrices ( rspm ) test than they do on the wechsler iq , in contrast to relatively similar performance on both tests in non - autistic individuals . however , the cerebral processes underlying these differences are not well understood . this study investigated whether activity in the fluid reasoning network , which includes frontal , parietal , temporal and occipital regions , is differently modulated by task complexity in autistic and non - autistic individuals during the rspm . in this purpose , we used fmri to study autistic and non - autistic participants solving the 60 rspm problems focussing on regions and networks involved in reasoning complexity . as complexity increased , activity in the left superior occipital gyrus and the left middle occipital gyrus increased for autistic participants , whereas non - autistic participants showed increased activity in the left middle frontal gyrus and bilateral precuneus . using psychophysiological interaction analyses ( ppi ) , we then verified in which regions did functional connectivity increase as a function of reasoning complexity . ppi analyses revealed greater connectivity in autistic , compared to non - autistic participants , between the left inferior occipital gyrus and areas in the left superior frontal gyrus , right superior parietal lobe , right middle occipital gyrus and right inferior temporal gyrus . we also observed generally less modulation of the reasoning network as complexity increased in autistic participants . these results suggest that autistic individuals , when confronted with increasing task complexity , rely mainly on visuospatial processes when solving more complex matrices . in addition to the now well - established enhanced activity observed in visual areas in a range of tasks , these results suggest that the enhanced reliance on visual perception has a central role in autistic cognition .

Introduction Methods Results Discussion

one of the most stable and intriguing properties of autistic intelligence is that the relative level of difficulty of the different tasks used to estimate intelligence is not the same for autistic and non - autistic people . for instance , autistic individuals obtain a better score when evaluated with the raven 's progressive matrices ( rspm ) test ( raven , 1976 ) than with the wechsler iq test , whereas non - autistic individuals obtain similar scores on both tests ( dawson et al . , 2007 ; in parallel , autistic individuals tend to exhibit a relative advantage on visuospatial tasks in comparison to verbal tasks , as reflected by their ability on visual search , embedded figures , wechsler 's block design , pattern discrimination and mental imagery tasks ( soulires et al . , 2012 ; stronger recruitment of visual perceptual brain regions can be found in a wide array of tasks using faces , objects and words as stimuli ( samson et al . , 2012 ) , including higher order tasks such as fluid reasoning ( sahyoun et al . , 2012 ) and that perceptual long - range functional connectivity is sometimes stronger in autistic relative to non - autistic participants ( keehn et al . , 2013 ) , and fluid reasoning abilities ( ellmore et al . fluid reasoning relates to the ability to infer logical solutions when solving novel problems ( cattell , 1987 ) . activity in the fronto - parietal reasoning network is modulated by reasoning complexity and is associated with individual reasoning ability levels ( gray et al . increasing reasoning complexity is associated with widespread increases in activity in the reasoning network , with marked increases in dorsolateral prefrontal cortex ( kalbfleisch et al . higher reasoning abilities are associated with higher activity in frontal and parietal regions ( perfetti , 2009 ) , and particularly in posterior parietal cortex ( lee et al . in autistic individuals , solving fluid reasoning problems is accompanied by higher activity in occipital and temporal regions , but lower activity in some frontal ( middle frontal gyrus ) and parietal regions ( precuneus ) , compared to non - autistic individuals ( soulires et al . , 2009 ) , we recorded brain activity while participants performed the rspm , the emblematic fluid reasoning test ( see examples in fig . during the resolution of the matrices , left middle occipital gyrus ( ba18 ) was disproportionally engaged in autistic participants , suggesting that autistic reasoning might rely more heavily on the involvement of occipital regions and their associated perceptual processes during fluid reasoning . however , in light of recent findings identifying increased activity in perceptual areas in autistic individuals in a wide range of tasks ( see samson et al . , 2012 for a meta - analysis ) , one can question whether the increased activity in occipital areas during a reasoning task contributes to reasoning processes per se . we inferred that observing connectivity between this region and other elements of the reasoning network that was modulated by problem complexity would provide further evidence that occipital areas genuinely contribute to autistic reasoning . the objective of this study was to characterize how regional cerebral activity and connectivity are modulated by task complexity in autistic individuals during fluid reasoning . we were specifically interested in verifying whether the increased activity previously observed in visuospatial areas in autistic individuals during matrix reasoning would be associated with activity and connectivity modulations according to problem complexity . we therefore conducted psychophysiological interaction analyses ( ppi ) on data from our previous rspm study ( soulires et al . , 2009 ) , with two sets of seed regions . the first set was based on areas of maximal activity common to both groups while solving the rspm . often in studies with clinical samples , the selection of seed regions is based on task - related activity patterns observed in the control group , with those seed regions possibly being slightly displaced from activity peaks in the clinical group . this selection approach could result in a bias towards detecting reduced functional connectivity in the clinical sample . here , we employed a more neutral approach , selecting seed regions based on the conjunction of task - related activity observed in both groups . then , using a second set of seed regions based on areas of between - group differences in task - related activity , we more specifically addressed our primary goal to examine the contribution of occipital regions to autistic reasoning . we predicted that autistic participants would exhibit higher functional connectivity involving the occipital seeds with increasing reasoning complexity , relative to non - autistic participants , as suggested by previous observations of stronger reasoning - related activity and anatomical local connectivity in occipital regions ( sahyoun et al . given numerous previous findings of lower parieto - frontal functional connectivity in autism and the lower frontal activity seen during reasoning in autistic individuals in our previous study , we also predicted lower functional connectivity between prefrontal and posterior parietal regions during the resolution of the more complex reasoning items in autistic relative to non - autistic participants . mri data were collected from 15 autistic participants ( aged 1435 , m = 22.40 , sd = 5.95 , 2f ) and 18 non - autistic participants ( aged 1436 , m = 21.72 , sd = 5.20 , 3f ) ( dataset from soulires et al . autistic participants received a diagnosis according to dsm - iv criteria and were evaluated with two diagnostic instruments , the autism diagnosis observation schedule ( ados - g ; lord et al . a computerized version of the rspm test was used ( see fig . 1 for examples similar to the items in the rspm test ) . for each of the 60 test items , participants selected the missing entry in a matrix among 8 possible choices , with no time limit . the second modification made to the original test was to present the 60 items in a randomized order ( instead of ascending order of complexity as in the original test ) , to avoid any presentation order / difficulty confound in the analyses . the rspm task took between 14 to 35 min , as the participants were instructed to take as much time as needed to solve each item . the 60 rspm test items were divided into 3 complexity levels : figural ( 16 items ) , analytical ( 23 items ) and complex analytical ( 21 items ) . the analytical items were then further divided into two complexity levels based on rspm accuracy data from 26 non - autistic adults in our research database . group ( autistic , non - autistic ) complexity ( figural , analytical , complex analytical ) analysis of variance ( anova ) were conducted on accuracy and response times using spss ( ibm corp . slice timing was corrected to the middle slice of the volume , and then a two - pass realignment involving first registration to the first image and then to the mean of the realigned images , followed by reslicing with 4th degree b - spline interpolation . to verify the presence of atypical oculomotor movement sometimes reported in autism we compared saccade density during the reasoning task in the two groups . we extracted the time course of the bold contrast signal activity within two 12.5 mm spherical roi ( one for each eye ) , for each participant . an estimate of the net saccade density during the rspm was obtained by computing the standard deviation of the activity time course , and compared between groups with a t test . head motion data ( head translation and rotation estimates ) was extracted during the preprocessing realignment and included as covariates in the first - level models . moreover , as to insure that there was no group difference in head movement , head motion data was directly compared between groups . mean displacement rate ( mm / s ) and rotation rate ( degrees / s ) , as well as peak - to - peak translation ( x , y , z ) and rotation ( pitch , roll , yaw ) for each axis , were computed and compared with independent samples t tests . individual contrasts for each of the three levels of complexity were used in the subsequent second - level model . additional analyses were conducted in each group to verify the influence of response times on task - related activity , by entering response times as a covariate in a t - test . a first set of seed regions was derived from a conjunction analysis of the overall rspm task - related activity across all levels of complexity , allowing identification of areas of activity common to both groups . using conjunction analysis for seed selection ensured having high levels of activity in both groups in order to guide the seed selection for the connectivity analyses . coordinates of local task - related maximal activity were identified in each of the frontal , parietal and occipital lobes . these seeds were located in left inferior occipital gyrus ( ba18 ) , left superior parietal lobule ( ba7 ) and right inferior frontal gyrus ( ba9 ) . a second set of seed regions was selected using the results of the between - group contrast analysis exploring the complexity factor , in order to represent areas maximizing activity differences between the two groups . peak coordinates of clusters more active in autistic participants ( autistic > non - autistic ) , as well as in non - autistic participants ( non - autistic > autistic ) , were identified on the complexity contrast ( figural < analytical < complex analytical ) , yielding seeds in the left superior occipital gyrus , left middle frontal gyrus and left precuneus . the modulation of functional connectivity by task complexity was assessed using a ppi approach ( friston , 2011 ; friston et al . each extracted time series was then entered in a ppi analysis model in order to form the interaction between the seed region activity and task complexity ( figural < analytical < complex analytical ) . this step created 3 vectors : the interaction term , the volume of interest ( voi ) eigenvariate and the relative complexity contrast vector . finally , 6 s - level factorial models , one per seed region , were computed to illustrate the interaction between functional connectivity involving the seed regions and task complexity . autistic participants ( 12.52 s ) were significantly faster than non - autistic participants ( 17.29 s ) by an average of 4.77 s. response times were longer for complex analytical ( 23.87 s ) than for analytical problems ( 13.03 s ) , p < 0.001 , and analytical than figural problems ( 7.82 s ) , p = 0.002 ( see table 2 ) . a linear contrast examining reasoning complexity ( figural < analytical < complex analytical ) revealed an extended network involving frontal , parietal , occipital , cerebellar and basal ganglia regions , all associated with increasing reasoning complexity ( table 3 ) . entering response times as a covariate in within - group analyses did not result in any significant changes in the findings . first , increased complexity - related activity was observed in the left superior occipital gyrus and the left middle occipital gyrus for autistic participants relative to non - autistic participants . second , non - autistic participants showed increased activity in the left middle frontal gyrus ( mfg ) and bilateral precuneus , relative to autistic participants ( fig . 2c and d further illustrates that left mfg was only recruited for complex analytical problems in autistic participants , and that precuneus was not significantly recruited during the task in the autistic group . these results suggest a stronger reliance on occipital areas for autistic participants as task complexity increased . the eye movement analysis showed similar levels of net saccade density during the rspm task between the autistic participants ( 3.96 ) and non - autistic participants ( 4.44 ; t = 0.89 , p = 0.38 ) . similar mean displacement rates were observed in autistic ( 0.032 mm / s ) and non - autistic participants ( 0.038 mm / s ; t = 0.72 , p = 0.48 ) . similar mean rotation rates were observed in the two groups ( autistic group : 0.025/s ; non - autistic group : 0.036/s ; t = 1.28 , p = 0.21 ) . no significant differences were observed in the peak - to - peak translations or rotations ( all p > 0.05 ) . conjunction seeds ( right ifg ( ba9 ) , left spl ( ba7 ) and left inferior occipital gyrus ( ba18 ) ) represent the common areas of activity in both groups during the rspm task . their associated time series were used in ppi analyses in order to assess how these key fluid reasoning regions interact with other brain regions as complexity increases . within - group analyses in the autistic group revealed an interaction between the frontal seed ( right ifg ) and the left ifg . the occipital seed showed an interaction with frontal , temporal , occipital , parietal regions as well as the cerebellum . moreover , the parietal seed interacted with the left ifg , left ipl , left angular gyrus , right postcentral gyrus and left cerebellum . the within - group analyses in the non - autistic group showed interaction between the 3 seeds and multiple regions in frontal , parietal , temporal and occipital areas . a between - group contrast ( aut > non - aut ) showed that autistic participants had greater connectivity than non - autistic participants ( p < 0.001 , unc . ) between the occipital seed ( left inferior occipital gyrus ) and areas in the left superior frontal gyrus , right spl , right middle occipital gyrus and right inferior temporal gyrus . in contrast , non - autistic participants had greater connectivity than autistic participants between all 3 seeds and multiple other regions involved in reasoning ( frontal , parietal , temporal and occipital regions ) as complexity increased ( table 4 ) . overall , non - autistic participants exhibited more complexity - related connectivity than autistic participants in the selected frontal , parietal and occipital seeds ( fig . 3 ) , demonstrating that autistic individuals exhibit reduced modulation of connectivity by reasoning complexity in those areas . in order to explore group - specific patterns of connectivity in relation to complexity , maximum difference seeds were used to identify the key areas associated with reasoning complexity that are unique to each group in within - group analyses . in the autistic group , reasoning complexity modulated connectivity between the occipital seed ( left superior occipital gyrus ) and circumscribed frontal , temporal , occipital and subcortical regions ( table 5 ) . in the non - autistic group , reasoning complexity modulated connectivity between each of the 3 seeds and an extended network of cortical and subcortical regions ( fig . this second set of ppi analyses also suggests less extended network interactions involved in reasoning in autistic individuals relative to non - autistic individuals . our objective was to investigate how task complexity modulates the level of activity and connectivity among brain regions during fluid reasoning in autism . while both groups showed similar accuracy in solving rspm problems , autistic participants performed faster than non - autistic participants at all complexity levels , as we previously reported in a study identifying occipital cortex as being more active in autistic individuals during matrix reasoning ( soulires et al . moreover , while autistic participants exhibited less modulation of fronto - parietal activity and connectivity as reasoning complexity increased , connectivity involving occipital areas was more consistently modulated by task complexity in autistic than non - autistic participants . occipital regions are not only known for their involvement in visuospatial processes , but also in the identification of correct answers with abstract rules ( skosnik et al . the greater use of these regions as complexity increases in a reasoning task has an equivalent in typical individuals , albeit to a lesser extent ( goel , 2007 ; soulires et al . , in contrast , compared to autistic participants , non - autistic participants more strongly engaged a combination of regions related to verbal and visuospatial processing as task complexity increased . the left middle frontal gyrus is known to be involved in inner speech ( geva et al . in this group an occipital to frontal / parietal activation shift is seen when participants inhibit perceptual information to engage in logical reasoning , and by extension with increasing reasoning complexity ( houde et al . the present results suggest that this shift might not be as visible in autistic individuals , as perceptual processes able to support this type of reasoning are engaged more strongly . thus , we suggest that autistic individuals rely more specifically on visuospatial processes to resolve complex matrices whereas non - autistic individuals rely more prominently on a combination of verbal and visuospatial processes . our hypothesis that autistic participants would exhibit higher cortical connectivity in the posterior parts of the reasoning network as task complexity increased , compared to non - autistic participants , was partially confirmed . indeed , a stronger long - range functional connectivity was identified in autistic participants , relative to non - autistic participants , between the left inferior occipital gyrus and the left superior frontal gyrus and the right spl as task complexity increased . in combination with the demonstration of enhanced activity associated with visual processing in many contexts in autism ( samson et al . , 2012 ) , we believe that autism is characterized , not only by enhanced perceptual performance , but also by enhanced role of perceptual processing in higher order cognitive processes ( mottron et al . by contrast , none of the analyses involving seeds in prefrontal cortex revealed increased functional connectivity in autistic participants relative to non - autistic participants . to the contrary , autistic participants generally exhibited less connectivity in the anterior elements of the network , a finding also reported by previous studies ( sahyoun et al . mcgrath and colleagues also obtained similar results in a mental rotation task , as they observed a general decrease in functional connectivity as task complexity increased in autistic individuals , the visual cortex being the only exception to this decrease ( mcgrath et al . this decreased modulation of the fronto - parietal network could suggest that autistic individuals are less influenced by task complexity than non - autistic individuals , as they showed less change in the network 's connectivity as complexity increased , while maintaining equal accuracy . considering that both groups showed similar accuracy , and autistic individuals responded more quickly during the task , we can not conclude that our results are best interpreted as altered network activity in the network as suggested by yamada et al . rather , they may reflect lower complexity - induced modulation of the network in autistic individuals , with potential positive consequences on performance . as the enhanced perceptual functioning ( epf ) model suggests , autistic cognition is , among other elements , characterized by a stronger engagement of perceptual processes and a different equation between neuronal engagement and task difficulty ( mottron et al . our results of a stronger engagement of visuospatial processes and lower modulation of the reasoning network as complexity increases during reasoning directly contribute to this model and add to the body of work describing a stronger engagement of perceptual processes in higher cognition in autistic individuals . this study focussed on the influence of visuospatial processes on fluid reasoning in autistic individuals and may not reflect all the cognitive process involved in other aspects of intelligence . also , we chose to use a liberal threshold ( p < 0.001 uncorrected ) in order to observe the full extent of between - group differences in the cerebral activity and connectivity underlying reasoning , as a more stringent threshold may smooth out some interesting differences . finally , our sample was composed of participants with measured intelligence in the normal range only ; hence , these results may or may not apply to the whole spectrum of autistic intelligence . nevertheless , these results provide us with a better understanding of the atypical , yet not dysfunctional , reasoning abilities of autistic individuals ( dawson et al . indeed , the relative advantage of autistic individuals at the rspm might be linked to a more efficient recruitment of posterior brain regions analyzing the visuospatial information provided in the problems . further studies could explore the effect of complexity in other domains of autistic cognition , as well as how the modulation of cognitive processes in relation to complexity occurs in autistic individuals with lower reasoning abilities . this could lead to the application of these findings to optimizing learning and work environments for autistic individuals .

for the past 35 years , hybridoma technology has enhanced our capacity for research and diagnostics by providing monoclonal antibody reagents allowing tracking , detection and quantitation of target molecules in cells and serum . recently , a number of more advanced methods to harness the immune response have also been developed1,2,3 that significantly increase the number of antibody producing cells that can be screened . alongside these traditional method of making monoclonal antibodies , a quiet revolution has been brewing in the generation of antibodies using in vitro display technologies , which offer a number of advantages , including a greater degree of control over the nature of the derived antibodies . the success of these technologies has relied upon many previous advances , including the conception and implementation of phage display4,5 , the expression of antibody fragments in bacteria6 and pcr - mediated amplification of antibody genes and libraries7,8,9,10,11 . the most popular technologies , antibody phage8,12,13 and yeast display14,15 , which are complementary in their properties , can be used with nave , immunized or synthetic repertoires . as a direct consequence of genome sequencing , and the advent of high throughput biology , the demand for large numbers of renewable high quality affinity reagents , recognizing ever - greater numbers of proteins , for affinity reagent based proteomic scale experiments , furthermore , it is generally accepted that , irrespective of the source , there is an urgent need to improve antibody quality , as reflected by a raft of recent papers16,17,18,19,20,21,22 showing an alarmingly high proportion of commercial antibodies demonstrating poor specificity , or even failing to recognize their targets at all . given that much of modern biological research relies on the fidelity of commercially supplied antibodies , there is an urgent need to resolve this problem . the high throughput potential of in vitro technologies make them ideal platforms for large scale projects to derive antibodies for all human proteins , which once completed are likely to have impacts perhaps as great as the completion of the human genome . by carefully controlling selection and screening conditions , display technologies allow the generation of antibodies to defined antigen conformations or epitopes , for example , by the presentation of specific antigen conformations , or the inclusion of competitors to direct selection towards specific targets or epitopes ( figure 1 ) . moreover , when variable regions from immunized sources are used with display technologies , specificities not detectable by traditional immunological techniques can often be selected23 . during the process of in vitro antibody selection , the gene encoding the antibody is cloned at the same time as the antibody is selected , providing many advantages to the recombinant approach ( fig . 1 ) . the availability of the antibody gene allows the creation of alternative constructs with added functionality by simple subcloning ( see below ) . libraries of mutagenized variants can be created and the same selection process repeated to yield variants that are improved , both in terms of specificity and affinity . the improvement of antibody affinity to picomolar levels24,25,26,27,28 has become relatively routine , with one study describing an antibody in the femtomolar range29 . these affinities are far higher than those of antibodies obtained by immunization , which are limited to ~100 pm by the physiological mechanism of b cell activation30,31 . in addition , antibody specificities can be broadened or narrowed by appropriate selection and screening . as these in vitro methods are based on microbial systems , selection and screening also overcome immunological tolerance , allowing the selection of affinity reagents that recognize highly conserved targets such as ubiquitin34 , histones35 , hemoglobins36 and post - translational modifications37,38,39 . while tricks can be used to overcome tolerance during immunization40,41 , none are required to select antibodies against conserved proteins using in vitro display methods . remarkably , the selection of hundreds of different antibodies from nave human antibody repertoires against many different individual human targets has not been problematic32,42,43 . most of the examples described below relate to antibody fragments . however , display technologies have allowed the development of alternative , non - antibody scaffolds and they too will provide affinity reagents with similar , or in some applications , superior properties to those described here . selection platforms44,45,46 and different scaffold proteins47,48,49 , including antibody fragments50 , have been widely dealt with in previous reviews . our goal here is not to reiterate the ways in which such libraries are made or used , but to illustrate how in vitro display methods have yielded antibodies with remarkable properties some of which have rarely , if ever , been obtained by immunization . this will be carried out by describing a number of different classes of unique and interesting antibodies , as well as outlining the enormous advantages provided by immediate access to cloned antibody genes . monoclonal and polyclonal antibodies with specificities for small molecules have been obtained by traditional immunization51,52,53,54,55 . however , the ability of display methods to tailor both affinity and specificity has led to significantly better antibodies than can be obtained by immunization ( table 1 and fig . sulfotyrosine is a post - translational modification ( ptm ) predicted to occur in 30% of all secretory and membrane proteins65 . despite decades of immunization this is probably due to the innate tolerance immune systems have for such ubiquitous protein modifications , as well as the presence of the recognized target in the secretory pathway , resulting in retention and an inability to secrete the antibody . using phage display , two groups recently selected antibodies recognizing proteins containing sulfotyrosine ( but not tyrosine phosphate ) , independently of protein context or sequence38,39 . these antibodies recognized sulfotyrosinated proteins in western blotting , immunofluorescence , elisa and immunoprecipitation , and recognition could be abolished by sulfatase treatment or preincubation with free tyrosine sulfate . this represents an enormous advance in the analysis of this modification , which has traditionally required thin - layer chromatography of radiolabeled protein hydrolysates66 or mass spectrometry ( ms)67 , with the presence of sulfate groups often inferred , rather than proven . phage display allows the generation of antibodies against nearly any target , including toxins , pathogens , non - immunogenic , or highly conserved antigens . with respect to protein targets , antibodies have been selected with exquisite specificity , differentiating , for example , between chicken and quail lysozyme68 that differ by a single surface amino acid , and the sh2 domains of abl1 and abl269,70 . these include antibodies that discriminate between different strains of hantavirus71 , dengue virus72 , influenza73,74 , ebola75 and venezuelan equine encephalitis virus76 . given that many of these viruses are classified serologically , the ability to select phage antibodies with similar specificities is not surprising , but unlike antibodies generated by immunization , these have the potential to be used therapeutically . human antibodies , some of which are protective in animal models77,78,79 , have also been selected against a number of bacterial biothreat targets , including brucella melitensis80 , burkholderia mallei and burkholderia pseudomallei81 , anthrax toxins77,82,83 and spores84 , and botulinum toxin24,85 . one library79 was generated from servicemen vaccinated against a plethora of different biothreat agents , reflecting the additional ability of display technologies to exploit antibodies generated during traditional immunization . the in vitro nature of phage display technology has been exploited to target particular features of blood cells . in one study36 , antibodies recognizing fetal hemoglobin but not adult hemoglobin , were selected by depleting high affinity cross - reactive antibodies followed by a selection against the fetal protein . notably , the selected discriminatory antibody was of much lower affinity than cross - reactive antibodies , demonstrating the power of negative selections to favor clones with desirable binding specificities , even if their affinity is lower . similar methods applied to cells have been used to select antibodies specifically recognizing fetal nucleated red blood cells95 . protein allostery is a common means for the regulation of protein function , and many signaling proteins exist in alternative conformational states that mediate different cellular responses . antibodies that recognize specific proteins conformers are powerful tools for probing the details of cell signaling . however , the generation of such antibodies by immunization is complicated by the difficulty of maintaining a particular protein conformation in an immunized animal . in contrast , in vitro selection technologies are ideally suited for these applications . negative selections can be used to deplete non - specific binders , and affinity maturation strategies can be employed to fine - tune specificity . in one study , scfvs specific to the gtp - bound form of the small guanosine triphosphatase ( gtpase ) rab6 were generated by performing selections against a gtp - locked mutant96 . in another study97 , small molecules were used to covalently lock caspase-1 in either the active or inactive form and the locked antigens were used to select fabs that were highly selective for either the on or off form of the protease . the concept of using in vitro selections to generate conformation specific antibodies has also been combined with selections on whole cells in a powerful strategy that enables the probing the cell surfaces for conformational changes in response to various stimuli101 . finally , phage display has enabled the generation of antibodies recognizing structured rna molecules100 , which are essentially non - immunogenic , and not as amenable to simple nucleotide probes . by ensuring a nuclease - free in vitro environment and selecting under conditions optimized for the structural stability of the rna , high affinity fabs were isolated against a structured domain from the tetrahymena group i intron . these results hold great promise , as they establish general methods applicable to the generation of antibodies against other structured rnas , and will be useful to decipher the biological roles of the vast numbers of noncoding rnas found in metazoan transcriptomes . communication between cells is driven and controlled by interactions between cell surface receptors and the ligands they recognize . antibodies can modify such interactions and many therapeutic antibodies exert their effect by interfering in communications at the cell surface using different mechanisms ( fig . 3 ) . in vitro display technologies provide a powerful route to generating functional antibodies that interfere in normal or pathological extracellular signaling . although it is usually difficult to select for function directly , display technologies have the ability to generate thousands of independent binders , each of which can then be screened for functional activity . for example , over 1200 different antibodies directed towards b lymphocyte stimulator ( blys ) were generated by phage display102 . this large panel was subsequently screened in biochemical and cellular assays to identify antibodies that bound to blys , preventing its interaction with the receptor ( fig . 3a ) , and thereby blocking b cell activation . in some cases blocking antibodies with sub - nanomolar affinities one of these antibodies , specific only for the secreted form of blys ( benlysta ) , was affinity matured103 and is close to approval for treatment of systemic lupus erythematosus . similar results have been reported for the selection of phage antibodies against a panel of 28 different potentially therapeutic targets , with an average of 120 functionally active ( i.e. antagonistic or agonistic ) antibodies selected per target43 . an alternative strategy to block receptor signaling is to target the ligand binding sites on the receptors , thereby preventing the natural ligand from acting ( fig . this was used in a recent study , where antibodies were selected that prevented the interaction of insulin - like growth factor type 1 ( igf-1 ) with the igf-1 receptor106 . several groups of receptor binders were generated that competed with ligand binding and blocked cell growth in vitro and in vivo . studies on a panel of therapeutic antibodies targeting the egf receptor ( erb - b1 ) have also shown competition with ligand binding . erb - b1 has four extracellular domains , which adopt a mainly closed conformation in the absence of ligand , and a more extended conformation allowing dimerization , and subsequent phosphorylation of the intracellular domain , in the presence of ligand . structural studies have shown that antibodies such as cetuximab stabilize the receptor in the closed conformation ( fig . , pertuzumab appears to work by preventing dimerization while trastuzumab ( herceptin ) prevents receptor shedding and forms inactive tetramers120 . while the original blocking antibodies in these examples were generated from mice , they demonstrate the therapeutic approaches that could benefit from human antibodies isolated directly from display technologies . following ligand binding , a conformational change occurs at the juxta - membrane negative regulatory region ( nrr ) exposing a protease cleavage site resulting in the release and translocation to the nucleus of the intracellular domain . in addition to generating antibodies that block the interaction with ligand , antibodies recognizing the nrr domains stabilized the closed dimeric antibodies targeting ligand - binding domains sometimes mimic the natural ligand , causing receptor activation rather than inhibition . this is the case for antibodies recognizing met109 , with monomeric antibodies being antagonistic . however , in the case of trail receptor 1 ( trail - r1 ) and trail receptor 2 ( trail - r2)119 , an analysis of over 500 distinct selected antibodies , revealed some that were agonistic even as monomeric scfvs or fabs . this is difficult to reconcile with the mode of action of trail , which is a homotrimeric ligand that causes multimerization of the trail receptor leading to apoptosis , particularly in tumor cells over - expressing the receptor . antibodies also have great potential in blocking protein interactions associated with viral entry into target cells , illustrated by antibodies selected from nave antibody libraries against recombinant h5 hemagglutinin influenza ectodomain112,113 . structural analysis of one of the antibodies showed it bound to hemagglutinin at a highly conserved previously unrecognized pocket , found in many different influenza viruses . although antibodies have not been generated against this epitope by traditional immunization or infection , antibodies with similar vh gene usage and neutralizing activity have been selected from phage antibody libraries created from recently infected individuals23 , showing that phage display can access the diversity of immune responses in ways not possible by traditional immunological means . in vitro selection schemes this was carried out by incubating phage libraries with target cells and isolating those phage antibodies found within the cell after removing phage antibodies bound to the cell surface . however , the use of mammalian cells transfected with the target of interest 118 , or yeast displaying targets of interest on their surface114 , provides a means of carrying this out on predetermined targets . this approach is particularly suitable for the selection of antibodies used for specific targeting of chemotherapeutics121,122 . in summary , antibodies and other binding molecules provide a means of modulating biological function by specifically interfering in protein interactions . in vitro display systems provide a means of presenting targets in appropriate conformations , including on cell surfaces , which facilitate rapid screening for potentially rare functional binders . while initial leads can be used directly as affinity reagents , a major advantage of in vitro methods is that it is possible to further improve function by constructing secondary libraries that introduce additional mutations . the most prevalent application of secondary libraries is the improvement of affinity , and all three major display formats ( phage , yeast and ribosome ) have been applied to develop extremely tight affinities that exceed those possible with natural antibodies ( table 4 ) . both stepwise123 and computational124 methods have also been developed that are able to generate similarly high affinity antibodies , but they have not been as widely used as the in vitro display methods . there are many examples of in vitro affinity maturation , and here we highlight some key studies . in ribosome display , each selection cycles involves a pcr amplification step , which is ideal for introducing additional mutations by error prone pcr . this strategy has been used to simultaneously select and affinity mature anti - insulin antibodies with affinities in the sub - nanomolar range26 . while yeast - displayed libraries are smaller than phage and ribosome libraries , they allow quantitative and exhaustive screening by fluorescence activated cell sorting ( facs ) . coupled with sequential rounds of error - prone pcr , modest libraries of 1010 unique clones were sufficient to affinity mature an anti - fluoroscein scfv into the low femtomolar range29 . species cross - reactivity allows better assessment of therapeutic efficacy and toxicity in animal models . unfortunately , cross - reactive antibodies are often difficult to obtain by hybridoma methods , due to tolerance . in contrast , in vitro libraries are unaffected by immune tolerance and antibodies targeting conserved sites across species have proven to be the rule rather than the exception . in the case of highly conserved proteins , such as vascular endothelial growth factor ( vegf ) , human / mouse cross - reactive antibodies have been obtained directly from nave libraries128,129 . in the case of less conserved orthologs , such as baff / blys receptor 3 ( br3 ) , initial anti - human antibodies with weak cross - reactivity to the mouse protein have been obtained from nave libraries and evolved to be highly cross - reactive130 . similar approaches have been used to generate antibodies recognizing two closely related chemokines ( cxlc10 and cxcl9)125 thereby permitting neutralization of 2 human chemokines with a single antibody . most specificity engineering examples involve the improvement of pre - existing weak recognition , due to homology between the recognized targets . in perhaps the most extreme case of engineered cross - reactivity , herceptin has been evolved to recognize a very different protein , vegf , as well as its original target , erb - b2126 . after significant evolution the affinities for both targets were comparable to those of therapeutic antibodies ( kd = 3/0.2 nm for vegf / erb - b2 ) . the antibody inhibited both vegf and erb - b2-mediated cell proliferation in vitro and tumor progression in mouse models . the structures of the bispecific fab in complex with erb - b2 or vegf revealed a common paratope , with the erb - b2 functional paratope located predominantly on vh , and that for vegf on vl ( fig . 4 ) . the ability to design antigen - binding sites with dual specificity against structurally unrelated antigens may be important in therapeutic strategies targeting two distinct signaling pathways with a single antibody . the ability to improve affinity and broaden specificity also has major implications for the development of antibodies against pathogens . for the effective inhibition of viral infection and bacterial toxins , antibodies would ideally recognize a variety of antigen subtypes with high affinity , to afford broad protection against pathogen variants . furthermore , several studies have shown that multiple antibodies targeting distinct epitopes provide synergistic effects necessary for effective neutralization of pathogens131,132 . in vitro antibody technologies provide an effective means for achieving these demanding criteria , as exemplified by a long - term study of neutralizing antibodies against the botulinum neurotoxin ( bont ) . phage antibody libraries from immunized mice and humans resulted in the isolation of three antibodies recognizing non - overlapping epitopes on bont133 . the use of these three antibodies together as an oligoclonal igg provided strong synergy that dramatically increased toxin neutralization . a long series of affinity and specificity maturation cycles using yeast display , resulted in the final development of a remarkable antibody able to recognize botulinum toxins a , b , e and f , all the serotypes afflicting man127 . all in vitro selection systems immediately provide the genes , and corresponding sequences , of antibodies selected against a particular target , providing ready access to additional antibody formats by simple sub - cloning . functions adopted using this gene - based approach include dimerization134 , multimerization135,136 , and fusions to enzymes137 , tags138 or fluorescent proteins139 . as this is a dimeric enzyme , fusing antibodies , either individually or as libraries , to alkaline phosphatase simultaneously provides dimerization and alkaline phosphatase activity , greatly facilitating screening32,137 . short peptides acting as in vivo biotinylation tags138 , placed at the carboxy terminus of antibody fragments , allow stoichiometrically defined site specific antibody biotinylation , as well as a straightforward multimerization method140 . antibody fragments can also be transformed into full - length antibodies141 , or scfv - fc fusions , which are very similar in many aspects142 . the use of engineered fc regions can result in improved pharmacokinetics and effector functions ( for reviews see 143,144 ) , including bispecific igg , in which engineering of two different fc regions allow only heterologous pairing145,146 . other approaches to generate bispecific antibodies build upon the observation that some scfv fragments form bivalent dimers ( diabodies)147 , trimers148,149 and even tetramers150 when the vh / vl linker is shortened . various other bispecific antibody designs have also been created ( see 151 for a review ) . even more radically , completely novel biochemical entities have been added to antigen binding fragments . fusions of scfv and fab fragments to heterologous proteins , such as interleukins and cytokines152,153 , apoptotic ligands , enzymes , toxins or rnases ( see 154,155 for reviews ) have allowed novel therapeutic paradigms . many of the above candidate therapeutic antibody constructs arose from antibody genes initially isolated from mouse hybridomas , but this is expected to change as more human antibodies are made available from engineered repertoires . antibody fragments can be expressed within target cells and targeted to various subcellular compartments141,157 by adding suitable signal sequences , allowing visualization or functional modification of proteins in different compartments . removing the standard leader sequence results in cytoplasmic expression while the addition of a nuclear localization signal targets to the nucleus . the combination of a leader sequence and the endoplasmic reticulum ( er ) retention sequence retains expressed antibodies in the er and has been used to prevent the expression of membrane proteins by sequestration in the er . these include human interleukin 2 receptor , the erbb-2 receptor , s - amyloid precursor protein , vascular adhesion molecule 1 and many others158,159,160,161 . the advantage of this strategy is that it requires antibodies that bind to any accessible epitope to provide the functional knockout , as opposed to the functional activity required of cytoplasmically expressed antibodies . functional studies of membrane receptors or secreted proteins can thus be attempted by a single standardized subcloning step immediately after in vitro antibody selection , providing equivalence to rnai knockdowns at the protein level . while expression in the secretory pathway is straightforward , folding of antibody fragments in the cytoplasm is far more challenging , due to the absence of specific chaperones , and the reducing environment , which prevents disulfide bond formation162 . despite these problems , the success of this approach has been improved by the creation of libraries of particularly stable scfvs164,165,166 , preselecting antibodies for functional cytoplasmic expression167,168 , or by using binder libraries based on molecular scaffolds that do not rely on disulphide bond formation , such as engineered ankyrin repeat proteins169,170 . one major advantage of such protein based allosteric blockers is the ability to generate very specific binders , able to distinguish between closely related family members . while the need to genetically modify the target cell is a major disadvantage , this has been partly alleviated by fusion to internalizing sequences that allow antibodies to enter the cell from the outside171 . the ease with which antibodies can be selected , screened and produced by in vitro display technologies , makes generation and screening of antibodies rapid and simple compared to hybridomas . typically a panel of elisa positive monoclonal antibody fragments can be generated within two weeks . early experiments demonstrated the feasibility of semi - automated selection / screening of phage antibody libraries172,173,174 on small numbers of targets . more recently , selections on over 400 different antigens were successful with 54% of bacterial , and 88% of mammalian - produced antigens32 yielding antibodies , with the differences between the two protein classes probably due to the levels of correct folding . in a recent international comparative study antibodies were raised to 20 different human sh2 domains using hybridoma or phage display . results from two of the participating phage display labs69,70 , show that antibodies ( some with sub - nanomolar affinities ) were generated to all antigens , with 55% of positive antibodies specific for target sh2 domains when assessed against the entire sh2 panel . these antibodies were validated in a broad range of assays , including microarrays , immunoblots , immunofluorescence and immunoprecipitation . if antibodies selected by in vitro methods are so powerful , why are they not more widely perceived as valuable research reagents ? part of the answer lies in the difficult patent situation , which resulted in restriction of this technology to the high margin therapeutic markets for commercial use . it is perhaps significant in this regard that hybridoma technology was never patented , and achieved relatively wide acceptance within a short period . the situation for some of the core phage display patents is in the process of rapid change as most platform patents have either expired , or will do so over the next few years175 , and it is possible that the technology will become more widely disseminated as a result . are actually recombinant antibodies selected by phage display , reformatted to look like traditional murine antibodies by the fusion of fc regions to human variable regions ( e.g. the sulfotyrosine antibody described above38 ) . it therefore seems that the most important impediments to widespread adoption are a lack of knowledge of the capabilities of this technology , coupled with limited expertise and library availability . furthermore , the number of companies willing to carry out in vitro selection as a fee for service is vanishingly small compared to the 180 companies willing to generate antibodies by immunization16 . while the specificities obtained , and described herein , are remarkable , the expression and stability of antibody fragments varies enormously , from exceptionally stable scfv fragments used in clinical trials176 to other fragments that are poorly expressed . a typical selection almost always generates a number of different binders to any well - folded antigen . among these there is usually at least one that is sufficiently stable and well produced for research use . furthermore , it is expected that stability and expression levels will improve as libraries are based on more stable scaffolds177 . the studies described above indicate that this goal can now be met in highly parallelized screening setups with low effort per antigen69,70 , provided that libraries of sufficient diversity and optimized protocols are used . furthermore , stability and expression screening can be easily included as part of the ht screening process . an additional issue with in vitro derived antibodies is that they are either not glycosylated if expressed in bacteria , or incorrectly glycosylated if expressed in standard yeast strains . if correct glycosylation is necessary , this can be overcome by expression in human cells or yeast modified to give human glycosylation patterns178 . once an antibody is generated , it can be defined precisely by sequence and even distributed in this way . gene synthesis is progressing at a remarkable pace , with the cost per base of synthesized genes falling dramatically . in fact , genes corresponding to the sequences of specific antibody fragments can now be synthesized for less than the cost of purchase of some antibodies from traditional vendors . the present state of this field can be compared to the situation with sequencing technologies at the start of the human genome project . just as enormous technical advances occurred in the human genome project once it was started and rigorous industrial processes were applied , so we anticipate dramatic improvement in all aspects of selection , screening , downstream use and distribution of in vitro derived affinity reagents once a proteomic scale project is initiated and financed . in summary , in vitro display technologies permit the facile generation of antibodies by providing access to billions of potential binders in large universal , or immune , display libraries . the technologies facilitate production , screening and maturation of selected binders , allowing selection on target conformations and formats not possible by more traditional routes based on immunization . furthermore , the easy availability of the gene sequence not only provides a definitive description of the product but also allows electronic sharing and recreation of the binding molecule through gene synthesis . over the last 20 years display technologies have been applied successfully to the development of therapeutic antibody candidates . in the coming decade we expect to see increased realization of the benefits of this technology within the research and diagnostic markets as well .

in vitro display technologies , best exemplified by phage and yeast display , were first described for the selection of antibodies some twenty years ago . since that time a large number of antibodies , some with remarkable properties , have been selected and improved upon using these methods . the first antibodies derived using in vitro display methods are now in the clinic , with many more waiting in the wings . here we discuss the scope of the technology , some of the powerful antibodies selected , and the future potential in a post - genomic world . unique advantages offered by in vitro display technologies include the ability to carefully define selection conditions , allowing the derivation of antibodies recognizing predefined epitopes or conformations , the further improvement of selected antibodies , the potential for high throughput applications and the immediate availability of genes encoding the selected antibody . we anticipate that the high throughput potential of these technologies will soon lead to their use to select antibodies against all human proteins .

Introduction Recognition of chemical modifications and small molecules Specificity for protein sequences and conformations Antibodies to cell surface receptors Improving antibody affinity and specificity Exploiting the recombinant nature of in vitro selected antibodies High throughput selection by in vitro display methods The future vision of affinity reagents generated by display technologies

alongside these traditional method of making monoclonal antibodies , a quiet revolution has been brewing in the generation of antibodies using in vitro display technologies , which offer a number of advantages , including a greater degree of control over the nature of the derived antibodies . the success of these technologies has relied upon many previous advances , including the conception and implementation of phage display4,5 , the expression of antibody fragments in bacteria6 and pcr - mediated amplification of antibody genes and libraries7,8,9,10,11 . the most popular technologies , antibody phage8,12,13 and yeast display14,15 , which are complementary in their properties , can be used with nave , immunized or synthetic repertoires . as a direct consequence of genome sequencing , and the advent of high throughput biology , the demand for large numbers of renewable high quality affinity reagents , recognizing ever - greater numbers of proteins , for affinity reagent based proteomic scale experiments , furthermore , it is generally accepted that , irrespective of the source , there is an urgent need to improve antibody quality , as reflected by a raft of recent papers16,17,18,19,20,21,22 showing an alarmingly high proportion of commercial antibodies demonstrating poor specificity , or even failing to recognize their targets at all . the high throughput potential of in vitro technologies make them ideal platforms for large scale projects to derive antibodies for all human proteins , which once completed are likely to have impacts perhaps as great as the completion of the human genome . by carefully controlling selection and screening conditions , display technologies allow the generation of antibodies to defined antigen conformations or epitopes , for example , by the presentation of specific antigen conformations , or the inclusion of competitors to direct selection towards specific targets or epitopes ( figure 1 ) . moreover , when variable regions from immunized sources are used with display technologies , specificities not detectable by traditional immunological techniques can often be selected23 . during the process of in vitro antibody selection , the gene encoding the antibody is cloned at the same time as the antibody is selected , providing many advantages to the recombinant approach ( fig . the availability of the antibody gene allows the creation of alternative constructs with added functionality by simple subcloning ( see below ) . libraries of mutagenized variants can be created and the same selection process repeated to yield variants that are improved , both in terms of specificity and affinity . the improvement of antibody affinity to picomolar levels24,25,26,27,28 has become relatively routine , with one study describing an antibody in the femtomolar range29 . as these in vitro methods are based on microbial systems , selection and screening also overcome immunological tolerance , allowing the selection of affinity reagents that recognize highly conserved targets such as ubiquitin34 , histones35 , hemoglobins36 and post - translational modifications37,38,39 . while tricks can be used to overcome tolerance during immunization40,41 , none are required to select antibodies against conserved proteins using in vitro display methods . remarkably , the selection of hundreds of different antibodies from nave human antibody repertoires against many different individual human targets has not been problematic32,42,43 . however , display technologies have allowed the development of alternative , non - antibody scaffolds and they too will provide affinity reagents with similar , or in some applications , superior properties to those described here . selection platforms44,45,46 and different scaffold proteins47,48,49 , including antibody fragments50 , have been widely dealt with in previous reviews . our goal here is not to reiterate the ways in which such libraries are made or used , but to illustrate how in vitro display methods have yielded antibodies with remarkable properties some of which have rarely , if ever , been obtained by immunization . this will be carried out by describing a number of different classes of unique and interesting antibodies , as well as outlining the enormous advantages provided by immediate access to cloned antibody genes . monoclonal and polyclonal antibodies with specificities for small molecules have been obtained by traditional immunization51,52,53,54,55 . however , the ability of display methods to tailor both affinity and specificity has led to significantly better antibodies than can be obtained by immunization ( table 1 and fig . sulfotyrosine is a post - translational modification ( ptm ) predicted to occur in 30% of all secretory and membrane proteins65 . despite decades of immunization this is probably due to the innate tolerance immune systems have for such ubiquitous protein modifications , as well as the presence of the recognized target in the secretory pathway , resulting in retention and an inability to secrete the antibody . using phage display , two groups recently selected antibodies recognizing proteins containing sulfotyrosine ( but not tyrosine phosphate ) , independently of protein context or sequence38,39 . these antibodies recognized sulfotyrosinated proteins in western blotting , immunofluorescence , elisa and immunoprecipitation , and recognition could be abolished by sulfatase treatment or preincubation with free tyrosine sulfate . this represents an enormous advance in the analysis of this modification , which has traditionally required thin - layer chromatography of radiolabeled protein hydrolysates66 or mass spectrometry ( ms)67 , with the presence of sulfate groups often inferred , rather than proven . phage display allows the generation of antibodies against nearly any target , including toxins , pathogens , non - immunogenic , or highly conserved antigens . with respect to protein targets , antibodies have been selected with exquisite specificity , differentiating , for example , between chicken and quail lysozyme68 that differ by a single surface amino acid , and the sh2 domains of abl1 and abl269,70 . given that many of these viruses are classified serologically , the ability to select phage antibodies with similar specificities is not surprising , but unlike antibodies generated by immunization , these have the potential to be used therapeutically . human antibodies , some of which are protective in animal models77,78,79 , have also been selected against a number of bacterial biothreat targets , including brucella melitensis80 , burkholderia mallei and burkholderia pseudomallei81 , anthrax toxins77,82,83 and spores84 , and botulinum toxin24,85 . one library79 was generated from servicemen vaccinated against a plethora of different biothreat agents , reflecting the additional ability of display technologies to exploit antibodies generated during traditional immunization . the in vitro nature of phage display technology has been exploited to target particular features of blood cells . in one study36 , antibodies recognizing fetal hemoglobin but not adult hemoglobin , were selected by depleting high affinity cross - reactive antibodies followed by a selection against the fetal protein . notably , the selected discriminatory antibody was of much lower affinity than cross - reactive antibodies , demonstrating the power of negative selections to favor clones with desirable binding specificities , even if their affinity is lower . similar methods applied to cells have been used to select antibodies specifically recognizing fetal nucleated red blood cells95 . protein allostery is a common means for the regulation of protein function , and many signaling proteins exist in alternative conformational states that mediate different cellular responses . however , the generation of such antibodies by immunization is complicated by the difficulty of maintaining a particular protein conformation in an immunized animal . in contrast , in vitro selection technologies are ideally suited for these applications . negative selections can be used to deplete non - specific binders , and affinity maturation strategies can be employed to fine - tune specificity . in one study , scfvs specific to the gtp - bound form of the small guanosine triphosphatase ( gtpase ) rab6 were generated by performing selections against a gtp - locked mutant96 . in another study97 , small molecules were used to covalently lock caspase-1 in either the active or inactive form and the locked antigens were used to select fabs that were highly selective for either the on or off form of the protease . the concept of using in vitro selections to generate conformation specific antibodies has also been combined with selections on whole cells in a powerful strategy that enables the probing the cell surfaces for conformational changes in response to various stimuli101 . finally , phage display has enabled the generation of antibodies recognizing structured rna molecules100 , which are essentially non - immunogenic , and not as amenable to simple nucleotide probes . by ensuring a nuclease - free in vitro environment and selecting under conditions optimized for the structural stability of the rna , high affinity fabs were isolated against a structured domain from the tetrahymena group i intron . these results hold great promise , as they establish general methods applicable to the generation of antibodies against other structured rnas , and will be useful to decipher the biological roles of the vast numbers of noncoding rnas found in metazoan transcriptomes . in vitro display technologies provide a powerful route to generating functional antibodies that interfere in normal or pathological extracellular signaling . although it is usually difficult to select for function directly , display technologies have the ability to generate thousands of independent binders , each of which can then be screened for functional activity . 3a ) , and thereby blocking b cell activation . in some cases blocking antibodies with sub - nanomolar affinities one of these antibodies , specific only for the secreted form of blys ( benlysta ) , was affinity matured103 and is close to approval for treatment of systemic lupus erythematosus . similar results have been reported for the selection of phage antibodies against a panel of 28 different potentially therapeutic targets , with an average of 120 functionally active ( i.e. antagonistic or agonistic ) antibodies selected per target43 . this was used in a recent study , where antibodies were selected that prevented the interaction of insulin - like growth factor type 1 ( igf-1 ) with the igf-1 receptor106 . erb - b1 has four extracellular domains , which adopt a mainly closed conformation in the absence of ligand , and a more extended conformation allowing dimerization , and subsequent phosphorylation of the intracellular domain , in the presence of ligand . following ligand binding , a conformational change occurs at the juxta - membrane negative regulatory region ( nrr ) exposing a protease cleavage site resulting in the release and translocation to the nucleus of the intracellular domain . this is the case for antibodies recognizing met109 , with monomeric antibodies being antagonistic . however , in the case of trail receptor 1 ( trail - r1 ) and trail receptor 2 ( trail - r2)119 , an analysis of over 500 distinct selected antibodies , revealed some that were agonistic even as monomeric scfvs or fabs . antibodies also have great potential in blocking protein interactions associated with viral entry into target cells , illustrated by antibodies selected from nave antibody libraries against recombinant h5 hemagglutinin influenza ectodomain112,113 . structural analysis of one of the antibodies showed it bound to hemagglutinin at a highly conserved previously unrecognized pocket , found in many different influenza viruses . although antibodies have not been generated against this epitope by traditional immunization or infection , antibodies with similar vh gene usage and neutralizing activity have been selected from phage antibody libraries created from recently infected individuals23 , showing that phage display can access the diversity of immune responses in ways not possible by traditional immunological means . however , the use of mammalian cells transfected with the target of interest 118 , or yeast displaying targets of interest on their surface114 , provides a means of carrying this out on predetermined targets . this approach is particularly suitable for the selection of antibodies used for specific targeting of chemotherapeutics121,122 . in vitro display systems provide a means of presenting targets in appropriate conformations , including on cell surfaces , which facilitate rapid screening for potentially rare functional binders . the most prevalent application of secondary libraries is the improvement of affinity , and all three major display formats ( phage , yeast and ribosome ) have been applied to develop extremely tight affinities that exceed those possible with natural antibodies ( table 4 ) . both stepwise123 and computational124 methods have also been developed that are able to generate similarly high affinity antibodies , but they have not been as widely used as the in vitro display methods . there are many examples of in vitro affinity maturation , and here we highlight some key studies . in ribosome display , each selection cycles involves a pcr amplification step , which is ideal for introducing additional mutations by error prone pcr . this strategy has been used to simultaneously select and affinity mature anti - insulin antibodies with affinities in the sub - nanomolar range26 . in contrast , in vitro libraries are unaffected by immune tolerance and antibodies targeting conserved sites across species have proven to be the rule rather than the exception . in the case of highly conserved proteins , such as vascular endothelial growth factor ( vegf ) , human / mouse cross - reactive antibodies have been obtained directly from nave libraries128,129 . in the case of less conserved orthologs , such as baff / blys receptor 3 ( br3 ) , initial anti - human antibodies with weak cross - reactivity to the mouse protein have been obtained from nave libraries and evolved to be highly cross - reactive130 . similar approaches have been used to generate antibodies recognizing two closely related chemokines ( cxlc10 and cxcl9)125 thereby permitting neutralization of 2 human chemokines with a single antibody . most specificity engineering examples involve the improvement of pre - existing weak recognition , due to homology between the recognized targets . the antibody inhibited both vegf and erb - b2-mediated cell proliferation in vitro and tumor progression in mouse models . the structures of the bispecific fab in complex with erb - b2 or vegf revealed a common paratope , with the erb - b2 functional paratope located predominantly on vh , and that for vegf on vl ( fig . the ability to design antigen - binding sites with dual specificity against structurally unrelated antigens may be important in therapeutic strategies targeting two distinct signaling pathways with a single antibody . the ability to improve affinity and broaden specificity also has major implications for the development of antibodies against pathogens . for the effective inhibition of viral infection and bacterial toxins , antibodies would ideally recognize a variety of antigen subtypes with high affinity , to afford broad protection against pathogen variants . in vitro antibody technologies provide an effective means for achieving these demanding criteria , as exemplified by a long - term study of neutralizing antibodies against the botulinum neurotoxin ( bont ) . phage antibody libraries from immunized mice and humans resulted in the isolation of three antibodies recognizing non - overlapping epitopes on bont133 . a long series of affinity and specificity maturation cycles using yeast display , resulted in the final development of a remarkable antibody able to recognize botulinum toxins a , b , e and f , all the serotypes afflicting man127 . all in vitro selection systems immediately provide the genes , and corresponding sequences , of antibodies selected against a particular target , providing ready access to additional antibody formats by simple sub - cloning . the combination of a leader sequence and the endoplasmic reticulum ( er ) retention sequence retains expressed antibodies in the er and has been used to prevent the expression of membrane proteins by sequestration in the er . these include human interleukin 2 receptor , the erbb-2 receptor , s - amyloid precursor protein , vascular adhesion molecule 1 and many others158,159,160,161 . while expression in the secretory pathway is straightforward , folding of antibody fragments in the cytoplasm is far more challenging , due to the absence of specific chaperones , and the reducing environment , which prevents disulfide bond formation162 . despite these problems , the success of this approach has been improved by the creation of libraries of particularly stable scfvs164,165,166 , preselecting antibodies for functional cytoplasmic expression167,168 , or by using binder libraries based on molecular scaffolds that do not rely on disulphide bond formation , such as engineered ankyrin repeat proteins169,170 . one major advantage of such protein based allosteric blockers is the ability to generate very specific binders , able to distinguish between closely related family members . the ease with which antibodies can be selected , screened and produced by in vitro display technologies , makes generation and screening of antibodies rapid and simple compared to hybridomas . more recently , selections on over 400 different antigens were successful with 54% of bacterial , and 88% of mammalian - produced antigens32 yielding antibodies , with the differences between the two protein classes probably due to the levels of correct folding . in a recent international comparative study antibodies were raised to 20 different human sh2 domains using hybridoma or phage display . results from two of the participating phage display labs69,70 , show that antibodies ( some with sub - nanomolar affinities ) were generated to all antigens , with 55% of positive antibodies specific for target sh2 domains when assessed against the entire sh2 panel . these antibodies were validated in a broad range of assays , including microarrays , immunoblots , immunofluorescence and immunoprecipitation . if antibodies selected by in vitro methods are so powerful , why are they not more widely perceived as valuable research reagents ? part of the answer lies in the difficult patent situation , which resulted in restriction of this technology to the high margin therapeutic markets for commercial use . it is perhaps significant in this regard that hybridoma technology was never patented , and achieved relatively wide acceptance within a short period . the situation for some of the core phage display patents is in the process of rapid change as most platform patents have either expired , or will do so over the next few years175 , and it is possible that the technology will become more widely disseminated as a result . are actually recombinant antibodies selected by phage display , reformatted to look like traditional murine antibodies by the fusion of fc regions to human variable regions ( e.g. it therefore seems that the most important impediments to widespread adoption are a lack of knowledge of the capabilities of this technology , coupled with limited expertise and library availability . furthermore , the number of companies willing to carry out in vitro selection as a fee for service is vanishingly small compared to the 180 companies willing to generate antibodies by immunization16 . while the specificities obtained , and described herein , are remarkable , the expression and stability of antibody fragments varies enormously , from exceptionally stable scfv fragments used in clinical trials176 to other fragments that are poorly expressed . an additional issue with in vitro derived antibodies is that they are either not glycosylated if expressed in bacteria , or incorrectly glycosylated if expressed in standard yeast strains . just as enormous technical advances occurred in the human genome project once it was started and rigorous industrial processes were applied , so we anticipate dramatic improvement in all aspects of selection , screening , downstream use and distribution of in vitro derived affinity reagents once a proteomic scale project is initiated and financed . in summary , in vitro display technologies permit the facile generation of antibodies by providing access to billions of potential binders in large universal , or immune , display libraries . the technologies facilitate production , screening and maturation of selected binders , allowing selection on target conformations and formats not possible by more traditional routes based on immunization . furthermore , the easy availability of the gene sequence not only provides a definitive description of the product but also allows electronic sharing and recreation of the binding molecule through gene synthesis . over the last 20 years display technologies have been applied successfully to the development of therapeutic antibody candidates . in the coming decade we expect to see increased realization of the benefits of this technology within the research and diagnostic markets as well .

conivaptan is a nonpeptide antagonist with a high affinity for the human vasopressin receptor subtypes v1a and v2 . the other vaptans mentioned earlier , lixivaptan , satavaptan , and tolvaptan , are selective v2 receptor antagonists , and do not have appreciable binding to the v1a receptors . the v1a receptor ( v1ar ) is ubiquitous and is located on the vascular smooth muscle cells , hepatocytes and platelets . stimulation of v1ar results in vasoconstriction , glycogenolysis , and platelet aggregation . the v1b receptor ( v1br ) is found in the pituitary , where it mediates the release of adrenocorticotropic hormone ( acth ) in conjunction with corticotrophin - releasing hormone ( crh ) . the v2 receptor ( v2r ) is located primarily in the kidney , where it mediates antidiuresis.4 the major pharmacological effect of conivaptan in hyponatremia is accomplished by potent antagonism of v2r . as mentioned earlier , the v2r are functionally coupled to aquaporin-2 channels in the apical membrane of the collecting ducts of the kidney . antagonism of v2r in the collecting duct of the kidney results in an increase in free water excretion or effective water clearance , net fluid loss , increased urine output , and decreased urine osmolality.10 but unlike diuretic agents , conivaptan enhances free water excretion without significantly increasing renal excretion of sodium or potassium . agents , ie , stimulating excretion of only water , to distinguish their renal effects from the increased solute and water excretion characteristically produced by diuretics . conivaptan is available as an iv formulation in 4 ml ampoules containing 20 mg of conivaptan hydrochloride . a loading dose of 20 mg is administered through a large vein over 30 minutes , followed by a maintenance dose of 2040 mg per day for up to four days by continuous infusion . peripheral intravenous infusion sites should be changed every 24 hours to reduce common infusion site complications such as irritation and phlebitis resulting from the use of propylene glycol as a solvent in the ampules . a new formulation pre - mixed in 100 ml infusion bags was approved by the fda in october 2008 , which should reduce complications from infusion site reactions since this formulation does not include propylene glycol . close monitoring of serum sodium and volume status ( every 46 hrs ) is recommended with dose adjustments as necessary to achieve desired correction rates of the serum sodium concentration.9 clinical trials of oral conivaptan have been reported at doses ranging from 20 to 80 mg . however , further development of an oral formulation was discontinued due to effects at the cytochrome p450 isoenzyme 3a4 ( cyp3a4 ) , leading to potential prolongation of action of other drugs also metabolized by cyp3a4 . the pharmacokinetics of conivaptan have been studied in healthy and special populations using both the oral and the intravenous formulations . the peak diuretic effect of oral and intravenous conivaptan occurred between two to four hours,1114 resulting in a seven - fold increase in urine flow rate and a decrease in urinary osmolality . the duration of aquaretic effect following single doses both orally and intravenously was 12 hours.11 urine output returned to baseline value 12 hours following oral or intravenous administration of conivaptan . the effect of conivaptan on pulmonary capillary wedge pressure ( pcwp ) in patients with advanced heart failure was sustained for approximately eight hours following the administration of intravenous conivaptan , and pcwp remained below baseline values even after 12 hours.13 the pharmacokinetics of 40 mg / day and 80 mg / day of intravenous and oral conivaptan were nonlinear , because conivaptan inhibits its own metabolism at cyp3a4 . conivaptan is metabolized by the liver by cyp3a4 , thus it can interact with other drugs that are also metabolized by cyp3a4 . because of this , conivaptan is contraindicated with the concurrent use of other potent cyp3a4 inhibitors such as ketoconazole , itraconazole , clarithromycin , ritonavir , and indinavir . the effects of hepatic or renal impairments on the pharmacokinetics of conivaptan have not been systematically evaluated . conivaptan s efficacy for the treatment of hyponatremia has been assessed in three randomized double - blinded , placebo - controlled clinical trials and one open - label nonrandomized study . the three randomized studies include one pivotal study with intravenous conivaptan and two trials using oral conivaptan ; the non - randomized study used intravenous conivaptan . phase iii studies showed that conivaptan reliably increases the serum sodium concentration , beginning as early as one to two hours after administration . mmol / l within 5 hours and by 6 to 9 mmol / l within 24 hours after initiation of therapy at the 20-mg and 40-mg doses , respectively . in most patients , approximately 70% , the serum sodium normalized over a four - day continuous infusion , with the greatest increase in serum sodium occurring during the first 24 to 48 hours of treatment . the primary efficacy endpoint in these studies was the mean change in serum sodium over the duration of treatment , as measured by the area under the serum sodium effect curve ( auc ) , corrected for baseline serum sodium.1517 in the pivotal phase iii trial conducted with iv conivaptan,17 84 patients with euvolemic or hypervolemic hyponatremia ( serum sodium 115130 meq / l ) were randomized to receive conivaptan 40 mg / day , 80 mg / day or placebo for four days . of 84 total patients included in the intention - to - treat analysis , 66 completed treatment . both doses of conivaptan were associated with significant improvements in serum sodium compared to placebo as measured by both the primary and secondary efficacy endpoints ( table 1 ) . all patients also received standard care treatments for hyponatremia , which primarily consisted of fluid restriction . this was not a strict fluid restriction , ie , < 2 liters / day , because the primary goal was to prevent the patients on placebo from worsening , rather than as an active treatment for hyponatremia . a planned subgroup analysis of this trial was performed to evaluate the efficacy of intravenous conivaptan in patients with euvolemic hyponatremia.18 fifty - six of the 84 patients analyzed in the trial were euvolemic . conivaptan 40 and 80 mg / day effectively raised mean serum sodium on the first day of treatment . the mean increase in serum sodium with conivaptan remained significantly greater than that with placebo at the end of the four - day treatment period . the placebo group did not demonstrate any significant changes in serum sodium concentration ( figure 1 ) . of the two oral conivaptan trials , one has been published.16 the efficacy of oral conivaptan was evaluated in 74 euvolemic and hypervolemic hyponatremic patients who were randomized to receive oral conivaptan 40 mg / day , 80 mg / day , or placebo , all given in two divided doses / day for five days . both doses of conivaptan were associated with significant dose - dependent improvements in serum sodium compared to placebo ( table 2 ) . results from the second oral conivaptan study are available in abstract form only.15 the study , similar to the other oral conivaptan study , included 83 patients with euvolemic and hypovolemic hyponatremia , and revealed the same dose - dependent increase in serum sodium concentrations ( table 2 ) . results from the open - label multicenter trial using intravenous conivaptan were presented at the annual meeting of the endocrine society in june 2008.19 the study was conducted from february 2004 to june 2005 , and included 251 hospitalized patients with euvolemic and hypervolemic hyponatremia to assess the efficacy and safety of intravenous conivaptan 20 and 40 mg / day for four days and for up to 30 days after cessation of therapy . the most common underlying illness associated with hyponatremia was siadh ( 43% of patients ) . conivaptan was associated with significant increases in serum sodium concentration that were sustained throughout the study . at the cessation of active therapy , the mean serum sodium increased by 9.4 and 8.8 meq / l in patients given conivaptan 20 and 40 mg / day , respectively . the response did not appear to differ significantly among patients given 20 or 40 mg / day . very interestingly , the increases in serum sodium over baseline achieved through the end of treatment persisted through days 11 and 34 after initiation of aquaretic therapy even though the duration of active treatment was only four days . the reason for the persistence of the correction is unknown , but it suggests that limited infusions of conivaptan may be sufficient to improve hyponatremia not only during active treatment , but also for longer periods of time in patients who have improvement in their underlying comorbidities responsible for the hyponatremia . although conivaptan has been evaluated extensively in patients with congestive heart failure ( chf ) , efficacy has not been established other than for correction of hyponatremia ; thus , conivaptan is not indicated for primary treatment of chf at this time . vasopressin levels are often elevated in patients with heart failure and left ventricular ( lv ) dysfunction,20 and they are clearly associated with adverse cardiovascular outcomes in the setting of lv dysfunction after myocardial infarction.21 these data suggest that vasopressin may contribute to the circulatory response in patients with heart failure , and may also play a role in the development and progression of heart failure . combined v1ar / v2r antagonism with conivaptan therefore does offer a theoretical benefit in chf . if systemic hemodynamic improvement occurs with v1ar antagonism , renal hemodynamics would improve as well with v2r antagonism , leading to a greater clinical benefit . the hemodynamic effects of conivaptan were evaluated in a prospective , randomized , double - blind study of 142 patients with new york heart association ( nyha ) class iii or iv heart failure of at least three months duration.13 subjects were randomized to receive a single dose of intravenous ( iv ) conivaptan ( 10 , 20 , or 40 mg ) or placebo , administered over 30 minutes . conivaptan produced favorable hemodynamic changes in patients with advanced heart failure , without affecting blood pressure or heart rate . pulmonary capillary wedge pressure and right atrial pressure were significantly reduced in the conivaptan 20 mg and 40 mg groups at three to six hours post - dose as compared with the placebo groups . urine output was significantly higher in the conivaptan - treated patients as compared with placebo , and dose - dependent increases in urine output were observed with increasing conivaptan doses ( p < 0.001 ) . despite conivaptan s properties as a v1ar antagonist , it had no significant effects on systolic blood pressure and pulse rate . no statistically significant changes in cardiac index , mean arterial pressure , pulmonary artery pressure , systemic or pulmonary vascular resistance were observed among the treatment groups . serum osmolality , serum sodium , and potassium levels were similar among conivaptan and placebo groups . of note , the patients were fluid - restricted to 250 ml every two hours during the 12-hour study periods and background diuretics and other medications were held during the study period . it should be noted that this study only examined the acute alterations in hemodynamics following a single intravenous dose of conivaptan . despite the encouraging results of this study , other pilot and phase ii studies have not shown benefits in clinical outcomes with longer term conivaptan use . in a double - blind , dose - ranging pilot study,22 162 hospitalized patients with nyha class iii or iv heart failure were randomized to receive conivaptan 20 mg by intravenous bolus followed by continuous infusion of 40 mg / day , 80 mg / day , 120 mg / day , or placebo for two days . ten patients in the conivaptan groups and one in the placebo group discontinued the drug due to adverse events . significant increases in urine output and changes in body weight were observed with all doses of conivaptan compared to placebo , but conivaptan did not improve the severity of respiratory symptoms . a dose evaluation of vasopressin antagonist in chf patients undergoing exercise ( advance ) was a multi - center , double - blind , placebo - controlled , randomized trial designed to investigate the effect of conivaptan on functional capacity in patients with heart failure.23 the study enrolled 345 patients with nyha class ii to iv symptoms who were being treated with standard pharmacotherapy for heart failure . currently , only the methodology of this trial has been published , but additional results are anticipated in the future . conivaptan s efficacy for the treatment of hyponatremia has been assessed in three randomized double - blinded , placebo - controlled clinical trials and one open - label nonrandomized study . the three randomized studies include one pivotal study with intravenous conivaptan and two trials using oral conivaptan ; the non - randomized study used intravenous conivaptan . phase iii studies showed that conivaptan reliably increases the serum sodium concentration , beginning as early as one to two hours after administration . mmol / l within 5 hours and by 6 to 9 mmol / l within 24 hours after initiation of therapy at the 20-mg and 40-mg doses , respectively . in most patients , approximately 70% , the serum sodium normalized over a four - day continuous infusion , with the greatest increase in serum sodium occurring during the first 24 to 48 hours of treatment . the primary efficacy endpoint in these studies was the mean change in serum sodium over the duration of treatment , as measured by the area under the serum sodium effect curve ( auc ) , corrected for baseline serum sodium.1517 in the pivotal phase iii trial conducted with iv conivaptan,17 84 patients with euvolemic or hypervolemic hyponatremia ( serum sodium 115130 meq / l ) were randomized to receive conivaptan 40 mg / day , 80 mg / day or placebo for four days . of 84 total patients included in the intention - to - treat analysis , 66 completed treatment . both doses of conivaptan were associated with significant improvements in serum sodium compared to placebo as measured by both the primary and secondary efficacy endpoints ( table 1 ) . all patients also received standard care treatments for hyponatremia , which primarily consisted of fluid restriction . this was not a strict fluid restriction , ie , < 2 liters / day , because the primary goal was to prevent the patients on placebo from worsening , rather than as an active treatment for hyponatremia . a planned subgroup analysis of this trial was performed to evaluate the efficacy of intravenous conivaptan in patients with euvolemic hyponatremia.18 fifty - six of the 84 patients analyzed in the trial were euvolemic . conivaptan 40 and 80 mg / day effectively raised mean serum sodium on the first day of treatment . the mean increase in serum sodium with conivaptan remained significantly greater than that with placebo at the end of the four - day treatment period . the placebo group did not demonstrate any significant changes in serum sodium concentration ( figure 1 ) . of the two oral conivaptan trials , one has been published.16 the efficacy of oral conivaptan was evaluated in 74 euvolemic and hypervolemic hyponatremic patients who were randomized to receive oral conivaptan 40 mg / day , 80 mg / day , or placebo , all given in two divided doses / day for five days . both doses of conivaptan were associated with significant dose - dependent improvements in serum sodium compared to placebo ( table 2 ) . results from the second oral conivaptan study are available in abstract form only.15 the study , similar to the other oral conivaptan study , included 83 patients with euvolemic and hypovolemic hyponatremia , and revealed the same dose - dependent increase in serum sodium concentrations ( table 2 ) . results from the open - label multicenter trial using intravenous conivaptan were presented at the annual meeting of the endocrine society in june 2008.19 the study was conducted from february 2004 to june 2005 , and included 251 hospitalized patients with euvolemic and hypervolemic hyponatremia to assess the efficacy and safety of intravenous conivaptan 20 and 40 mg / day for four days and for up to 30 days after cessation of therapy . the most common underlying illness associated with hyponatremia was siadh ( 43% of patients ) . conivaptan was associated with significant increases in serum sodium concentration that were sustained throughout the study . at the cessation of active therapy , the mean serum sodium increased by 9.4 and 8.8 meq / l in patients given conivaptan 20 and 40 mg / day , respectively . the response did not appear to differ significantly among patients given 20 or 40 mg / day . very interestingly , the increases in serum sodium over baseline achieved through the end of treatment persisted through days 11 and 34 after initiation of aquaretic therapy even though the duration of active treatment was only four days . the reason for the persistence of the correction is unknown , but it suggests that limited infusions of conivaptan may be sufficient to improve hyponatremia not only during active treatment , but also for longer periods of time in patients who have improvement in their underlying comorbidities responsible for the hyponatremia . although conivaptan has been evaluated extensively in patients with congestive heart failure ( chf ) , efficacy has not been established other than for correction of hyponatremia ; thus , conivaptan is not indicated for primary treatment of chf at this time . vasopressin levels are often elevated in patients with heart failure and left ventricular ( lv ) dysfunction,20 and they are clearly associated with adverse cardiovascular outcomes in the setting of lv dysfunction after myocardial infarction.21 these data suggest that vasopressin may contribute to the circulatory response in patients with heart failure , and may also play a role in the development and progression of heart failure . combined v1ar / v2r antagonism with conivaptan therefore does offer a theoretical benefit in chf . if systemic hemodynamic improvement occurs with v1ar antagonism , renal hemodynamics would improve as well with v2r antagonism , leading to a greater clinical benefit . the hemodynamic effects of conivaptan were evaluated in a prospective , randomized , double - blind study of 142 patients with new york heart association ( nyha ) class iii or iv heart failure of at least three months duration.13 subjects were randomized to receive a single dose of intravenous ( iv ) conivaptan ( 10 , 20 , or 40 mg ) or placebo , administered over 30 minutes . conivaptan produced favorable hemodynamic changes in patients with advanced heart failure , without affecting blood pressure or heart rate . pulmonary capillary wedge pressure and right atrial pressure were significantly reduced in the conivaptan 20 mg and 40 mg groups at three to six hours post - dose as compared with the placebo groups . urine output was significantly higher in the conivaptan - treated patients as compared with placebo , and dose - dependent increases in urine output were observed with increasing conivaptan doses ( p < 0.001 ) . despite conivaptan s properties as a v1ar antagonist , it had no significant effects on systolic blood pressure and pulse rate . no statistically significant changes in cardiac index , mean arterial pressure , pulmonary artery pressure , systemic or pulmonary vascular resistance were observed among the treatment groups . serum osmolality , serum sodium , and potassium levels were similar among conivaptan and placebo groups . of note , the patients were fluid - restricted to 250 ml every two hours during the 12-hour study periods and background diuretics and other medications were held during the study period . it should be noted that this study only examined the acute alterations in hemodynamics following a single intravenous dose of conivaptan . despite the encouraging results of this study , other pilot and phase ii studies have not shown benefits in clinical outcomes with longer term conivaptan use . in a double - blind , dose - ranging pilot study,22 162 hospitalized patients with nyha class iii or iv heart failure were randomized to receive conivaptan 20 mg by intravenous bolus followed by continuous infusion of 40 mg / day , 80 mg / day , 120 mg / day , or placebo for two days . ten patients in the conivaptan groups and one in the placebo group discontinued the drug due to adverse events . significant increases in urine output and changes in body weight were observed with all doses of conivaptan compared to placebo , but conivaptan did not improve the severity of respiratory symptoms . a dose evaluation of vasopressin antagonist in chf patients undergoing exercise ( advance ) was a multi - center , double - blind , placebo - controlled , randomized trial designed to investigate the effect of conivaptan on functional capacity in patients with heart failure.23 the study enrolled 345 patients with nyha class ii to iv symptoms who were being treated with standard pharmacotherapy for heart failure . currently , only the methodology of this trial has been published , but additional results are anticipated in the future . the majority of adverse events were infusion site reactions during intravenous administration , likely as a result of propylene glycol , which is irritating to veins . these reactions occurred in over 50% of conivaptan patients compared with 2% of placebo - treated patients during clinical trials ( table 3 ) . a new formulation pre - mixed in 100 ml infusion bags was approved by the fda in october 2008 , and should reduce infusion site complications since this formulation does not include propylene glycol . although most infusion site complications were minor , including local inflammation , phlebitis , and pain at the infusion site , some serious infusion site reactions did occur . other side effects in decreasing order included headaches , hypokalemia , thirst , vomiting , polyuria , peripheral edema , diarrhea , and orthostatic hypotension . orthostatic hypotension generally occurred on days 1 and 2 of therapy , likely because of volume depletion from induced aquaresis . although v1ar antagonism on vascular smooth muscle could contribute to hypotension as well , this is less likely because : ( 1 ) avp is not necessary for blood pressure homeostasis,8 and ( 2 ) the orthostatic hypotension was mild , short lived , and did not result in withdrawal of patients from the studies . another safety concern that occurred in approximately 9% of the subjects during clinical trials was a too - rapid correction of serum sodium concentration with the administration of conivaptan.9 overly rapid correction of severe hyponatremia can produce pontine and extrapontine myelinolysis , also called osmotic demyelination syndrome ( ods ) , a brain demyelinating disease that can cause substantial neurological morbidity and mortality.24 however , no cases of ods were noted in any of the clinical trials , nor have been reported with subsequent clinical use of conivaptan since 2006 . this is likely the result of using titrated doses of conivaptan with careful monitoring of the serum sodium concentrations during the active phase of correction , thereby achieving a controlled and limited correction of hyponatremia.4 review of data from patients with adverse effects of hypokalemia revealed that the occurrence of a low serum potassium < 3.3 meq / l ( the protocol - defined low threshold for hypokalemia , normal reference range : 3.55.0 meq / l ) at the end of days 2 and 4 was not nearly as high as the reported occurrence of hypokalemia adverse events . overall changes from baseline in serum potassium during the four - day treatment period were mild , and did not reach a level of clinical significance . conivaptan is currently indicated for the treatment of euvolemic hyponatremia as a result of siadh , pulmonary disorders , hypothyroidism or adrenal insufficiency , and for the treatment of hypervolemic hyponatremia . a panel of experts in hyponatremia evaluated the situations in which aquaretic agents should be considered for therapy.18 in hypovolemic hyponatremia , induced aquaresis would aggravate the underlying volume depletion . consequently , conivaptan and all other vaptans are contraindicated in hypovolemic hyponatremia . for euvolemic hyponatremia however , careful clinical assessment of the patient and an accurate diagnosis of euvolemic hyponatremia are necessary before therapy is prescribed . in hypervolemic hyponatremia , antagonism of v2r represents the best approach to treating hyponatremia in most edema - forming states such as congestive heart failure and cirrhosis , because excess avp is the most important pathophysiological factor involved in the pathogenesis of hyponatremia under these conditions , and because therapies employing administration of sodium chloride are generally contraindicated in such patients . conivaptan is currently indicated for the treatment of euvolemic hyponatremia as a result of siadh , pulmonary disorders , hypothyroidism or adrenal insufficiency , and for the treatment of hypervolemic hyponatremia . a panel of experts in hyponatremia evaluated the situations in which aquaretic agents should be considered for therapy.18 in hypovolemic hyponatremia , induced aquaresis would aggravate the underlying volume depletion . consequently , conivaptan and all other vaptans are contraindicated in hypovolemic hyponatremia . for euvolemic hyponatremia however , careful clinical assessment of the patient and an accurate diagnosis of euvolemic hyponatremia are necessary before therapy is prescribed . in hypervolemic hyponatremia , antagonism of v2r represents the best approach to treating hyponatremia in most edema - forming states such as congestive heart failure and cirrhosis , because excess avp is the most important pathophysiological factor involved in the pathogenesis of hyponatremia under these conditions , and because therapies employing administration of sodium chloride are generally contraindicated in such patients . patients with chf often do not correct hypo - osmolar hyponatremia with conventional diuretic therapy . however , reversing impaired free water excretion with conivaptan normalizes dilutional hyponatremia in congestive heart failure patients.4,17 conivaptan and other v2r antagonists will likely prove to be superior to conventional diuretics in this setting , since they can induce free water diuresis without an accompanying solute diuresis . tolvaptan , an oral v2r antagonist , has been assessed in several trials for the management of acute and chronic heart failure . the acute and chronic therapeutic impact of vasopressin antagonist in congestive heart failure ( activ in chf ) trial was a multicenter , randomized controlled trial with 319 patients with acute exacerbation of chf.25 21.3% of the patients had hyponatremia ( serum sodium < 136 patients were randomized to receive one of three doses of oral tolvaptan or placebo for up to 60 days in addition to standard chf therapy . all tolvaptan groups had significant reductions in body weight compared with the placebo group and had significantly higher urine volumes than the placebo group . a large phase iii multi - center trial , efficacy of vasopressin antagonist in heart failure outcome study with tolvaptan ( everest ) , compared the short - term and long - term administration of tolvaptan with placebo in 4133 patients admitted with worsening heart failure . 26,27 in the short - term study , tolvaptan patients lost more weight on day 1 and day 7 or on discharge . more patients receiving tolvaptan reported improvement in dyspnea on day 1 and reduction in edema on day 7 , whereas improvement in general clinical status did not differ between groups.25 however in the long - term study , with a median follow - up of 9.9 months , tolvaptan had no effect on long - term mortality or heart failure - related morbidity.27 within the limitations inherent in this study , the long - standing association between adverse outcomes and hyponatremia in patients with chf does not appear to be due to adverse effects of the hyponatremia itself.28 patients with cirrhosis , intractable ascites , and peripheral edema are often ineffectively treated with aldosterone antagonists and beta - blockers , but it is currently not advisable to use conivaptan in patients with cirrhosis because v1ar inhibition may block the vasoconstrictive effects of avp in the portal and splanchnic circulation , possibly increasing portal blood flow . however , decreases in portal hypertension may offset this effect . consequently , the net effect of conivaptan on the risk of precipitating esophageal variceal bleeding is unknown . theoretically , selective v2r antagonists should not entail adverse effects on portal hemodynamics , but their safety in cirrhosis remains to be ascertained . one of the other oral v2r antagonists , lixivaptan , has been studied in patients with cirrhosis . one randomized controlled trial of 44 hospitalized patients with stable hyponatremia that included 33 patients with cirrhosis showed that a seven - day course of lixivaptan was well tolerated and increased free water clearance , urine output and serum sodium concentrations in a dose - dependent manner.29 another study of 60 patients with cirrhosis showed an increase in serum sodium concentration after seven days of treatment to a mean of 132 meq / l compared with 126 meq / l in the placebo group.30 lixivaptan had no significant effects on blood pressure or heart rate . most placebo - controlled studies using avpr antagonists to treat hyponatremia have been of relatively short duration . however , recent data from two multicenter 30-day placebo - controlled trials of tolvaptan resulted in sustained correction of hyponatremia and improved cognitive function.31 another study using satavaptan , an oral v2r antagonist , began as a five - day randomized , placebo - controlled trial in patients with siadh and continued as an open - label trial which showed continued efficacy and good tolerance for up to 12 months of treatment.32 under a fluid restriction of < 1500 ml / day , mean serum sodium levels remained within the normal range during the open - label period . long - term response rates to oral conivaptan and other avpr antagonists , and whether correction of chronic hyponatremia results in improved functional status and quality of life , will require additional studies . patients with chf often do not correct hypo - osmolar hyponatremia with conventional diuretic therapy . however , reversing impaired free water excretion with conivaptan normalizes dilutional hyponatremia in congestive heart failure patients.4,17 conivaptan and other v2r antagonists will likely prove to be superior to conventional diuretics in this setting , since they can induce free water diuresis without an accompanying solute diuresis . tolvaptan , an oral v2r antagonist , has been assessed in several trials for the management of acute and chronic heart failure . the acute and chronic therapeutic impact of vasopressin antagonist in congestive heart failure ( activ in chf ) trial was a multicenter , randomized controlled trial with 319 patients with acute exacerbation of chf.25 21.3% of the patients had hyponatremia ( serum sodium < 136 patients were randomized to receive one of three doses of oral tolvaptan or placebo for up to 60 days in addition to standard chf therapy . all tolvaptan groups had significant reductions in body weight compared with the placebo group and had significantly higher urine volumes than the placebo group . a large phase iii multi - center trial , efficacy of vasopressin antagonist in heart failure outcome study with tolvaptan ( everest ) , compared the short - term and long - term administration of tolvaptan with placebo in 4133 patients admitted with worsening heart failure . 26,27 in the short - term study , tolvaptan patients lost more weight on day 1 and day 7 or on discharge . more patients receiving tolvaptan reported improvement in dyspnea on day 1 and reduction in edema on day 7 , whereas improvement in general clinical status did not differ between groups.25 however in the long - term study , with a median follow - up of 9.9 months , tolvaptan had no effect on long - term mortality or heart failure - related morbidity.27 within the limitations inherent in this study , the long - standing association between adverse outcomes and hyponatremia in patients with chf does not appear to be due to adverse effects of the hyponatremia itself.28 patients with cirrhosis , intractable ascites , and peripheral edema are often ineffectively treated with aldosterone antagonists and beta - blockers , but it is currently not advisable to use conivaptan in patients with cirrhosis because v1ar inhibition may block the vasoconstrictive effects of avp in the portal and splanchnic circulation , possibly increasing portal blood flow . consequently , the net effect of conivaptan on the risk of precipitating esophageal variceal bleeding is unknown . theoretically , selective v2r antagonists should not entail adverse effects on portal hemodynamics , but their safety in cirrhosis remains to be ascertained . one of the other oral v2r antagonists , lixivaptan , has been studied in patients with cirrhosis . one randomized controlled trial of 44 hospitalized patients with stable hyponatremia that included 33 patients with cirrhosis showed that a seven - day course of lixivaptan was well tolerated and increased free water clearance , urine output and serum sodium concentrations in a dose - dependent manner.29 another study of 60 patients with cirrhosis showed an increase in serum sodium concentration after seven days of treatment to a mean of 132 meq / l compared with 126 meq / l in the placebo group.30 lixivaptan had no significant effects on blood pressure or heart rate . most placebo - controlled studies using avpr antagonists to treat hyponatremia have been of relatively short duration . however , recent data from two multicenter 30-day placebo - controlled trials of tolvaptan resulted in sustained correction of hyponatremia and improved cognitive function.31 another study using satavaptan , an oral v2r antagonist , began as a five - day randomized , placebo - controlled trial in patients with siadh and continued as an open - label trial which showed continued efficacy and good tolerance for up to 12 months of treatment.32 under a fluid restriction of < 1500 ml / day , mean serum sodium levels remained within the normal range during the open - label period . long - term response rates to oral conivaptan and other avpr antagonists , and whether correction of chronic hyponatremia results in improved functional status and quality of life , will require additional studies . conivaptan , along with the other avpr antagonists , produce an aquaretic effect by blocking the activity of endogenous avp at the v2r , which improves dilutional hyponatremia . the current data indicate that these agents are highly effective in producing a safe and predictable aquaresis , thereby increasing serum sodium levels in hyponatremic patients . conivaptan is the first avpr antagonist to receive fda approval , specifically for iv administration to hospitalized patients with euvolemic or hypervolemic hyponatremia . several investigational oral v2r - selective avpr antagonists are in late - stage clinical trials and hold promise for long - term therapy of chronic hyponatremia . however , the safe use of conivaptan and other aquaretics will require avoidance of their use in hypovolemic patients and caution with regard to the correction rate of hyponatremia . clinicians must also be aware of drug interactions , particularly drugs that are metabolized by cytochrome cyp3a4 .

introduction : the available treatment options for euvolemic and hypervolemic hyponatremia are limited , and consist mainly of fluid restriction , diuresis , or hypertonic solutions . most of these therapies are neither well tolerated nor totally effective , and many are associated with significant adverse effects . vasopressin receptor antagonists , also known as vaptans , are a new class of agents that now offer an additional treatment option for hyponatremic patients . conivaptan hydrochloride , a competitive antagonist of vasopressin v1a and v2 receptors , is the first agent in this class to be approved for treatment of euvolemic and hypervolemic hyponatremia in hospitalized patients.aims:this review critically assesses the evidence that support the use of conivaptan for the treatment of patients with euvolemic and hypervolemic hyponatremia.evidence review conclusion : conivaptan is effective in raising serum sodium levels in a predictable and safe fashion in euvolemic and hypervolemic hyponatremic patients . conivaptan provides the first molecularly targeted approach for correcting hyponatremia in hospitalized patients .

Mechanism of action Drug formulation and dosing Pharmacokinetics Clinical efficacy Hyponatremia Heart failure Tolerability Clinical indications Hyponatremia Potential therapeutic indications for conivaptan and similar agents Congestive heart failure Cirrhosis Long-term treatment of hyponatremia Conclusions

conivaptan is a nonpeptide antagonist with a high affinity for the human vasopressin receptor subtypes v1a and v2 . the other vaptans mentioned earlier , lixivaptan , satavaptan , and tolvaptan , are selective v2 receptor antagonists , and do not have appreciable binding to the v1a receptors . conivaptan is available as an iv formulation in 4 ml ampoules containing 20 mg of conivaptan hydrochloride . the effect of conivaptan on pulmonary capillary wedge pressure ( pcwp ) in patients with advanced heart failure was sustained for approximately eight hours following the administration of intravenous conivaptan , and pcwp remained below baseline values even after 12 hours.13 the pharmacokinetics of 40 mg / day and 80 mg / day of intravenous and oral conivaptan were nonlinear , because conivaptan inhibits its own metabolism at cyp3a4 . because of this , conivaptan is contraindicated with the concurrent use of other potent cyp3a4 inhibitors such as ketoconazole , itraconazole , clarithromycin , ritonavir , and indinavir . conivaptan s efficacy for the treatment of hyponatremia has been assessed in three randomized double - blinded , placebo - controlled clinical trials and one open - label nonrandomized study . the primary efficacy endpoint in these studies was the mean change in serum sodium over the duration of treatment , as measured by the area under the serum sodium effect curve ( auc ) , corrected for baseline serum sodium.1517 in the pivotal phase iii trial conducted with iv conivaptan,17 84 patients with euvolemic or hypervolemic hyponatremia ( serum sodium 115130 meq / l ) were randomized to receive conivaptan 40 mg / day , 80 mg / day or placebo for four days . both doses of conivaptan were associated with significant improvements in serum sodium compared to placebo as measured by both the primary and secondary efficacy endpoints ( table 1 ) . a planned subgroup analysis of this trial was performed to evaluate the efficacy of intravenous conivaptan in patients with euvolemic hyponatremia.18 fifty - six of the 84 patients analyzed in the trial were euvolemic . conivaptan 40 and 80 mg / day effectively raised mean serum sodium on the first day of treatment . of the two oral conivaptan trials , one has been published.16 the efficacy of oral conivaptan was evaluated in 74 euvolemic and hypervolemic hyponatremic patients who were randomized to receive oral conivaptan 40 mg / day , 80 mg / day , or placebo , all given in two divided doses / day for five days . both doses of conivaptan were associated with significant dose - dependent improvements in serum sodium compared to placebo ( table 2 ) . results from the second oral conivaptan study are available in abstract form only.15 the study , similar to the other oral conivaptan study , included 83 patients with euvolemic and hypovolemic hyponatremia , and revealed the same dose - dependent increase in serum sodium concentrations ( table 2 ) . results from the open - label multicenter trial using intravenous conivaptan were presented at the annual meeting of the endocrine society in june 2008.19 the study was conducted from february 2004 to june 2005 , and included 251 hospitalized patients with euvolemic and hypervolemic hyponatremia to assess the efficacy and safety of intravenous conivaptan 20 and 40 mg / day for four days and for up to 30 days after cessation of therapy . the most common underlying illness associated with hyponatremia was siadh ( 43% of patients ) . conivaptan was associated with significant increases in serum sodium concentration that were sustained throughout the study . the reason for the persistence of the correction is unknown , but it suggests that limited infusions of conivaptan may be sufficient to improve hyponatremia not only during active treatment , but also for longer periods of time in patients who have improvement in their underlying comorbidities responsible for the hyponatremia . although conivaptan has been evaluated extensively in patients with congestive heart failure ( chf ) , efficacy has not been established other than for correction of hyponatremia ; thus , conivaptan is not indicated for primary treatment of chf at this time . vasopressin levels are often elevated in patients with heart failure and left ventricular ( lv ) dysfunction,20 and they are clearly associated with adverse cardiovascular outcomes in the setting of lv dysfunction after myocardial infarction.21 these data suggest that vasopressin may contribute to the circulatory response in patients with heart failure , and may also play a role in the development and progression of heart failure . the hemodynamic effects of conivaptan were evaluated in a prospective , randomized , double - blind study of 142 patients with new york heart association ( nyha ) class iii or iv heart failure of at least three months duration.13 subjects were randomized to receive a single dose of intravenous ( iv ) conivaptan ( 10 , 20 , or 40 mg ) or placebo , administered over 30 minutes . in a double - blind , dose - ranging pilot study,22 162 hospitalized patients with nyha class iii or iv heart failure were randomized to receive conivaptan 20 mg by intravenous bolus followed by continuous infusion of 40 mg / day , 80 mg / day , 120 mg / day , or placebo for two days . a dose evaluation of vasopressin antagonist in chf patients undergoing exercise ( advance ) was a multi - center , double - blind , placebo - controlled , randomized trial designed to investigate the effect of conivaptan on functional capacity in patients with heart failure.23 the study enrolled 345 patients with nyha class ii to iv symptoms who were being treated with standard pharmacotherapy for heart failure . conivaptan s efficacy for the treatment of hyponatremia has been assessed in three randomized double - blinded , placebo - controlled clinical trials and one open - label nonrandomized study . the primary efficacy endpoint in these studies was the mean change in serum sodium over the duration of treatment , as measured by the area under the serum sodium effect curve ( auc ) , corrected for baseline serum sodium.1517 in the pivotal phase iii trial conducted with iv conivaptan,17 84 patients with euvolemic or hypervolemic hyponatremia ( serum sodium 115130 meq / l ) were randomized to receive conivaptan 40 mg / day , 80 mg / day or placebo for four days . both doses of conivaptan were associated with significant improvements in serum sodium compared to placebo as measured by both the primary and secondary efficacy endpoints ( table 1 ) . of the two oral conivaptan trials , one has been published.16 the efficacy of oral conivaptan was evaluated in 74 euvolemic and hypervolemic hyponatremic patients who were randomized to receive oral conivaptan 40 mg / day , 80 mg / day , or placebo , all given in two divided doses / day for five days . both doses of conivaptan were associated with significant dose - dependent improvements in serum sodium compared to placebo ( table 2 ) . results from the second oral conivaptan study are available in abstract form only.15 the study , similar to the other oral conivaptan study , included 83 patients with euvolemic and hypovolemic hyponatremia , and revealed the same dose - dependent increase in serum sodium concentrations ( table 2 ) . results from the open - label multicenter trial using intravenous conivaptan were presented at the annual meeting of the endocrine society in june 2008.19 the study was conducted from february 2004 to june 2005 , and included 251 hospitalized patients with euvolemic and hypervolemic hyponatremia to assess the efficacy and safety of intravenous conivaptan 20 and 40 mg / day for four days and for up to 30 days after cessation of therapy . conivaptan was associated with significant increases in serum sodium concentration that were sustained throughout the study . although conivaptan has been evaluated extensively in patients with congestive heart failure ( chf ) , efficacy has not been established other than for correction of hyponatremia ; thus , conivaptan is not indicated for primary treatment of chf at this time . vasopressin levels are often elevated in patients with heart failure and left ventricular ( lv ) dysfunction,20 and they are clearly associated with adverse cardiovascular outcomes in the setting of lv dysfunction after myocardial infarction.21 these data suggest that vasopressin may contribute to the circulatory response in patients with heart failure , and may also play a role in the development and progression of heart failure . the hemodynamic effects of conivaptan were evaluated in a prospective , randomized , double - blind study of 142 patients with new york heart association ( nyha ) class iii or iv heart failure of at least three months duration.13 subjects were randomized to receive a single dose of intravenous ( iv ) conivaptan ( 10 , 20 , or 40 mg ) or placebo , administered over 30 minutes . in a double - blind , dose - ranging pilot study,22 162 hospitalized patients with nyha class iii or iv heart failure were randomized to receive conivaptan 20 mg by intravenous bolus followed by continuous infusion of 40 mg / day , 80 mg / day , 120 mg / day , or placebo for two days . a dose evaluation of vasopressin antagonist in chf patients undergoing exercise ( advance ) was a multi - center , double - blind , placebo - controlled , randomized trial designed to investigate the effect of conivaptan on functional capacity in patients with heart failure.23 the study enrolled 345 patients with nyha class ii to iv symptoms who were being treated with standard pharmacotherapy for heart failure . another safety concern that occurred in approximately 9% of the subjects during clinical trials was a too - rapid correction of serum sodium concentration with the administration of conivaptan.9 overly rapid correction of severe hyponatremia can produce pontine and extrapontine myelinolysis , also called osmotic demyelination syndrome ( ods ) , a brain demyelinating disease that can cause substantial neurological morbidity and mortality.24 however , no cases of ods were noted in any of the clinical trials , nor have been reported with subsequent clinical use of conivaptan since 2006 . this is likely the result of using titrated doses of conivaptan with careful monitoring of the serum sodium concentrations during the active phase of correction , thereby achieving a controlled and limited correction of hyponatremia.4 review of data from patients with adverse effects of hypokalemia revealed that the occurrence of a low serum potassium < 3.3 meq / l ( the protocol - defined low threshold for hypokalemia , normal reference range : 3.55.0 meq / l ) at the end of days 2 and 4 was not nearly as high as the reported occurrence of hypokalemia adverse events . conivaptan is currently indicated for the treatment of euvolemic hyponatremia as a result of siadh , pulmonary disorders , hypothyroidism or adrenal insufficiency , and for the treatment of hypervolemic hyponatremia . for euvolemic hyponatremia however , careful clinical assessment of the patient and an accurate diagnosis of euvolemic hyponatremia are necessary before therapy is prescribed . in hypervolemic hyponatremia , antagonism of v2r represents the best approach to treating hyponatremia in most edema - forming states such as congestive heart failure and cirrhosis , because excess avp is the most important pathophysiological factor involved in the pathogenesis of hyponatremia under these conditions , and because therapies employing administration of sodium chloride are generally contraindicated in such patients . conivaptan is currently indicated for the treatment of euvolemic hyponatremia as a result of siadh , pulmonary disorders , hypothyroidism or adrenal insufficiency , and for the treatment of hypervolemic hyponatremia . for euvolemic hyponatremia however , careful clinical assessment of the patient and an accurate diagnosis of euvolemic hyponatremia are necessary before therapy is prescribed . in hypervolemic hyponatremia , antagonism of v2r represents the best approach to treating hyponatremia in most edema - forming states such as congestive heart failure and cirrhosis , because excess avp is the most important pathophysiological factor involved in the pathogenesis of hyponatremia under these conditions , and because therapies employing administration of sodium chloride are generally contraindicated in such patients . however , reversing impaired free water excretion with conivaptan normalizes dilutional hyponatremia in congestive heart failure patients.4,17 conivaptan and other v2r antagonists will likely prove to be superior to conventional diuretics in this setting , since they can induce free water diuresis without an accompanying solute diuresis . the acute and chronic therapeutic impact of vasopressin antagonist in congestive heart failure ( activ in chf ) trial was a multicenter , randomized controlled trial with 319 patients with acute exacerbation of chf.25 21.3% of the patients had hyponatremia ( serum sodium < 136 patients were randomized to receive one of three doses of oral tolvaptan or placebo for up to 60 days in addition to standard chf therapy . more patients receiving tolvaptan reported improvement in dyspnea on day 1 and reduction in edema on day 7 , whereas improvement in general clinical status did not differ between groups.25 however in the long - term study , with a median follow - up of 9.9 months , tolvaptan had no effect on long - term mortality or heart failure - related morbidity.27 within the limitations inherent in this study , the long - standing association between adverse outcomes and hyponatremia in patients with chf does not appear to be due to adverse effects of the hyponatremia itself.28 patients with cirrhosis , intractable ascites , and peripheral edema are often ineffectively treated with aldosterone antagonists and beta - blockers , but it is currently not advisable to use conivaptan in patients with cirrhosis because v1ar inhibition may block the vasoconstrictive effects of avp in the portal and splanchnic circulation , possibly increasing portal blood flow . one randomized controlled trial of 44 hospitalized patients with stable hyponatremia that included 33 patients with cirrhosis showed that a seven - day course of lixivaptan was well tolerated and increased free water clearance , urine output and serum sodium concentrations in a dose - dependent manner.29 another study of 60 patients with cirrhosis showed an increase in serum sodium concentration after seven days of treatment to a mean of 132 meq / l compared with 126 meq / l in the placebo group.30 lixivaptan had no significant effects on blood pressure or heart rate . however , recent data from two multicenter 30-day placebo - controlled trials of tolvaptan resulted in sustained correction of hyponatremia and improved cognitive function.31 another study using satavaptan , an oral v2r antagonist , began as a five - day randomized , placebo - controlled trial in patients with siadh and continued as an open - label trial which showed continued efficacy and good tolerance for up to 12 months of treatment.32 under a fluid restriction of < 1500 ml / day , mean serum sodium levels remained within the normal range during the open - label period . however , reversing impaired free water excretion with conivaptan normalizes dilutional hyponatremia in congestive heart failure patients.4,17 conivaptan and other v2r antagonists will likely prove to be superior to conventional diuretics in this setting , since they can induce free water diuresis without an accompanying solute diuresis . the acute and chronic therapeutic impact of vasopressin antagonist in congestive heart failure ( activ in chf ) trial was a multicenter , randomized controlled trial with 319 patients with acute exacerbation of chf.25 21.3% of the patients had hyponatremia ( serum sodium < 136 patients were randomized to receive one of three doses of oral tolvaptan or placebo for up to 60 days in addition to standard chf therapy . more patients receiving tolvaptan reported improvement in dyspnea on day 1 and reduction in edema on day 7 , whereas improvement in general clinical status did not differ between groups.25 however in the long - term study , with a median follow - up of 9.9 months , tolvaptan had no effect on long - term mortality or heart failure - related morbidity.27 within the limitations inherent in this study , the long - standing association between adverse outcomes and hyponatremia in patients with chf does not appear to be due to adverse effects of the hyponatremia itself.28 patients with cirrhosis , intractable ascites , and peripheral edema are often ineffectively treated with aldosterone antagonists and beta - blockers , but it is currently not advisable to use conivaptan in patients with cirrhosis because v1ar inhibition may block the vasoconstrictive effects of avp in the portal and splanchnic circulation , possibly increasing portal blood flow . one randomized controlled trial of 44 hospitalized patients with stable hyponatremia that included 33 patients with cirrhosis showed that a seven - day course of lixivaptan was well tolerated and increased free water clearance , urine output and serum sodium concentrations in a dose - dependent manner.29 another study of 60 patients with cirrhosis showed an increase in serum sodium concentration after seven days of treatment to a mean of 132 meq / l compared with 126 meq / l in the placebo group.30 lixivaptan had no significant effects on blood pressure or heart rate . however , recent data from two multicenter 30-day placebo - controlled trials of tolvaptan resulted in sustained correction of hyponatremia and improved cognitive function.31 another study using satavaptan , an oral v2r antagonist , began as a five - day randomized , placebo - controlled trial in patients with siadh and continued as an open - label trial which showed continued efficacy and good tolerance for up to 12 months of treatment.32 under a fluid restriction of < 1500 ml / day , mean serum sodium levels remained within the normal range during the open - label period . long - term response rates to oral conivaptan and other avpr antagonists , and whether correction of chronic hyponatremia results in improved functional status and quality of life , will require additional studies . the current data indicate that these agents are highly effective in producing a safe and predictable aquaresis , thereby increasing serum sodium levels in hyponatremic patients . conivaptan is the first avpr antagonist to receive fda approval , specifically for iv administration to hospitalized patients with euvolemic or hypervolemic hyponatremia .

nitric oxide ( no ) , a free radical with moderate reactivity , has emerged as an important signaling molecule in a multitude of physiological systems in the human body . in the gastrointestinal tract , no is involved in the regulation of regional blood flow , smooth muscle relaxation , secretory and immunological regulation . during inflammation the production of no rectal no concentrations in healthy volunteers ( range 325 ppbv ) are significantly lower than the concentrations in patients with active inflammatory bowel disease ( ibd ) ( range 718978 ppbv ) . no can be synthesized in eukaryotic cells through oxidation of l - arginine by no synthase ( nos ) [ 4 , 5 ] . depending on the isoform of nos catalyzing the reaction , nanomolar ( endothelial nos and neuronal nos ) or micromolar ( inducible nos ) no concentrations are produced serving as a messenger or as an antibacterial agent . besides eukaryotic no production , it is well known that no can be produced also by microorganisms . a first way of microbial no synthesis can be achieved from l - arginine by bacterial nos ( bnos ) with features resembling the eukaryotic nos ( euknos ) . bnos activity has been described in a group of gram - positive bacteria [ 614 ] and genome sequencing has revealed genes coding for similar proteins that are however shorter than the euknos . bnos from bacillus subtilis , bacillus anthracis , deinococcus radiodurans , and streptomyces spp . display no - forming activity in vivo dependent on arginine [ 1417 ] . besides l - arginine , nitrate and nitrite can also serve as an n - source for no . per day , between 0 and 10 mg no3-n can reach the large intestine in healthy persons . feces were shown to contain around 0.15 mg no3-n / l . in vitro studies showed a correlation between the concentration of the added nitrate or nitrite and the concentration of no produced by the fecal microbiota . the metabolic process best known for the microbial production of no , and extensively described in soils , sediments and water treatment plants , is denitrification . this dissimilatory process uses oxidized nitrogen species as final electron acceptors when oxygen levels are limiting . during the denitrification process nitrate or nitrite is reduced to n2 gas with no and n2o as intermediates . at steady - state conditions , the no concentrations range between 14 and 900 ng . another no producing mechanism is by the reduction of nitrite . for lactobacilli and bifidobacteria hence , acidic nonenzymatic reduction of nitrite was suggested as the predominant pathway for no production in vitro by lactic acid producing bacteria ( lactobacilli and bifidobacteria ) as the produced organic acids decrease the ph of the agar plates to 4 . whereas no production by lactic acid bacteria was considered to be chemical , no production from nitrite by escherichia coli and salmonella typhimurium was shown to be biological . the enzymes responsible for the no formation from nitrite in e. coli and s. typhimurium are periplasmic and cytoplasmic nitrite reductase and nitrate reductase , respectively [ 23 , 24 ] . to date , the relevance and mechanism of bacterial no production under gastrointestinal conditions remain elusive . with no being produced by inducible nos in epithelial cells as a response to cytokines and bacterial products , it is of interest to know whether bacterial no production can occur under gastrointestinal conditions . the objective of our study was to unravel the mechanisms by which human fecal microbiota are able to produce no in vitro . three possible pathways were considered ( i ) l - arginine as substrate for no synthase , ( ii ) denitrification to nitrogen gas and ( iii ) dissimilatory nitrate reduction to ammonium ( dnra ) . the latter was considered as fermentative nitrate reduction is expected under gastrointestinal conditions however no relation with no production has been considered [ 26 , 27 ] . l - cysteine was added to scavenge dissolved oxygen and to lower the initial redox potential . analyses of the feed medium with ion chromatography indicated that very low concentrations of nitrate , that is , 110 50 g fecal samples were obtained from 6 healthy women with a mean age of 55.5 ranging between 53 and 59 . the main group of organisms in fecal samples belongs to the anaerobic gram - positive bacteria . anaerobic populations belonging to the genera bacteroidetes and firmicutes dominate [ 31 , 32 ] . incubations were performed with fecal suspension of different healthy persons which all showed similar results . in the figures , the results of incubations of one representative person are shown . the bottles were filled with 50 ml of one of the growth media and the ph was set at 7.0 using a 1 m naoh solution . the headspace ( 70 ml ) was flushed for 45 minutes with n2-gas to create anaerobic conditions . different parameters ( growth media , concentrations of nitrate and l - arginine , ph , dilution of the fecal suspension and supernatant ) were varied to examine their effect on the production of no , as described in figure 1 . nitrate as a possible n - source for no was added in concentrations of 5 and 50 mg no3-n / l using a 14 g nano3-n / l solution . to study l - arginine as n - source , a stock solution of 3.5 g ( l - arginine - hcl)-n / l was added to final concentrations of 5 and 50 mg l - arginine - n / l . when incubations were performed in buffered feed medium , a 1 m phosphate buffer ( 88 g k2hpo4/l and 68 g kh2po4/l ) dilutions of the fecal suspension ( 1/2 , 1/10 , 1/20 , 1/200 , and 1/2000 ) were made in saline ( 8.5 g , 1 ml was inoculated in the growth medium . to study the effect of the supernatant of the fecal suspension on no production , the penicillin flasks were incubated at 37c for 24 hours while shaking at 130 rpm . to assess the production of no during dnra , escherichia coli k12 ( lmg 18221 ) ( bccm culture collection , ghent university , gent , belgium ) was grown anaerobically for 24 hours at 37c in buffered feed medium without additional nitrate . to determine the reduction of nitrate to n2 or ammonium by , respectively , denitrification or dnra , an experiment was set up as described in the section above but using a 14 g kno3-n / l ( 10 atom % ) solution to final concentrations of 5 and 50 mg no3-n / l ( 10 atom % ) . the reactor setup was adapted from the shime , representing the different parts of the adult human gut and consisting of a succession of 5 compartments . the first 2 compartments are a fill - and - draw setup and represent the stomach and small intestine . peristaltic pumps add a defined amount of feed medium ( 140 ml , 3 times / day ) and pancreatic and bile liquid ( 60 ml , 3 times / day ) , respectively , to the stomach and duodenum compartments and empty the respective compartments after specified intervals . the last 3 compartments are continuously stirred reactors with constant volume and ph control , specific for each compartment . upon inoculation with fecal microbiota , these reactors simulate the ascending , transverse , and descending colon . inoculum preparation , retention time , ph , temperature settings , and reactor feed composition were described previously . closing off the gas phase of the different colon compartments ( ascending , transverse , and descending ) upon n2 flushing allowed to study the no production in the respective colon regions separately . all the incubations were done anaerobically in penicillin flasks of 120 ml , as described previously . the suspensions were incubated as such or with 20 ml fresh feed medium . to test the effect of the biomass , the biomass of the suspensions was washed 3 times with saline and resuspended in feed medium . the supernatant was prepared by centrifuging the suspension at 10 000 g for 15 minutes . the supernatant was filtersterilized using 0.22 m membrane filters ( millipore , nyse : mil , usa ) or autoclaved . both no3 and no2 concentrations were measured before and after incubation of fecal suspension in growth media supplemented with no3 . a metrohm 761 compact ion chromatograph ( metrohm ag , herisau , switzerland ) equipped with a conductivity detector and a metrosep a supp 5 column was used . the operational parameters were as follows : eluent 1.06 g na2co3/l ; flow 0.7 ml / min ; sample loop 20 l . the samples were centrifuged at 4000 g for 3 minutes , filtered using 0.45 m membrane filters , and diluted in milli - q water before analysis ( millipore , nyse : mil , usa ) . during n experiments , the amount of no3 and no2 was measured by first reducing no3 to no2 by a copper - cadmium redactor coil at ph of 8.0 . no2 concentrations were determined colorimetrically by an imidazole buffered reaction with n-1-naphtylethylenediamine . the no3 concentration was quantified by subtraction of the concentrations of no2 from the concentration of ( no3 + no2 ) . the samples were centrifuged at 4000 g for 3 minutes , filtered using 0.45 m membrane filters , and diluted in milli - q water before analysis ( millipore , nyse : mil , usa ) . nh4 analyses were performed after conversion to n2o using a trace gas preconcentration unit ( anca - tgii , sercon , uk ) coupled to an isotope ratio mass spectrometer ( irms ) ( 20 - 20 , sercon , uk ) [ 35 , 36 ] . to determine the n / n ratio of the biomass , the samples were centrifuged at 1750 g for 15 minutes and the pellet was dried at 60c overnight . the n / n ratio was determined by an elemental analyzer ( ea ) ( anca - sl , sercon , uk ) coupled to the irms ( 20 - 20 , sercon , uk ) . no measurements were done based on the principle of chemiluminescence using eco physics cld 77 am ( eco physics ag , duernten , switzerland ) with a detection limit of 1 ppbv . gas samples of 25 ml were collected in syringes from the headspace of the penicillin flasks after 24 hours of anaerobic incubation . the concentrations were calculated by comparison with a standard curve prepared with no standard gas ( 98 0.5 ppmv ) diluted in air . for n2o measurements , 6 ml of gas from the headspace was collected in a vacutainer with a total volume of 12 ml ( labco limited , buckinghamshire , uk ) . one ml of this dilution was used for analysis with a shimadzu gc-14b gas chromatograph ( shimadzu , kyoto , japan ) with an electron capture detector ( ecd ) and two packed columns ( porapack q , 80/100 mesh , 1 and 2 m ) . the operating conditions were as follows : carrier gas n2 ( 55 ml / min ) , injector temperature 105c , column and oven temperature 35c , and detector temperature 250c . the chromatograph was calibrated using n2o standard gas ( 250 13 ppmv or 25.3 1.5 ppmv in he ) . for n2 analyses , 12 ml of gas from the headspace was collected in a vacutainer ( labco limited , buckinghamshire , uk ) . the gas was analyzed with a finnigan trace ultra gc ( interscience , breda , the netherlands ) with a thermal conductivity detector ( tcd ) , a precolumn ( hayesep q , 80/100 mesh , 0.25 m ) and 2 packed columns ( hayesep q , 80/100 mesh , 2 m and molsieve 5 , 80/100 mesh , 2 m ) . the injector temperature was 65c and oven temperature 90c . all no and n2o measurements were subsequently converted to concentrations present in the growth medium . the no and n2o concentrations measured in the gas phase ( ppbv ) were converted to molar concentrations using the ideal gas law pv = nrt with t = 294.65 k. taking into account [ no]g/[no]aq 20 and [ n2o]g/[n2o]aq 1.64 at equilibrium , the no and n2o concentrations in the gas phase were converted to the concentration in the liquid phase and expressed as g no - n or n2o - n per l. spss version 16.0 ( spss inc . , chicago , usa ) was used to carry out all statistical analyses . normality of the data and homogeneity of variances was assessed using the kolmogorov - smirnov and the levene test , respectively . comparison of normally distributed data was performed with anova or tamhane ; when anova indicated significant differences , means were compared using the bonferroni comparison test . comparison of means of not normally distributed data was evaluated with the nonparametric kruskal - wallis test ; when kruskal - wallis indicated significant differences , means were compared using the mann - whitney comparison test . the biological production of no with l - arginine as substrate was studied by supplementing mineral medium and feed medium with 0 , 5 , or 50 mg l - arginine - n / l . l - arginine - n / l but not inoculated with fecal suspension , served as a negative control . the ph was set at 7 and no and n2o concentrations were analyzed after 24 hours . when l - arginine was supplemented to the mineral medium , only traces of no ( 0.28 ng no - n / l medium ) could be detected for all the l - arginine concentrations . in feed medium , the mean no concentration after 24 hours was for all the samples around 75 ng no - n / l medium . fecal suspensions were inoculated anaerobically for 24 hours in mineral media ( with succinate as electron donor ) containing 0 , 5 , or 50 mg no3-n / l . none of the samples contained nitrite in the beginning or at the end of the incubation and after 24 hours the mean ph was 6.53 0.35 . after 24 hours of incubation , the no concentrations in the mineral media remained below 1 ng no - n / l medium in all experiments ( figure 2(a ) ) . a proportional effect was observed for n2o production and the amount of supplied nitrate ( figure 2(b ) ) . however , addition of 5 and 50 mg no3-n / l resulted in significant n2o production . to examine whether microbiological production of no and n2o was possible under gastrointestinal conditions , fecal microbiota were grown in the presence of a complex medium simulating the conditions in the colon ( figure 1 ) . the feed medium was analyzed for nitrate and a background of 0.11 mg no3-n / l was measured . after 24 hours of incubation , the ph dropped to 4.13 0.05 , 4.24 0.03 and 4.47 0.03 for 0.11 , 5 and 50 mg no3-n / l , respectively . nitrite at time 0 was below 0.1 mg no2-n / l but after 24 hours concentrations of 0.1 0.1 and 2.0 0.2 mg no2-n / l were detected for 5 and 50 mg no3-n / l , respectively . figures 2(c ) and 2(d ) show the concentrations of no and n2o measured after 24 hours . the no concentration measured in the medium without nitrate addition was 37.0 0.3 ng no - n / l . the addition of 5 mg no3-n / l increased the no concentration slightly to 44.1 2.5 ng no - n / l medium . however the addition of 50 mg / l no3-n increased the no concentration drastically to 298 33 g no - n / l medium . as for the mineral media , a positive effect for the n2o production with the supplemented nitrate was observed . the same experimental setup as described above was used but under buffered conditions ( measured ph was always between 6 and 6.8 ) , thus avoiding fermentative acidification during biotic incubations . as shown in figures 2(e ) and 2(f ) , the no concentrations increased slightly when nitrate was added . the concentration of n2o in buffered feed medium supplemented with 5 mg no3-n / l was comparable to the concentration measured in the nonbuffered test reported above . for the highest concentration of nitrate a lower n2o concentration was detected compared to the nonbuffered test ( figure 2 ) . a n tracer study was used to link nitrate consumption to denitrification or dnra by the fecal microbiota under gastrointestinal conditions . the setup was the same as for the buffered feed medium but kno3 ( 10 atom % ) was used as n - source in 2 different concentrations , that is , 5 and 50 mg no3-n / l ( figure 1 ) . table 1 gives an overview of the nitrogen concentrations measured and the recovery of n after 24 hours of anaerobic incubation . the amount of no and n2o produced per volume of feed medium was in the same range as what was found in the buffered feed medium experiment ( table 1 ) . after 24 hours , concentrations reached 182 4 , 210 1 , 275 6 , and 50.1 2.6 mg no3-n / l , and the chemical control , respectively . to verify if no can be produced as a side product or as an intermediate during dnra , e. coli lmg 18221 , a strain capable of dnra but not denitrification after 24 hours , no concentrations of 170 ng no - n / l medium were measured . this no concentration was twice as high compared to no concentrations produced by the fecal cultures . figure 3(a ) shows that diluting the fecal suspension 200 times before inoculation in buffered feed medium results in a 75% increase in the no concentration . there was a difference in the no concentration related to the fecal suspension and optimal dilution . the optimal dilution ranged between 1/20 and 1/200 and the increase of no between 50 and 75% , respectively . the addition of supernatant from a fecal suspension resulted in a decrease of the no concentration ( figure 3(b ) ) . a continuous model that simulates the gastrointestinal tract ( shime ) was used to measure no production by human gastrointestinal microbiota . the no concentrations in the headspace of the 3 colon vessels were analyzed after an incubation period of 24 hours . in the colon ascendens , 101 ng no - n / l shime suspension was measured but lower concentrations were found in the colon transversum and descendens ( 5.4 and 8.5 ng no - n / l shime suspension , respectively ) . a batch setup was performed to test whether this difference was due to ( i ) the microbial community or ( ii ) the availability of nutrients in the different colon compartments . the biomass of the different colon compartments was washed and grown anaerobically in fresh feed medium for 24 hours . as references , the microbial suspensions of the different colon vessels were incubated in parallel in penicillin bottles with or without the addition of fresh feed . to exclude a chemical process , cell - free supernatant of the shime suspension was filtersterilized or autoclaved and subsequently incubated . figure 4 shows that the addition of feed increases the no concentrations at least 3 times but the difference between the first colon vessel and the other 2 was maintained . incubation of the washed biomass showed that even higher concentrations of no were produced ( ranging between 220 and 250 ng no - n / l ) and that the supernatant that was removed might have an inhibitory effect on the no production as observed above . no chemical production of no was seen when the cell - free supernatant of the shime suspension was incubated . our results demonstrate that the fecal microbiota of healthy persons are able to produce no in concentrations up to 90 ng no - n / l medium when grown in conditions simulating the colon ( figure 2(e ) ) . adding l - arginine to the feed medium inoculated with fecal microbiota of a healthy person had no effect on the no production . high amounts of nitrate in combination with low ph resulted in substantial no and n2o production ( figures 2(c ) and 2(d ) ) . however , no and n2o were also formed under ph buffered conditions ( figures 2(e ) and 2(f ) ) . these data corroborate the metabolic capacity of the fecal microbiota to reduce nitrate and produce no . by using n tracer experiments , it was shown that nitrate was mainly reduced to ammonium by dnra suggesting that no was produced as a side product or intermediate of this pathway . no was detected not only in the batch tests but also in the in vitro shime model . indirect evidence from in vitro studies demonstrates that extracellular l - arginine increases the no production by euknos ( at concentrations at which the enzyme should be saturated ) , known as the l - arginine paradox [ 37 , 38 ] and some prokaryotic nos proteins have been shown to produce no in vivo [ 1417 ] . the reaction from l - arginine to no is oxygen dependent what might explain why l - arginine has no increasing effect during anaerobic growth of the fecal microbiota . therefore , we consider the arginine pathway for bacterial no production under gastrointestinal conditions as not important . in the lower parts of the gastrointestinal tract , nitrate originates from dietary products rich in nitrate , like spinach , beetroot , or fennel or simply from minor levels as present in drinking water . nitrate is reduced by the bacteria in the oral cavity or is absorbed in the small intestine . in healthy persons , levels in the order of mg no3-n / l can be estimated in the large intestine and feces were shown to contain around 0.15 mg no3-n / l . based on these data , 2 different concentrations of nitrate ( 5 and 50 mg no3-n very low concentrations of nitrate were measured in the feed medium receiving no additional nitrate ( 110 50 g no3-n / l ) , representative for the concentrations found in feces . no3-n / l very high concentrations of no ( 29.8 g no - n / l ) were measured . during the incubations , the ph dropped to a mean value of 4.24 due to the accumulation of organic acids produced by the microorganisms . at these low ph values , a chemical reduction of nitrite to no ( ph below 5.5 ) can not be excluded . in vivo , the ph increases over the different colon parts . considering a ph of 6.37 0.58 in the colon ascendens , 6.61 0.83 in the colon transversum and 7.04 0.67 in the colon descendens , a chemical reduction of nitrite to no in vivo is not likely . because it was unclear whether no was produced chemically or biologically the very high no concentrations produced at low ph in feed medium containing the highest nitrate concentration were not found at neutral ph . in a study of sobko the production of no by fecal microbiota cultivated on iso - sensitest agar supplemented with nitrate was demonstrated . for the plates supplemented with 1.4 mg no3-n / l , concentrations in the range 293600 ppbv no , corresponding to 1145 ng no - n / l in the microbial suspension , were measured but a high variability was seen between different fecal samples . as the ph of the plates decreased from 7 to 5 , a chemical reduction could not be excluded . however , it was concluded that the fecal no generation was not solely due to acidification of the plates . we can conclude that the no concentrations found in our study , which were between 90 and 100 ng no / l for low concentrations of nitrate , are in the same order of magnitude . under anaerobic conditions , bacteria can use nitrate as n - source in nitrate assimilation and 2 dissimilatory processes , denitrification and dnra . denitrification is assumed to be the major nitrate removal pathway in many anoxic ecosystems like soils , sediments , and water treatment plants but information about the occurrence of this process in the human colon is limited . denitrifying bacteria are found in alpha- , beta , gamma- and epsilonproteobacteria , firmicutes , and bacteroidetes . during denitrification no is produced in the nanomolar range , without reaching toxic levels [ 21 , 22 ] . when the supply of electron acceptor is limited , it is more beneficial for the bacteria to exploit dnra rather than denitrification because of the higher electron consumption . molecular and biochemical studies have described dnra in some enteric bacteria but information about the occurrence of this process in complex microbial cultures including the microbiota of the human gastrointestinal tract is limited . allison and macfarlane reported that dnra is the major pathway for nitrate reduction by the fecal microbiota . identification based on phenotype and genotype showed that clostridium ramosum , bacteroides vulgates , and the enterobacteriaceae were mainly capable of dnra . these species are representative for the dominant groups of the fecal microbiota [ 31 , 32 ] . by adding the stable isotope n in the form of nitrate , the occurrence of all nitrate - reducing processes under gastrointestinal conditions was studied and quantified . high ammonium concentrations in the medium repress nitrate assimilation and enhance the use of nitrate for the dissimilatory processes . under gastrointestinal conditions nitrate seems to be completely reduced to ammonium and not to n2 gas and during this reductive process , no is produced in low concentrations . to our knowledge , further proof for no production during dnra was given by e. coli k12 , a strain capable of dnra but not denitrification ( data not shown ) . this strain produced 170 ng no - n / l under gastrointestinal conditions with low concentrations of nitrate ( 110 g no3-n / l ) . evidence for no as an intermediate in dnra would be even more decisive when n - no was measured during the n tracer study . however , equipment for n - no analyses is very specialized and not in the scope of this paper . whether n2o is an intermediate as well or the reduction product of a defense mechanism of bacteria to no is not clear . the gaseous no concentration measured in the colon ascendens ( ca ) vessel of the in vitro shime model was around 15 times higher than the no concentrations measured in the colon transversum ( ct ) and colon descendens ( cd ) . during the batch experiments incubation of the microbial suspension from the different colon vessels showed that the no concentration of the ca doubled when adding fresh medium . the addition of fresh feed medium decreased the difference in no concentrations between the colon vessels . remarkably , when removing the supernatant of the suspension , a 6-fold increase of no was seen and the difference between the colon vessels disappeared , indicating that the availability of nutrients rather than a difference in the microbial community was responsible for the differences between the colon vessels . also , a similar inhibitory effect was seen by adding the supernatant of the fecal suspension to the inoculated buffered feed medium . diluting the fecal suspension to increase no production confirmed our observation . a possible explanation for this inhibitory effect can be the presence of bile salts . about 5% of the bile salts secreted in the duodenum is available to the microbiota in the colon . to protect them , the microbiota tend to hydrolyze the bile salts but around 5% of the nonconverted bile salts can be found in human feces and will have toxic effects on the bacteria . diluting the fecal suspension could decrease the toxic effect and thus increase the bacterial metabolism . the same explanation might be valid for the lower no concentrations in the nonbuffered compared to the buffered feed medium for the 2 lower concentrations of nitrate . as the ph drops during the nonbuffered incubations , the metabolism of the bacteria might be suppressed leading to a lower formation of no . epithelial cells are known to produce no through oxidation of l - arginine by nos [ 4 , 5 ] . although bacterial origin was considered , the no was believed to originate from the mucosa . several years later , no concentrations were measured in the rectum that was not cleared from luminal bacteria and again no concentrations in patients with active ibd were elevated . our results corroborate that the fecal microbiota actively produce substantial levels of no , even from traces of nitrate . in vitro the fecal microbiota no no was found in the rectum of healthy volunteers but 40 ng no - n / l was measured in the rectum of patients with active ibd . externally added no ( in the form of gas or no donor ) has been shown to alter the mucosal barrier function [ 4749 ] and to have an effect on the metabolism of colonocytes . as it was strongly suggested that several constitutive and/or inducible defense systems exist in mammalian cells that neutralize the damaging effects of no , the high rectal levels of no in ibd patients and the low levels in healthy persons might indicate that the expression of these defense mechanisms in ibd patients is ineffective . in this work , the human fecal microbiota were shown to produce no from trace levels of nitrate under gastrointestinal conditions . moreover , our data indicate that no was produced by dnra and not by denitrification or the l - arginine pathway . studying the interaction between bacterially produced no and the host epithelial cells might be pivotal in the etiology of inflammatory diseases of the gastrointestinal tract .

the free radical nitric oxide ( no ) is an important signaling molecule in the gastrointestinal tract . besides eukaryotic cells , gut microorganisms are also capable of producing no . however , the exact mechanism of no production by the gut microorganisms is unknown . microbial no production was examined under in vitro conditions simulating the gastrointestinal ecosystem using l - arginine or nitrate as substrates . l - arginine did not influence the microbial no production . however , no concentrations in the order of 90 ng no - n per l feed medium were produced by the fecal microbiota from nitrate . 15n tracer experiments showed that nitrate was mainly reduced to ammonium by the dissimilatory nitrate reduction to ammonium ( dnra ) pathway . to our knowledge , this is the first study showing that gastrointestinal microbiota can generate substantial amounts of no by dnra and not by the generally accepted denitrification or l - arginine pathway . further work is needed to elucidate the exact role between no produced by the gastrointestinal microbiota and host cells .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

nitric oxide ( no ) , a free radical with moderate reactivity , has emerged as an important signaling molecule in a multitude of physiological systems in the human body . in the gastrointestinal tract , no is involved in the regulation of regional blood flow , smooth muscle relaxation , secretory and immunological regulation . during inflammation the production of no rectal no concentrations in healthy volunteers ( range 325 ppbv ) are significantly lower than the concentrations in patients with active inflammatory bowel disease ( ibd ) ( range 718978 ppbv ) . no can be synthesized in eukaryotic cells through oxidation of l - arginine by no synthase ( nos ) [ 4 , 5 ] . besides eukaryotic no production , it is well known that no can be produced also by microorganisms . a first way of microbial no synthesis can be achieved from l - arginine by bacterial nos ( bnos ) with features resembling the eukaryotic nos ( euknos ) . besides l - arginine , nitrate and nitrite can also serve as an n - source for no . in vitro studies showed a correlation between the concentration of the added nitrate or nitrite and the concentration of no produced by the fecal microbiota . the metabolic process best known for the microbial production of no , and extensively described in soils , sediments and water treatment plants , is denitrification . during the denitrification process nitrate or nitrite is reduced to n2 gas with no and n2o as intermediates . at steady - state conditions , the no concentrations range between 14 and 900 ng . for lactobacilli and bifidobacteria hence , acidic nonenzymatic reduction of nitrite was suggested as the predominant pathway for no production in vitro by lactic acid producing bacteria ( lactobacilli and bifidobacteria ) as the produced organic acids decrease the ph of the agar plates to 4 . whereas no production by lactic acid bacteria was considered to be chemical , no production from nitrite by escherichia coli and salmonella typhimurium was shown to be biological . to date , the relevance and mechanism of bacterial no production under gastrointestinal conditions remain elusive . with no being produced by inducible nos in epithelial cells as a response to cytokines and bacterial products , it is of interest to know whether bacterial no production can occur under gastrointestinal conditions . the objective of our study was to unravel the mechanisms by which human fecal microbiota are able to produce no in vitro . three possible pathways were considered ( i ) l - arginine as substrate for no synthase , ( ii ) denitrification to nitrogen gas and ( iii ) dissimilatory nitrate reduction to ammonium ( dnra ) . the latter was considered as fermentative nitrate reduction is expected under gastrointestinal conditions however no relation with no production has been considered [ 26 , 27 ] . l - cysteine was added to scavenge dissolved oxygen and to lower the initial redox potential . in the figures , the results of incubations of one representative person are shown . different parameters ( growth media , concentrations of nitrate and l - arginine , ph , dilution of the fecal suspension and supernatant ) were varied to examine their effect on the production of no , as described in figure 1 . nitrate as a possible n - source for no was added in concentrations of 5 and 50 mg no3-n / l using a 14 g nano3-n / l solution . to study l - arginine as n - source , a stock solution of 3.5 g ( l - arginine - hcl)-n / l was added to final concentrations of 5 and 50 mg l - arginine - n / l . when incubations were performed in buffered feed medium , a 1 m phosphate buffer ( 88 g k2hpo4/l and 68 g kh2po4/l ) dilutions of the fecal suspension ( 1/2 , 1/10 , 1/20 , 1/200 , and 1/2000 ) were made in saline ( 8.5 g , 1 ml was inoculated in the growth medium . to study the effect of the supernatant of the fecal suspension on no production , the penicillin flasks were incubated at 37c for 24 hours while shaking at 130 rpm . to assess the production of no during dnra , escherichia coli k12 ( lmg 18221 ) ( bccm culture collection , ghent university , gent , belgium ) was grown anaerobically for 24 hours at 37c in buffered feed medium without additional nitrate . to determine the reduction of nitrate to n2 or ammonium by , respectively , denitrification or dnra , an experiment was set up as described in the section above but using a 14 g kno3-n / l ( 10 atom % ) solution to final concentrations of 5 and 50 mg no3-n / l ( 10 atom % ) . closing off the gas phase of the different colon compartments ( ascending , transverse , and descending ) upon n2 flushing allowed to study the no production in the respective colon regions separately . to test the effect of the biomass , the biomass of the suspensions was washed 3 times with saline and resuspended in feed medium . to determine the n / n ratio of the biomass , the samples were centrifuged at 1750 g for 15 minutes and the pellet was dried at 60c overnight . all no and n2o measurements were subsequently converted to concentrations present in the growth medium . the no and n2o concentrations measured in the gas phase ( ppbv ) were converted to molar concentrations using the ideal gas law pv = nrt with t = 294.65 k. taking into account [ no]g/[no]aq 20 and [ n2o]g/[n2o]aq 1.64 at equilibrium , the no and n2o concentrations in the gas phase were converted to the concentration in the liquid phase and expressed as g no - n or n2o - n per l. spss version 16.0 ( spss inc . the biological production of no with l - arginine as substrate was studied by supplementing mineral medium and feed medium with 0 , 5 , or 50 mg l - arginine - n / l . l - arginine - n / l but not inoculated with fecal suspension , served as a negative control . when l - arginine was supplemented to the mineral medium , only traces of no ( 0.28 ng no - n / l medium ) could be detected for all the l - arginine concentrations . in feed medium , the mean no concentration after 24 hours was for all the samples around 75 ng no - n / l medium . none of the samples contained nitrite in the beginning or at the end of the incubation and after 24 hours the mean ph was 6.53 0.35 . after 24 hours of incubation , the no concentrations in the mineral media remained below 1 ng no - n / l medium in all experiments ( figure 2(a ) ) . however , addition of 5 and 50 mg no3-n / l resulted in significant n2o production . to examine whether microbiological production of no and n2o was possible under gastrointestinal conditions , fecal microbiota were grown in the presence of a complex medium simulating the conditions in the colon ( figure 1 ) . the feed medium was analyzed for nitrate and a background of 0.11 mg no3-n / l was measured . after 24 hours of incubation , the ph dropped to 4.13 0.05 , 4.24 0.03 and 4.47 0.03 for 0.11 , 5 and 50 mg no3-n / l , respectively . figures 2(c ) and 2(d ) show the concentrations of no and n2o measured after 24 hours . the no concentration measured in the medium without nitrate addition was 37.0 0.3 ng no - n / l . the addition of 5 mg no3-n / l increased the no concentration slightly to 44.1 2.5 ng no - n / l medium . however the addition of 50 mg / l no3-n increased the no concentration drastically to 298 33 g no - n / l medium . as shown in figures 2(e ) and 2(f ) , the no concentrations increased slightly when nitrate was added . the concentration of n2o in buffered feed medium supplemented with 5 mg no3-n / l was comparable to the concentration measured in the nonbuffered test reported above . a n tracer study was used to link nitrate consumption to denitrification or dnra by the fecal microbiota under gastrointestinal conditions . the setup was the same as for the buffered feed medium but kno3 ( 10 atom % ) was used as n - source in 2 different concentrations , that is , 5 and 50 mg no3-n / l ( figure 1 ) . the amount of no and n2o produced per volume of feed medium was in the same range as what was found in the buffered feed medium experiment ( table 1 ) . to verify if no can be produced as a side product or as an intermediate during dnra , e. coli lmg 18221 , a strain capable of dnra but not denitrification after 24 hours , no concentrations of 170 ng no - n / l medium were measured . this no concentration was twice as high compared to no concentrations produced by the fecal cultures . figure 3(a ) shows that diluting the fecal suspension 200 times before inoculation in buffered feed medium results in a 75% increase in the no concentration . there was a difference in the no concentration related to the fecal suspension and optimal dilution . the optimal dilution ranged between 1/20 and 1/200 and the increase of no between 50 and 75% , respectively . a continuous model that simulates the gastrointestinal tract ( shime ) was used to measure no production by human gastrointestinal microbiota . the no concentrations in the headspace of the 3 colon vessels were analyzed after an incubation period of 24 hours . in the colon ascendens , 101 ng no - n / l shime suspension was measured but lower concentrations were found in the colon transversum and descendens ( 5.4 and 8.5 ng no - n / l shime suspension , respectively ) . a batch setup was performed to test whether this difference was due to ( i ) the microbial community or ( ii ) the availability of nutrients in the different colon compartments . as references , the microbial suspensions of the different colon vessels were incubated in parallel in penicillin bottles with or without the addition of fresh feed . figure 4 shows that the addition of feed increases the no concentrations at least 3 times but the difference between the first colon vessel and the other 2 was maintained . incubation of the washed biomass showed that even higher concentrations of no were produced ( ranging between 220 and 250 ng no - n / l ) and that the supernatant that was removed might have an inhibitory effect on the no production as observed above . no chemical production of no was seen when the cell - free supernatant of the shime suspension was incubated . our results demonstrate that the fecal microbiota of healthy persons are able to produce no in concentrations up to 90 ng no - n / l medium when grown in conditions simulating the colon ( figure 2(e ) ) . adding l - arginine to the feed medium inoculated with fecal microbiota of a healthy person had no effect on the no production . however , no and n2o were also formed under ph buffered conditions ( figures 2(e ) and 2(f ) ) . these data corroborate the metabolic capacity of the fecal microbiota to reduce nitrate and produce no . by using n tracer experiments , it was shown that nitrate was mainly reduced to ammonium by dnra suggesting that no was produced as a side product or intermediate of this pathway . no was detected not only in the batch tests but also in the in vitro shime model . indirect evidence from in vitro studies demonstrates that extracellular l - arginine increases the no production by euknos ( at concentrations at which the enzyme should be saturated ) , known as the l - arginine paradox [ 37 , 38 ] and some prokaryotic nos proteins have been shown to produce no in vivo [ 1417 ] . the reaction from l - arginine to no is oxygen dependent what might explain why l - arginine has no increasing effect during anaerobic growth of the fecal microbiota . therefore , we consider the arginine pathway for bacterial no production under gastrointestinal conditions as not important . in the lower parts of the gastrointestinal tract , nitrate originates from dietary products rich in nitrate , like spinach , beetroot , or fennel or simply from minor levels as present in drinking water . nitrate is reduced by the bacteria in the oral cavity or is absorbed in the small intestine . in healthy persons , levels in the order of mg no3-n / l can be estimated in the large intestine and feces were shown to contain around 0.15 mg no3-n / l . based on these data , 2 different concentrations of nitrate ( 5 and 50 mg no3-n very low concentrations of nitrate were measured in the feed medium receiving no additional nitrate ( 110 50 g no3-n / l ) , representative for the concentrations found in feces . no3-n / l very high concentrations of no ( 29.8 g no - n / l ) were measured . during the incubations , the ph dropped to a mean value of 4.24 due to the accumulation of organic acids produced by the microorganisms . in vivo , the ph increases over the different colon parts . because it was unclear whether no was produced chemically or biologically the very high no concentrations produced at low ph in feed medium containing the highest nitrate concentration were not found at neutral ph . in a study of sobko the production of no by fecal microbiota cultivated on iso - sensitest agar supplemented with nitrate was demonstrated . for the plates supplemented with 1.4 mg no3-n / l , concentrations in the range 293600 ppbv no , corresponding to 1145 ng no - n / l in the microbial suspension , were measured but a high variability was seen between different fecal samples . however , it was concluded that the fecal no generation was not solely due to acidification of the plates . we can conclude that the no concentrations found in our study , which were between 90 and 100 ng no / l for low concentrations of nitrate , are in the same order of magnitude . under anaerobic conditions , bacteria can use nitrate as n - source in nitrate assimilation and 2 dissimilatory processes , denitrification and dnra . during denitrification no is produced in the nanomolar range , without reaching toxic levels [ 21 , 22 ] . allison and macfarlane reported that dnra is the major pathway for nitrate reduction by the fecal microbiota . identification based on phenotype and genotype showed that clostridium ramosum , bacteroides vulgates , and the enterobacteriaceae were mainly capable of dnra . these species are representative for the dominant groups of the fecal microbiota [ 31 , 32 ] . by adding the stable isotope n in the form of nitrate , the occurrence of all nitrate - reducing processes under gastrointestinal conditions was studied and quantified . high ammonium concentrations in the medium repress nitrate assimilation and enhance the use of nitrate for the dissimilatory processes . under gastrointestinal conditions nitrate seems to be completely reduced to ammonium and not to n2 gas and during this reductive process , no is produced in low concentrations . to our knowledge , further proof for no production during dnra was given by e. coli k12 , a strain capable of dnra but not denitrification ( data not shown ) . this strain produced 170 ng no - n / l under gastrointestinal conditions with low concentrations of nitrate ( 110 g no3-n / l ) . however , equipment for n - no analyses is very specialized and not in the scope of this paper . whether n2o is an intermediate as well or the reduction product of a defense mechanism of bacteria to no is not clear . the gaseous no concentration measured in the colon ascendens ( ca ) vessel of the in vitro shime model was around 15 times higher than the no concentrations measured in the colon transversum ( ct ) and colon descendens ( cd ) . during the batch experiments incubation of the microbial suspension from the different colon vessels showed that the no concentration of the ca doubled when adding fresh medium . the addition of fresh feed medium decreased the difference in no concentrations between the colon vessels . remarkably , when removing the supernatant of the suspension , a 6-fold increase of no was seen and the difference between the colon vessels disappeared , indicating that the availability of nutrients rather than a difference in the microbial community was responsible for the differences between the colon vessels . also , a similar inhibitory effect was seen by adding the supernatant of the fecal suspension to the inoculated buffered feed medium . diluting the fecal suspension to increase no production confirmed our observation . about 5% of the bile salts secreted in the duodenum is available to the microbiota in the colon . the same explanation might be valid for the lower no concentrations in the nonbuffered compared to the buffered feed medium for the 2 lower concentrations of nitrate . as the ph drops during the nonbuffered incubations , the metabolism of the bacteria might be suppressed leading to a lower formation of no . epithelial cells are known to produce no through oxidation of l - arginine by nos [ 4 , 5 ] . several years later , no concentrations were measured in the rectum that was not cleared from luminal bacteria and again no concentrations in patients with active ibd were elevated . our results corroborate that the fecal microbiota actively produce substantial levels of no , even from traces of nitrate . in vitro the fecal microbiota no no was found in the rectum of healthy volunteers but 40 ng no - n / l was measured in the rectum of patients with active ibd . externally added no ( in the form of gas or no donor ) has been shown to alter the mucosal barrier function [ 4749 ] and to have an effect on the metabolism of colonocytes . as it was strongly suggested that several constitutive and/or inducible defense systems exist in mammalian cells that neutralize the damaging effects of no , the high rectal levels of no in ibd patients and the low levels in healthy persons might indicate that the expression of these defense mechanisms in ibd patients is ineffective . in this work , the human fecal microbiota were shown to produce no from trace levels of nitrate under gastrointestinal conditions . moreover , our data indicate that no was produced by dnra and not by denitrification or the l - arginine pathway . studying the interaction between bacterially produced no and the host epithelial cells might be pivotal in the etiology of inflammatory diseases of the gastrointestinal tract .

surviving out - of - hospital cardiac arrest ( ohca ) is highly dependent on time to treatment . accordingly , early cardiopulmonary resuscitation ( cpr ) and immediate post - resuscitation care are crucial . coronary angiography ( cag ) is recommended for ohca patients of presumed cardiac etiology . however , it remains elusive whether distance to an invasive heart centre affects survival in ohca patients and whether longer transportation to distant invasive heart centres is acceptable for providing specialized intervention and critical care . in denmark , a centralized strategy has been implemented for the treatment of patients with st - elevation myocardial infarction ( stemi ) . prehospital electrocardiographic diagnosis and field - triage of these patients directly to invasive heart centres for primary percutaneous coronary intervention ( primary pci ) are routine , whereby local hospitals are bypassed . it is unknown if a stemi - like approach with field - triage directly to invasive heart centres implying a longer prehospital transportation time is associated with lower mortality in patients with ohca . the purpose of the present study was to investigate if survival in ohca patients is associated with : ( i ) distance to an invasive heart centre ; ( ii ) performance of acute cag / pci within 6 h of the first contact with the health care system , and ( iii ) hospital - level - of care ( field - triage to invasive heart centre vs. admission to local hospital ) . this was a historical follow - up study of an unselected ohca population based on the danish cardiac arrest registry . the danish data protection agency ( file number 2013 - 41 - 1758 ) and the national board of health approved the study . in denmark , ethics approval is not required for register - based studies . we conducted a nationwide register - based study in denmark , which has a population of approximately 5.6 million inhabitants . the emergency telephone number ( 11 - 2 ) is used nationwide , with five regional emergency medical dispatch centres ( emdc ) providing telephone - guided cpr . the danish emergency medical service ( ems ) system is two - tiered , and operates as a double dispatch service . the first tier involves an ambulance with basic life support ( bls ) equipment including a defibrillator . the second tier consists of an anaesthetist - staffed prehospital critical care team operating a separate vehicle with the capability to provide advanced life support ( als ) and endotracheal intubation . in 2010 , a helicopter emergency medical service ( hems ) was launched with one helicopter serving the eastern part of denmark and in 2011 one more helicopter was put into action serving the western part of denmark . in emergency ohca calls , the emdc activates the ems system and the nearest available ambulance and the prehospital critical care team are dispatched . the ems treatment protocol adheres to the esc guidelines for resuscitation . a national consensus on triage of patients with ohca was established in 2013 by the danish society of cardiology , recommending acute cag in patients achieving rosc and with signs of stemi , severe ischaemia , cardiogenic shock or hemodynamic instability , need of pacing , and in patients without rosc when a cardiac cause was suspected . a lack of a consensus before 2013 implies that various referral patterns prevailed in the 5 regions during the study period . performance of acute cag / pci was only possible when patients were field - triaged directly to or transferred to one of the 5 high - volume invasive heart centres with a 24-h cag / pci service ; aarhus university hospital , aarhus ; rigshospitalet , copenhagen ; odense university hospital , odense ; aalborg university hospital , aalborg and gentofte hospital , gentofte ( until 2012 ) . in denmark , therapeutic hypothermia was gradually implemented between 2004 and 2006 in the intensive care units ( invasive heart centres and local hospitals ) . since 2006 , therapeutic hypothermia has been used universally in denmark as recommended by esc guidelines for resuscitation . patients were included whenever a clinical condition was followed by a resuscitation attempt either by a layperson or by the emts supported by the pre - hospital critical care team . the danish cardiac arrest register does not register patients with obvious late signs of death in whom resuscitation efforts are not initiated , and such patients were therefore not included in the study . furthermore , patients were excluded if their civil registration number was invalid or missing and if they were foreign citizens or had emigrated . following every resuscitation effort , the prehospital critical care team and emts completes an utstein template and a medical registration form . the specific time points for the emergency call , arrival on scene , departure from scene and arrival at the local hospital or invasive centre are registered by the emdc and the emts in a separate ems database . the following data were derived from the danish cardiac arrest register and the ems database ; date / time of cardiac arrest and ems call ; location ( private home or public ) ; address of ohca ; whether the event was witnessed by a layperson , healthcare personnel or ems ; and whether bystander cpr and/or defibrillation was performed , initially recorded rhythm [ asystole , pulseless electrical activity ( pea ) , ventricular fibrillation ( vf ) or ventricular tachycardia ( vt ) ] , time of first rhythm analysis by the ems and a status of the patient s condition upon arrival to hospital , e.g. rosc or dead . hospital level of care ( centre with acute cag / pci facilities or local hospital ) and the patient s home address were obtained from the danish national patient registry . the western denmark heart registry ( vdhd ) and the eastern denmark heart registries ( pats = patient analysis & tracking system ) databases provided individual baseline characteristics and procedure - specific information on all cags and pcis . mortality data were acquired from the danish civil registration system , which contains daily updated vital status on all danish citizens . valid individual linkages between all registries were obtained by use of the unique ten - digit civil registration number assigned to all danish citizens . the driving distance from the scene of the event to the nearest invasive centre was calculated in kilometres ( km ) using the danish geographic grid combined with the danish address web application programming interface routing machine . binary data are presented as percentages and continuous variables as medians with inter - quartile range ( iqr ) . follow - up was initiated on the date of cardiac arrest and ended on the date of death or april 27 2015 according to precedence . for temporal trends , we compared binary variables and continuous variables using non - parametric test of trend an extension of the wilcoxon rank - sum test . univariable and multivariable cox proportional hazards regression analyses were performed for calculation of hazard ratios ( hr ) , and 95% confidence intervals ( cis ) to examine the association between the covariates and mortality and to adjust for potential confounders . variables associated with mortality in the univariable cox regression analyses were included in the multivariable cox regression models . performance of cag and pci are a priori associated with improved outcome , i.e. only performed in those achieving rosc or being admitted with ongoing cpr . accordingly , in the cox regression analyses each patient were assigned a cag / pci index defined as the proportion of patients undergoing acute cag / pci ( within 6 h of the first contact with the health care system ) annually in each region to reflect the current use of cag / pci in the region where the patient lived . logistic regression modelling was used for binary covariates , ordinal logistic regression modelling was used for categorical variables with more than two values , and linear regression modelling was used for continuous covariates . data management and statistical analyses were performed using stata se 14.0 ( statacorp , college station , tx , usa ) . this was a historical follow - up study of an unselected ohca population based on the danish cardiac arrest registry . the danish data protection agency ( file number 2013 - 41 - 1758 ) and the national board of health approved the study . in denmark , ethics approval is not required for register - based studies . we conducted a nationwide register - based study in denmark , which has a population of approximately 5.6 million inhabitants . the emergency telephone number ( 11 - 2 ) is used nationwide , with five regional emergency medical dispatch centres ( emdc ) providing telephone - guided cpr . the danish emergency medical service ( ems ) system is two - tiered , and operates as a double dispatch service . the first tier involves an ambulance with basic life support ( bls ) equipment including a defibrillator . the second tier consists of an anaesthetist - staffed prehospital critical care team operating a separate vehicle with the capability to provide advanced life support ( als ) and endotracheal intubation . in 2010 , a helicopter emergency medical service ( hems ) was launched with one helicopter serving the eastern part of denmark and in 2011 one more helicopter was put into action serving the western part of denmark . in emergency ohca calls , the emdc activates the ems system and the nearest available ambulance and the prehospital critical care team are dispatched . a national consensus on triage of patients with ohca was established in 2013 by the danish society of cardiology , recommending acute cag in patients achieving rosc and with signs of stemi , severe ischaemia , cardiogenic shock or hemodynamic instability , need of pacing , and in patients without rosc when a cardiac cause was suspected . a lack of a consensus before 2013 implies that various referral patterns prevailed in the 5 regions during the study period . performance of acute cag / pci was only possible when patients were field - triaged directly to or transferred to one of the 5 high - volume invasive heart centres with a 24-h cag / pci service ; aarhus university hospital , aarhus ; rigshospitalet , copenhagen ; odense university hospital , odense ; aalborg university hospital , aalborg and gentofte hospital , gentofte ( until 2012 ) . in denmark , therapeutic hypothermia was gradually implemented between 2004 and 2006 in the intensive care units ( invasive heart centres and local hospitals ) . since 2006 , therapeutic hypothermia has been used universally in denmark as recommended by esc guidelines for resuscitation . patients were included whenever a clinical condition was followed by a resuscitation attempt either by a layperson or by the emts supported by the pre - hospital critical care team . the danish cardiac arrest register does not register patients with obvious late signs of death in whom resuscitation efforts are not initiated , and such patients were therefore not included in the study . furthermore , patients were excluded if their civil registration number was invalid or missing and if they were foreign citizens or had emigrated . following every resuscitation effort , the prehospital critical care team and emts completes an utstein template and a medical registration form . the specific time points for the emergency call , arrival on scene , departure from scene and arrival at the local hospital or invasive centre are registered by the emdc and the emts in a separate ems database . the following data were derived from the danish cardiac arrest register and the ems database ; date / time of cardiac arrest and ems call ; location ( private home or public ) ; address of ohca ; whether the event was witnessed by a layperson , healthcare personnel or ems ; and whether bystander cpr and/or defibrillation was performed , initially recorded rhythm [ asystole , pulseless electrical activity ( pea ) , ventricular fibrillation ( vf ) or ventricular tachycardia ( vt ) ] , time of first rhythm analysis by the ems and a status of the patient s condition upon arrival to hospital , e.g. rosc or dead . hospital level of care ( centre with acute cag / pci facilities or local hospital ) and the patient s home address were obtained from the danish national patient registry . the western denmark heart registry ( vdhd ) and the eastern denmark heart registries ( pats = patient analysis & tracking system ) databases provided individual baseline characteristics and procedure - specific information on all cags and pcis . mortality data were acquired from the danish civil registration system , which contains daily updated vital status on all danish citizens . valid individual linkages between all registries were obtained by use of the unique ten - digit civil registration number assigned to all danish citizens . the driving distance from the scene of the event to the nearest invasive centre was calculated in kilometres ( km ) using the danish geographic grid combined with the danish address web application programming interface routing machine . binary data are presented as percentages and continuous variables as medians with inter - quartile range ( iqr ) . follow - up was initiated on the date of cardiac arrest and ended on the date of death or april 27 2015 according to precedence . for temporal trends , we compared binary variables and continuous variables using non - parametric test of trend an extension of the wilcoxon rank - sum test . univariable and multivariable cox proportional hazards regression analyses were performed for calculation of hazard ratios ( hr ) , and 95% confidence intervals ( cis ) to examine the association between the covariates and mortality and to adjust for potential confounders . variables associated with mortality in the univariable cox regression analyses were included in the multivariable cox regression models . performance of cag and pci are a priori associated with improved outcome , i.e. only performed in those achieving rosc or being admitted with ongoing cpr . accordingly , in the cox regression analyses each patient were assigned a cag / pci index defined as the proportion of patients undergoing acute cag / pci ( within 6 h of the first contact with the health care system ) annually in each region to reflect the current use of cag / pci in the region where the patient lived . logistic regression modelling was used for binary covariates , ordinal logistic regression modelling was used for categorical variables with more than two values , and linear regression modelling was used for continuous covariates . data management and statistical analyses were performed using stata se 14.0 ( statacorp , college station , tx , usa ) . we excluded 2020 patients because they had an invalid or missing civil registration number , had emigrated or were foreign citizens . the cumulative 30-day survival was 9% ( 95% ci : 89% ) ( n = 3550 ) . the median follow - up time for patients with rosc was 28 ( 21404 ) days . a total of 8.419 ( 20% ) patients achieved rosc of which 605 ( 7% ) were conscious / awake at admission to hospital . table 1 shows geographical characteristics , patient characteristics and survival stratified according to the five danish regions . furthermore , the proportion of patients admitted directly to an invasive heart centre , acute cag / pci and 30-day survival differed . a total of 1.785 ( 21% ) patients with rosc had an acute cag performed and 1.262 ( 15% ) patients had an acute pci performed after the cag . more than 90% of the cag and pci procedures were performed within 6 h after ems call . the highest 30-day survival rates were seen in the capital region ( 10% ) and in the central denmark region ( 9% ) . table 1geographical characteristics , patient characteristics and outcome for ohca patients stratified according to regionabcdecapital region of denmarkcentral denmark regionsouth denmark regionzealand regionnorthern denmark regionp - valuepopulation size1 749 0001 271 0001 202 000816 270580 273area , km256813 05312 19172737933population density per km , official681979911273<0.001population density per km observed in the study population ( median)236613623712678<0.001population density per km observed in the study population among 30-day survivors ( median)307013627113981<0.001study population , no.15 7457751811957263845age , median ( iqr ) , years71 ( 6081)70 ( 5879)70 ( 5980)70 ( 6079)70 ( 5879)<0.001male gender , % ( n / valid cases)6366666668(9864/15 745)(5138/7751)(5344/8119)(3770/5726)(2608/3845)<0.001bystander cpr , % ( n / valid cases)3539343536(4236/12 136)(2885/7426)(2762/8040)(1952/5635)(1366/3802)<0.001distance to invasive centre , median ( iqr ) , km9 ( 517)51 ( 3082)71 ( 34122)82 ( 6199)51 ( 3068)<0.001initial shockable rhythm , % ( n / valid cases)2122212121(2851/13 624)(1629/7227)(1634/7691)(1110/5349)(773/3626)0.07witnessed arrest , % ( n / valid cases)5556545254(6786/12 276)(4242/7570)(4331/8037)(2953/5640)(2056/3799)<0.001charlson comorbidity index score , % ( n / valid cases)049 ( 7668/15 745)54 ( 4200/7751)53 ( 4325/8119)53 ( 3017/5726)55 ( 2101/3845)+151 ( 8077/15 745)46 ( 3551/7751)47 ( 3794/8119)47 ( 2709/5726)45 ( 1744/3845)<0.001previous myocardial infarction , % ( n / valid cases)1113121312(1761/15 688)(980/7745)(973/8117)(746/5726)(458/3845)0.001history of congestive heart failure , % ( n / valid cases)1412121311(2274/15 688)(933/7745)(1008/8117)(752/5726)(422/3845)<0.001previous stroke , % ( n / valid cases)107897(1577/15 688)(584/7745)(627/8117)(491/5726)(281/3845)<0.001hypertension , % ( n / valid cases)1615151616(2571/15 688)(1181/7745)(1270/8117)(928/5726)(619/3845)0.20admission to invasive centre , % ( n / valid cases)333038324(2958/8823)(1828/6059)(1981/5237)(102/3182)(504/2062)<0.001acute cag / pci in patients with rosc , % ( n / valid cases)1138321126(373/3475)(600/1594)(490/1541)(103/982)(219/827)<0.001acute pci in patients with rosc , % ( n / valid cases)1023201016(348/3475)(362/1594)(314/1541)(103/982)(135/827)<0.00130-day survival , % ( n / valid cases)109868(1515/15 745)(720/7751)(658/8119)(367/5726)(290/3845)<0.001cpr , cardiopulmonary resuscitation ; cag / pci , coronary angiography / percutaneous intervention ; rosc , return of spontaneous circulation . geographical characteristics , patient characteristics and outcome for ohca patients stratified according to region cpr , cardiopulmonary resuscitation ; cag / pci , coronary angiography / percutaneous intervention ; rosc , return of spontaneous circulation . throughout the study period , we observed a large increase in the overall 30-day survival ( 5% in 2001 , 12% in 2013 , p < 0.001 ) ( figure 1a ) . thirty - day survival rose significantly over time in patients with and without bystander cpr ( p < 0.001 ) with the largest increase being observed in those without bystander cpr ( 3% in 2001 , 10% in 2013 , p < 0.001 ) ( figure 1b ) . temporal changes in thirty - day survival stratified according to ( a ) bystander cpr , ( b ) overall 30-day survival , and ( c ) admission to invasive heart centre . a major temporal increase was also observed in the proportion of patients receiving bystander cpr ( 18% in 2001 , 60% in 2013 , p < 0.001 ) ( figure 2a ) and in the proportion of patients achieving rosc ( 10% in 2001 , 29% in 2013 ) ( figure 2b ) . bystander cpr was associated with lower mortality in the multivariable analyses , ( hr of 0.97 ( 95% ci : 0.950.99 , p = 0.005 ) ) ( table 2 ) . table 2hazard ratios of covariates associated with mortality in univariable cox regression analyses and multivariable analysis ( based on imputed datasets)covariates included in the analysesno.univariable analysisp - valuemultivariable analysisp - valuehr ( 95% ci)hr ( 95% ci)male gender41 1860.92 ( 0.910.94)<0.0010.99 ( 0.971.01)0.46age , per 10 year increase41 1861.06 ( 1.051.06)<0.0011.04 ( 1.031.05)<0.001bystander cpr37 0390.87 ( 0.850.89)<0.0010.97 ( 0.950.99)0.005non - shockable heart rhythm37 5171.68 ( 1.631.72)<0.0011.48 ( 1.431.52)<0.001witnessed ohca37 3220.79 ( 0.780.81)<0.0010.87 ( 0.850.89)<0.001comorbidity index 1 ( 0 reference)41 1861.14 ( 1.121.16)<0.0011.08 ( 1.061.11)<0.001admission to invasive centre ( reference : local hospital)25 3640.78 ( 0.760.81)<0.0010.91 ( 0.890.93)<0.001acute cag / pci per region and year ( index)41 1860.33 ( 0.250.42)<0.0010.33 ( 0.250.45)<0.001population density per zip code per km41 1860100referencereference1003000.98 ( 0.951.01)0.170.97 ( 0.951.01)0.1530020000.98 ( 0.951.00)0.100.97 ( 0.941.01)0.17200020 0000.95 ( 0.920.97)<0.0010.94 ( 0.890.98)0.007distance to invasive centre , kilometres41 18605 ( n = 5414)0.93 ( 0.890.96)<0.0011.01 ( 0.961.07)0.70510 ( n = 5749)0.97 ( 0.941.01)0.141.02 ( 0.971.08)0.371020 ( n = 4779)0.98 ( 0.941.02)0.231.00 ( 0.961.05)0.902050 ( n = 8513)0.99 ( 0.961.03)0.861.03 ( 0.991.07)0.1450100 ( n = 10 315)1.01 ( 0.981.05)0.401.02 ( 0.991.05)0.23100210 ( n = 6416)referencereferencecpr , cardiopulmonary resuscitation ; ohca , out - of - hospital cardiac arrest ; cag / pci , coronary angiography / percutaneous intervention ; rosc , return of spontaneous circulation . hazard ratios of covariates associated with mortality in univariable cox regression analyses and multivariable analysis ( based on imputed datasets ) cpr , cardiopulmonary resuscitation ; ohca , out - of - hospital cardiac arrest ; cag / pci , coronary angiography / percutaneous intervention ; rosc , return of spontaneous circulation . temporal changes in ( a ) bystander cpr ( b ) achievement of rosc ( c ) admission to invasive heart centre and ( d ) acute cag / pci in patients with ohca . cpr , cardiopulmonary resuscitation ; rosc , return of spontaneous circulation ; cag , coronary angiography ; pci , percutaneous coronary intervention ; ohca , out - of - hospital cardiac arrest . a total of 38% ( n = 15 822 ) of patients with ohca were not admitted to hospital but declared dead at the site of event . subgroup analyses revealed considerable heterogeneity between the regions with regard to the proportions of patients declared dead at the site of event . among the remaining patients ( n = 25 364 ) , the majority was admitted to a local hospital ( n = 17 991 ) . an increase in survival over time was observed in those admitted directly to an invasive heart centre ( figure 1c ) . however , the proportion of patients admitted directly to invasive heart centres differed significantly between regions ( figure 2c ) . admission directly to an invasive centre was independently associated with lower mortality ( adjusted hr = 0.91 ( 95% ci : 0.890.93 , p < 0.001 ) ) ( table 2 ) . among patients achieving rosc , the use of acute cag / pci increased significantly during the study period , with significant differences between the regions ( figure 2d ) . regional performance of acute cag / pci following ohca ( cag / pci index ) was associated with a lower mortality , revealing an adjusted hr of 0.33 ( 95% ci : 0.250.45 , p < 0.001 ) ( table 2 ) . the proportion of patients achieving rosc declined significantly at distances above 5 kilometres from the nearest invasive centre ( p < 0.001 ) ( figure 3a ) . increasing distance from the scene of the event to the invasive heart centre was associated with a lower proportion of patients admitted directly to an invasive heart centre ( p < 0.001 ) ( figure 3b ) and a lower use of acute cag / pci ( p = 0.005 ) ( figure 3c ) . a distance below 5 km from the site of the event to the invasive heart centre was associated with lower mortality in the univariable analysis ( unadjusted hr of 0.93 ( 95% ci : 0.890.96 , p < 0.01 ) , but not in the multivariable analysis ( adjusted hr 1.01 ( 95% ci : 0.961.07 , p = 0.70 ) ( table 2 ) . association between distance to invasive heart centre and ( a ) achievement of rosc , ( b ) direct admission to invasive heart centre , ( c ) acute cag / pci , and ( d ) 30-day survival . cpr , cardiopulmonary resuscitation ; rosc , return of spontaneous circulation ; cag , coronary angiography ; pci , percutaneous coronary intervention . the population density determined from the zip code of the site of the event the capital region of denmark had the highest population density and the shortest distance to an invasive centre ( table 1 ) . in the univariable analyses , a population density above 2000 per zip code per m was associated with lower mortality ( unadjusted hr of 0.95 ( 95% ci : 0.920.97 , p < 0.001 ) ( table 2 ) . population density remained independently associated with lower mortality , corresponding to an adjusted hr of 0.94 ( 95% ci : 0.890.98 , p < throughout the study period , we observed a large increase in the overall 30-day survival ( 5% in 2001 , 12% in 2013 , p < 0.001 ) ( figure 1a ) . thirty - day survival rose significantly over time in patients with and without bystander cpr ( p < 0.001 ) with the largest increase being observed in those without bystander cpr ( 3% in 2001 , 10% in 2013 , p < 0.001 ) ( figure 1b ) . temporal changes in thirty - day survival stratified according to ( a ) bystander cpr , ( b ) overall 30-day survival , and ( c ) admission to invasive heart centre . a major temporal increase was also observed in the proportion of patients receiving bystander cpr ( 18% in 2001 , 60% in 2013 , p < 0.001 ) ( figure 2a ) and in the proportion of patients achieving rosc ( 10% in 2001 , 29% in 2013 ) ( figure 2b ) . bystander cpr was associated with lower mortality in the multivariable analyses , ( hr of 0.97 ( 95% ci : 0.950.99 , p = 0.005 ) ) ( table 2 ) . table 2hazard ratios of covariates associated with mortality in univariable cox regression analyses and multivariable analysis ( based on imputed datasets)covariates included in the analysesno.univariable analysisp - valuemultivariable analysisp - valuehr ( 95% ci)hr ( 95% ci)male gender41 1860.92 ( 0.910.94)<0.0010.99 ( 0.971.01)0.46age , per 10 year increase41 1861.06 ( 1.051.06)<0.0011.04 ( 1.031.05)<0.001bystander cpr37 0390.87 ( 0.850.89)<0.0010.97 ( 0.950.99)0.005non - shockable heart rhythm37 5171.68 ( 1.631.72)<0.0011.48 ( 1.431.52)<0.001witnessed ohca37 3220.79 ( 0.780.81)<0.0010.87 ( 0.850.89)<0.001comorbidity index 1 ( 0 reference)41 1861.14 ( 1.121.16)<0.0011.08 ( 1.061.11)<0.001admission to invasive centre ( reference : local hospital)25 3640.78 ( 0.760.81)<0.0010.91 ( 0.890.93)<0.001acute cag / pci per region and year ( index)41 1860.33 ( 0.250.42)<0.0010.33 ( 0.250.45)<0.001population density per zip code per km41 1860100referencereference1003000.98 ( 0.951.01)0.170.97 ( 0.951.01)0.1530020000.98 ( 0.951.00)0.100.97 ( 0.941.01)0.17200020 0000.95 ( 0.920.97)<0.0010.94 ( 0.890.98)0.007distance to invasive centre , kilometres41 18605 ( n = 5414)0.93 ( 0.890.96)<0.0011.01 ( 0.961.07)0.70510 ( n = 5749)0.97 ( 0.941.01)0.141.02 ( 0.971.08)0.371020 ( n = 4779)0.98 ( 0.941.02)0.231.00 ( 0.961.05)0.902050 ( n = 8513)0.99 ( 0.961.03)0.861.03 ( 0.991.07)0.1450100 ( n = 10 315)1.01 ( 0.981.05)0.401.02 ( 0.991.05)0.23100210 ( n = 6416)referencereferencecpr , cardiopulmonary resuscitation ; ohca , out - of - hospital cardiac arrest ; cag / pci , coronary angiography / percutaneous intervention ; rosc , return of spontaneous circulation . hazard ratios of covariates associated with mortality in univariable cox regression analyses and multivariable analysis ( based on imputed datasets ) cpr , cardiopulmonary resuscitation ; ohca , out - of - hospital cardiac arrest ; cag / pci , coronary angiography / percutaneous intervention ; rosc , return of spontaneous circulation . temporal changes in ( a ) bystander cpr ( b ) achievement of rosc ( c ) admission to invasive heart centre and ( d ) acute cag / pci in patients with ohca . cpr , cardiopulmonary resuscitation ; rosc , return of spontaneous circulation ; cag , coronary angiography ; pci , percutaneous coronary intervention ; ohca , out - of - hospital cardiac arrest . a total of 38% ( n = 15 822 ) of patients with ohca were not admitted to hospital but declared dead at the site of event . subgroup analyses revealed considerable heterogeneity between the regions with regard to the proportions of patients declared dead at the site of event . among the remaining patients ( n = 25 364 ) , the majority was admitted to a local hospital ( n = 17 991 ) . an increase in survival over time was observed in those admitted directly to an invasive heart centre ( figure 1c ) . however , the proportion of patients admitted directly to invasive heart centres differed significantly between regions ( figure 2c ) . admission directly to an invasive centre was independently associated with lower mortality ( adjusted hr = 0.91 ( 95% ci : 0.890.93 , p < 0.001 ) ) ( table 2 ) . among patients achieving rosc , the use of acute cag / pci increased significantly during the study period , with significant differences between the regions ( figure 2d ) . regional performance of acute cag / pci following ohca ( cag / pci index ) was associated with a lower mortality , revealing an adjusted hr of 0.33 ( 95% ci : 0.250.45 , p < 0.001 ) ( table 2 ) . the proportion of patients achieving rosc declined significantly at distances above 5 kilometres from the nearest invasive centre ( p < 0.001 ) ( figure 3a ) . increasing distance from the scene of the event to the invasive heart centre was associated with a lower proportion of patients admitted directly to an invasive heart centre ( p < 0.001 ) ( figure 3b ) and a lower use of acute cag / pci ( p = 0.005 ) ( figure 3c ) . a distance below 5 km from the site of the event to the invasive heart centre was associated with lower mortality in the univariable analysis ( unadjusted hr of 0.93 ( 95% ci : 0.890.96 , p < 0.01 ) , but not in the multivariable analysis ( adjusted hr 1.01 ( 95% ci : 0.961.07 , p = 0.70 ) ( table 2 ) . association between distance to invasive heart centre and ( a ) achievement of rosc , ( b ) direct admission to invasive heart centre , ( c ) acute cag / pci , and ( d ) 30-day survival . cpr , cardiopulmonary resuscitation ; rosc , return of spontaneous circulation ; cag , coronary angiography ; pci , percutaneous coronary intervention . the population density determined from the zip code of the site of the event was incorporated in the analyses for each patient . the capital region of denmark had the highest population density and the shortest distance to an invasive centre ( table 1 ) . in the univariable analyses , a population density above 2000 per zip code per m was associated with lower mortality ( unadjusted hr of 0.95 ( 95% ci : 0.920.97 , p < population density remained independently associated with lower mortality , corresponding to an adjusted hr of 0.94 ( 95% ci : 0.890.98 , p < 0.001 ) ( table 2 ) . this nationwide study is currently the largest to investigate the association between ohca patients survival and distance to invasive heart centre , performance of acute cag / pci and hospital - level of care . the three main results of the study were , first , that admission to an invasive heart centre and regional use of acute cag / pci were associated with improved 30-day survival ; second , distance from the site of the event to the invasive heart centre was not associated with survival ; third , survival was associated with population density , bystander cpr , witnessed arrest and shockable rhythm . these results support a strategy that prioritizes the establishment of an efficient prehospital organization over the establishment of multiple geographically distributed invasive heart centres . few studies have assessed the association between distance to hospital and survival in ohca patients , and all of these studies were based on selected study populations small in number . we expected that a longer distance from the site of event to an invasive heart centre would be associated with a lower survival in ohca patients . nevertheless , a higher survival rate was seen among ohca patients only within 5 kilometres of the invasive heart centre . more importantly , in multivariable analyses the distance from the site of event to the nearest invasive heart centre was not associated with survival . our results reflect that successful prehospital cpr is the key to survival , regardless of whether it is provided by a bystander or ems , which should ideally be independent of the distance to the invasive centre . however , one would still expect an association between distance and outcome in those treated with pci , consistent with the findings in stemi patients . most likely , a larger sample size is needed to prove this association in ohca patients . however , several small observational studies have assessed this issue using the actual performance of cag / pci at patient level . it is unsurprising that a strong association then appears simply because cag / pci is performed mainly in patients achieving rosc , with the few exceptions of selected patients transferred to acute cag / pci during ongoing cpr . in the present study , we used an index for cag / pci activity defined by the proportion of patients having acute cag / pci performed annually in each region . this index accordingly reflects the regional level of care with regard to the performance of acute cag / pci , and it was significantly associated with improved survival . whether admission to an invasive heart centre is a surrogate for a higher level of care , performance of acute cag / pci or both , our results underline the importance of a centralized post - resuscitation care strategy in ohca patients . this is in consensus with the statement from the european association for percutaneous cardiovascular interventions ( eapci)/stent for life ( sfl ) groups as well as recent esc guidelines for resuscitation . it is hardly surprising that population density is associated with survival ; though , the present study is the first to implement population density in a 12-year survival analysis among ohca patients . our results are consistent with those of previous studies documenting that survival is higher among ohca patients in crowded areas , e.g. airports , than in rural areas . . we can not rule out that population density is , in part , a proxy for the region and a proxy for distance to the invasive heart centre . accordingly , one would assume that the region with the highest population density and the shortest distance to an invasive heart centre , i.e. the capital region of denmark ( a ) , would perform significantly better than the other regions . nonetheless , the survival rates were comparable in three other regions ( b , c , e ) with significantly lower population densities and longer distances to the nearest invasive heart centre . one explanation may be that the longer distance and lower population density were counter - balanced by a significantly higher rate of acute cag / pci in those regions ( b , c , e ) . hence , if the association between rate of acute cag / pci and survival is causal , a more frequent use of acute cag / pci in the capital region of denmark should produce an even higher survival rate in the future . this is , of course , entirely speculative , and it remains to be evaluated in a randomized study whether or not the association between acute cag / pci and survival is causal . however , the increase in bystander cpr has partially been used to explain the overall increase in survival . the novelty of our study is that the relative increase in survival was even higher among those not receiving bystander cpr and that the overall number of patients achieving rosc rose . more importantly , our results indicate that the improved bystander cpr rate is not the only reason for improved survival following ohca , which is in accordance with a previous study by chan et al . whether improved survival observed in patients admitted directly to invasive heart centres is caused by increased use of acute cag / pci , therapeutic hypothermia , overall advanced care or selection bias nevertheless , the totality of components available for optimization by high - volume invasive heart centres represents an argument in favour of centralized post - resuscitation care in ohca patients . the strength of this study is the combination of large registries and databases , by far the largest study conducted to date in denmark in ohca patients , providing a detailed description of the association between the distance from the site of the event to an invasive heart centre , acute cag / pci , hospital level - of - care and survival in patients with ohca . first , it is observational in nature and therefore we can not rule out some element of selection bias . in this context , the decision to discontinue a resuscitation attempt as well as the decision to transport the patient to an invasive heart centre or a local hospital was left at the discretion of the anaesthetist . however , due to the fact that this was an unselected ohca population , it is important to emphasize that the potential benefits of centralization may be even better in a population of ohca patients of presumed cardiac origin . further , the non - randomized study design does not allow the demonstration of any causal relationship between survival and hospital - level - of care as well as acute cag / pci . second , it would have been interesting if ecg data were available , which would enable stratified analyses in patients with and without st - elevations . however , several studies are questioning the value of the ecg in the early post - arrest course as regard to the stemi diagnosis and patients triage for acute cag / pci . furthermore , important factors about cpr quality , no - flow time ( interval between the arrest and the onset of basic life support ) and low - flow time ( interval between the arrest and rosc ) were not taken into account , due to lack of data . additionally , data on advanced outcome measurements as target temperature hypothermia and the cerebral performance category score were not available . thus , it was not possible to take these factors into account and this must be considered as an important limitation , even though the evidence to support hypothermia treatment is sparse . mild hypothermia is standard care in comatose survivors after cardiac arrest with shockable rhythm and has been extrapolated to survivors with non - shockable rhythm . however , few randomised trials have reported improved outcomes with mild hypothermia compared with normothermia in ohca patients . recently , the ttm - trial found no difference in overall mortality or cpc score at a targeted temperature of 33 c vs. 36 c . third , the site of ohca was unavailable in 23% of the cases and therefore substituted by the home address when calculating the distance from scene of event to the invasive centre . however , approximately 80% of ohca occur at home , indicating that the distance calculation was correct in 95% of cases ( 0.77 + 0.23 * 0.8 ) . finally , a number of patients had missing data , but comparing estimates from the observed dataset with estimates from the imputed datasets did not significantly change our results . the strength of this study is the combination of large registries and databases , by far the largest study conducted to date in denmark in ohca patients , providing a detailed description of the association between the distance from the site of the event to an invasive heart centre , acute cag / pci , hospital level - of - care and survival in patients with ohca . first , it is observational in nature and therefore we can not rule out some element of selection bias . in this context , the decision to discontinue a resuscitation attempt as well as the decision to transport the patient to an invasive heart centre or a local hospital was left at the discretion of the anaesthetist . however , due to the fact that this was an unselected ohca population , it is important to emphasize that the potential benefits of centralization may be even better in a population of ohca patients of presumed cardiac origin . further , the non - randomized study design does not allow the demonstration of any causal relationship between survival and hospital - level - of care as well as acute cag / pci . second , it would have been interesting if ecg data were available , which would enable stratified analyses in patients with and without st - elevations . however , several studies are questioning the value of the ecg in the early post - arrest course as regard to the stemi diagnosis and patients triage for acute cag / pci . furthermore , important factors about cpr quality , no - flow time ( interval between the arrest and the onset of basic life support ) and low - flow time ( interval between the arrest and rosc ) were not taken into account , due to lack of data . additionally , data on advanced outcome measurements as target temperature hypothermia and the cerebral performance category score were not available . thus , it was not possible to take these factors into account and this must be considered as an important limitation , even though the evidence to support hypothermia treatment is sparse . mild hypothermia is standard care in comatose survivors after cardiac arrest with shockable rhythm and has been extrapolated to survivors with non - shockable rhythm . however , few randomised trials have reported improved outcomes with mild hypothermia compared with normothermia in ohca patients . recently , the ttm - trial found no difference in overall mortality or cpc score at a targeted temperature of 33 c vs. 36 c . third , the site of ohca was unavailable in 23% of the cases and therefore substituted by the home address when calculating the distance from scene of event to the invasive centre . however , approximately 80% of ohca occur at home , indicating that the distance calculation was correct in 95% of cases ( 0.77 + 0.23 * 0.8 ) . finally , a number of patients had missing data , but comparing estimates from the observed dataset with estimates from the imputed datasets did not significantly change our results . immediate admission to an invasive heart centre and acute cag / pci are associated with improved survival in patients with ohca , whereas distance from site of the event to the invasive heart centre was not . our results support the establishment of few high - volume invasive heart centres and suggest that ohca patients should field - triage directly to these centres for optimal post - resuscitation care , regardless of the distance . clinical randomized studies are needed to clarify the causality between acute cag / pci and survival . the danish heart foundation , the savvaerksejer foundation and the danish cardiac arrest registry supported this study . the danish cardiac arrest registry is supported by trygfonden and has no commercial interests in the field of ohca . the study founders had no role in the design and conduct of the study ; collection , management , analysis , and interpretation of the data ; and preparation , review , or approval of the manuscript , or the decision to submit the manuscript for publication .

aimsto evaluate whether the distance from the site of event to an invasive heart centre , acute coronary angiography ( cag)/percutaneous coronary intervention ( pci ) and hospital - level of care ( invasive heart centre vs. local hospital ) is associated with survival in out - of - hospital cardiac arrest ( ohca ) patients.methods and resultsnationwide historical follow - up study of 41 186 unselected ohca patients , in whom resuscitation was attempted between 2001 and 2013 , identified through the danish cardiac arrest registry . we observed an increase in the proportion of patients receiving bystander cpr ( 18% in 2001 , 60% in 2013 , p < 0.001 ) , achieving return of spontaneous circulation ( rosc ) ( 10% in 2001 , 29% in 2013 , p < 0.001 ) and being admitted directly to an invasive centre ( 26% in 2001 , 45% in 2013 , p < 0.001 ) . simultaneously , 30-day survival rose from 5% in 2001 to 12% in 2013 , p < 0.001 . among patients achieving rosc , a larger proportion underwent acute cag / pci ( 5% in 2001 , 27% in 2013 , p < 0.001 ) . the proportion of patients undergoing acute cag / pci annually in each region was defined as the cag / pci index . the following variables were associated with lower mortality in multivariable analyses : direct admission to invasive heart centre ( hr 0.91 , 95% ci : 0.890.93 ) , cag / pci index ( hr 0.33 , 95% ci : 0.250.45 ) , population density above 2000 per square kilometre ( hr 0.94 , 95% ci : 0.890.98 ) , bystander cpr ( hr 0.97 , 95% ci : 0.950.99 ) and witnessed ohca ( hr 0.87 , 95% ci : 0.850.89 ) , whereas distance to the nearest invasive centre was not associated with survival.conclusionadmission to an invasive heart centre and regional performance of acute cag / pci were associated with improved survival in ohca patients , whereas distance to the invasive centre was not . these results support a centralized strategy for immediate post - resuscitation care in ohca patients .

Introduction Methods Design Setting Population Registries Distance to invasive heart centres Statistical analyses Results Temporal changes in 30-day survival, bystander CPR and ROSC Admission to invasive centre and performance of acute CAG and PCI Return of spontaneous circulation, hospital level-of-care, acute CAG/PCI and 30-day survival according to distance from the site of event to the invasive heart centre Population density and 30-day survival Discussion Strengths and limitations Conclusion Funding

the purpose of the present study was to investigate if survival in ohca patients is associated with : ( i ) distance to an invasive heart centre ; ( ii ) performance of acute cag / pci within 6 h of the first contact with the health care system , and ( iii ) hospital - level - of care ( field - triage to invasive heart centre vs. admission to local hospital ) . accordingly , in the cox regression analyses each patient were assigned a cag / pci index defined as the proportion of patients undergoing acute cag / pci ( within 6 h of the first contact with the health care system ) annually in each region to reflect the current use of cag / pci in the region where the patient lived . accordingly , in the cox regression analyses each patient were assigned a cag / pci index defined as the proportion of patients undergoing acute cag / pci ( within 6 h of the first contact with the health care system ) annually in each region to reflect the current use of cag / pci in the region where the patient lived . furthermore , the proportion of patients admitted directly to an invasive heart centre , acute cag / pci and 30-day survival differed . table 1geographical characteristics , patient characteristics and outcome for ohca patients stratified according to regionabcdecapital region of denmarkcentral denmark regionsouth denmark regionzealand regionnorthern denmark regionp - valuepopulation size1 749 0001 271 0001 202 000816 270580 273area , km256813 05312 19172737933population density per km , official681979911273<0.001population density per km observed in the study population ( median)236613623712678<0.001population density per km observed in the study population among 30-day survivors ( median)307013627113981<0.001study population , no.15 7457751811957263845age , median ( iqr ) , years71 ( 6081)70 ( 5879)70 ( 5980)70 ( 6079)70 ( 5879)<0.001male gender , % ( n / valid cases)6366666668(9864/15 745)(5138/7751)(5344/8119)(3770/5726)(2608/3845)<0.001bystander cpr , % ( n / valid cases)3539343536(4236/12 136)(2885/7426)(2762/8040)(1952/5635)(1366/3802)<0.001distance to invasive centre , median ( iqr ) , km9 ( 517)51 ( 3082)71 ( 34122)82 ( 6199)51 ( 3068)<0.001initial shockable rhythm , % ( n / valid cases)2122212121(2851/13 624)(1629/7227)(1634/7691)(1110/5349)(773/3626)0.07witnessed arrest , % ( n / valid cases)5556545254(6786/12 276)(4242/7570)(4331/8037)(2953/5640)(2056/3799)<0.001charlson comorbidity index score , % ( n / valid cases)049 ( 7668/15 745)54 ( 4200/7751)53 ( 4325/8119)53 ( 3017/5726)55 ( 2101/3845)+151 ( 8077/15 745)46 ( 3551/7751)47 ( 3794/8119)47 ( 2709/5726)45 ( 1744/3845)<0.001previous myocardial infarction , % ( n / valid cases)1113121312(1761/15 688)(980/7745)(973/8117)(746/5726)(458/3845)0.001history of congestive heart failure , % ( n / valid cases)1412121311(2274/15 688)(933/7745)(1008/8117)(752/5726)(422/3845)<0.001previous stroke , % ( n / valid cases)107897(1577/15 688)(584/7745)(627/8117)(491/5726)(281/3845)<0.001hypertension , % ( n / valid cases)1615151616(2571/15 688)(1181/7745)(1270/8117)(928/5726)(619/3845)0.20admission to invasive centre , % ( n / valid cases)333038324(2958/8823)(1828/6059)(1981/5237)(102/3182)(504/2062)<0.001acute cag / pci in patients with rosc , % ( n / valid cases)1138321126(373/3475)(600/1594)(490/1541)(103/982)(219/827)<0.001acute pci in patients with rosc , % ( n / valid cases)1023201016(348/3475)(362/1594)(314/1541)(103/982)(135/827)<0.00130-day survival , % ( n / valid cases)109868(1515/15 745)(720/7751)(658/8119)(367/5726)(290/3845)<0.001cpr , cardiopulmonary resuscitation ; cag / pci , coronary angiography / percutaneous intervention ; rosc , return of spontaneous circulation . throughout the study period , we observed a large increase in the overall 30-day survival ( 5% in 2001 , 12% in 2013 , p < 0.001 ) ( figure 1a ) . thirty - day survival rose significantly over time in patients with and without bystander cpr ( p < 0.001 ) with the largest increase being observed in those without bystander cpr ( 3% in 2001 , 10% in 2013 , p < 0.001 ) ( figure 1b ) . a major temporal increase was also observed in the proportion of patients receiving bystander cpr ( 18% in 2001 , 60% in 2013 , p < 0.001 ) ( figure 2a ) and in the proportion of patients achieving rosc ( 10% in 2001 , 29% in 2013 ) ( figure 2b ) . bystander cpr was associated with lower mortality in the multivariable analyses , ( hr of 0.97 ( 95% ci : 0.950.99 , p = 0.005 ) ) ( table 2 ) . table 2hazard ratios of covariates associated with mortality in univariable cox regression analyses and multivariable analysis ( based on imputed datasets)covariates included in the analysesno.univariable analysisp - valuemultivariable analysisp - valuehr ( 95% ci)hr ( 95% ci)male gender41 1860.92 ( 0.910.94)<0.0010.99 ( 0.971.01)0.46age , per 10 year increase41 1861.06 ( 1.051.06)<0.0011.04 ( 1.031.05)<0.001bystander cpr37 0390.87 ( 0.850.89)<0.0010.97 ( 0.950.99)0.005non - shockable heart rhythm37 5171.68 ( 1.631.72)<0.0011.48 ( 1.431.52)<0.001witnessed ohca37 3220.79 ( 0.780.81)<0.0010.87 ( 0.850.89)<0.001comorbidity index 1 ( 0 reference)41 1861.14 ( 1.121.16)<0.0011.08 ( 1.061.11)<0.001admission to invasive centre ( reference : local hospital)25 3640.78 ( 0.760.81)<0.0010.91 ( 0.890.93)<0.001acute cag / pci per region and year ( index)41 1860.33 ( 0.250.42)<0.0010.33 ( 0.250.45)<0.001population density per zip code per km41 1860100referencereference1003000.98 ( 0.951.01)0.170.97 ( 0.951.01)0.1530020000.98 ( 0.951.00)0.100.97 ( 0.941.01)0.17200020 0000.95 ( 0.920.97)<0.0010.94 ( 0.890.98)0.007distance to invasive centre , kilometres41 18605 ( n = 5414)0.93 ( 0.890.96)<0.0011.01 ( 0.961.07)0.70510 ( n = 5749)0.97 ( 0.941.01)0.141.02 ( 0.971.08)0.371020 ( n = 4779)0.98 ( 0.941.02)0.231.00 ( 0.961.05)0.902050 ( n = 8513)0.99 ( 0.961.03)0.861.03 ( 0.991.07)0.1450100 ( n = 10 315)1.01 ( 0.981.05)0.401.02 ( 0.991.05)0.23100210 ( n = 6416)referencereferencecpr , cardiopulmonary resuscitation ; ohca , out - of - hospital cardiac arrest ; cag / pci , coronary angiography / percutaneous intervention ; rosc , return of spontaneous circulation . hazard ratios of covariates associated with mortality in univariable cox regression analyses and multivariable analysis ( based on imputed datasets ) cpr , cardiopulmonary resuscitation ; ohca , out - of - hospital cardiac arrest ; cag / pci , coronary angiography / percutaneous intervention ; rosc , return of spontaneous circulation . admission directly to an invasive centre was independently associated with lower mortality ( adjusted hr = 0.91 ( 95% ci : 0.890.93 , p < 0.001 ) ) ( table 2 ) . regional performance of acute cag / pci following ohca ( cag / pci index ) was associated with a lower mortality , revealing an adjusted hr of 0.33 ( 95% ci : 0.250.45 , p < 0.001 ) ( table 2 ) . the proportion of patients achieving rosc declined significantly at distances above 5 kilometres from the nearest invasive centre ( p < 0.001 ) ( figure 3a ) . increasing distance from the scene of the event to the invasive heart centre was associated with a lower proportion of patients admitted directly to an invasive heart centre ( p < 0.001 ) ( figure 3b ) and a lower use of acute cag / pci ( p = 0.005 ) ( figure 3c ) . a distance below 5 km from the site of the event to the invasive heart centre was associated with lower mortality in the univariable analysis ( unadjusted hr of 0.93 ( 95% ci : 0.890.96 , p < 0.01 ) , but not in the multivariable analysis ( adjusted hr 1.01 ( 95% ci : 0.961.07 , p = 0.70 ) ( table 2 ) . association between distance to invasive heart centre and ( a ) achievement of rosc , ( b ) direct admission to invasive heart centre , ( c ) acute cag / pci , and ( d ) 30-day survival . in the univariable analyses , a population density above 2000 per zip code per m was associated with lower mortality ( unadjusted hr of 0.95 ( 95% ci : 0.920.97 , p < 0.001 ) ( table 2 ) . population density remained independently associated with lower mortality , corresponding to an adjusted hr of 0.94 ( 95% ci : 0.890.98 , p < throughout the study period , we observed a large increase in the overall 30-day survival ( 5% in 2001 , 12% in 2013 , p < 0.001 ) ( figure 1a ) . thirty - day survival rose significantly over time in patients with and without bystander cpr ( p < 0.001 ) with the largest increase being observed in those without bystander cpr ( 3% in 2001 , 10% in 2013 , p < 0.001 ) ( figure 1b ) . a major temporal increase was also observed in the proportion of patients receiving bystander cpr ( 18% in 2001 , 60% in 2013 , p < 0.001 ) ( figure 2a ) and in the proportion of patients achieving rosc ( 10% in 2001 , 29% in 2013 ) ( figure 2b ) . bystander cpr was associated with lower mortality in the multivariable analyses , ( hr of 0.97 ( 95% ci : 0.950.99 , p = 0.005 ) ) ( table 2 ) . table 2hazard ratios of covariates associated with mortality in univariable cox regression analyses and multivariable analysis ( based on imputed datasets)covariates included in the analysesno.univariable analysisp - valuemultivariable analysisp - valuehr ( 95% ci)hr ( 95% ci)male gender41 1860.92 ( 0.910.94)<0.0010.99 ( 0.971.01)0.46age , per 10 year increase41 1861.06 ( 1.051.06)<0.0011.04 ( 1.031.05)<0.001bystander cpr37 0390.87 ( 0.850.89)<0.0010.97 ( 0.950.99)0.005non - shockable heart rhythm37 5171.68 ( 1.631.72)<0.0011.48 ( 1.431.52)<0.001witnessed ohca37 3220.79 ( 0.780.81)<0.0010.87 ( 0.850.89)<0.001comorbidity index 1 ( 0 reference)41 1861.14 ( 1.121.16)<0.0011.08 ( 1.061.11)<0.001admission to invasive centre ( reference : local hospital)25 3640.78 ( 0.760.81)<0.0010.91 ( 0.890.93)<0.001acute cag / pci per region and year ( index)41 1860.33 ( 0.250.42)<0.0010.33 ( 0.250.45)<0.001population density per zip code per km41 1860100referencereference1003000.98 ( 0.951.01)0.170.97 ( 0.951.01)0.1530020000.98 ( 0.951.00)0.100.97 ( 0.941.01)0.17200020 0000.95 ( 0.920.97)<0.0010.94 ( 0.890.98)0.007distance to invasive centre , kilometres41 18605 ( n = 5414)0.93 ( 0.890.96)<0.0011.01 ( 0.961.07)0.70510 ( n = 5749)0.97 ( 0.941.01)0.141.02 ( 0.971.08)0.371020 ( n = 4779)0.98 ( 0.941.02)0.231.00 ( 0.961.05)0.902050 ( n = 8513)0.99 ( 0.961.03)0.861.03 ( 0.991.07)0.1450100 ( n = 10 315)1.01 ( 0.981.05)0.401.02 ( 0.991.05)0.23100210 ( n = 6416)referencereferencecpr , cardiopulmonary resuscitation ; ohca , out - of - hospital cardiac arrest ; cag / pci , coronary angiography / percutaneous intervention ; rosc , return of spontaneous circulation . hazard ratios of covariates associated with mortality in univariable cox regression analyses and multivariable analysis ( based on imputed datasets ) cpr , cardiopulmonary resuscitation ; ohca , out - of - hospital cardiac arrest ; cag / pci , coronary angiography / percutaneous intervention ; rosc , return of spontaneous circulation . admission directly to an invasive centre was independently associated with lower mortality ( adjusted hr = 0.91 ( 95% ci : 0.890.93 , p < 0.001 ) ) ( table 2 ) . regional performance of acute cag / pci following ohca ( cag / pci index ) was associated with a lower mortality , revealing an adjusted hr of 0.33 ( 95% ci : 0.250.45 , p < 0.001 ) ( table 2 ) . the proportion of patients achieving rosc declined significantly at distances above 5 kilometres from the nearest invasive centre ( p < 0.001 ) ( figure 3a ) . increasing distance from the scene of the event to the invasive heart centre was associated with a lower proportion of patients admitted directly to an invasive heart centre ( p < 0.001 ) ( figure 3b ) and a lower use of acute cag / pci ( p = 0.005 ) ( figure 3c ) . a distance below 5 km from the site of the event to the invasive heart centre was associated with lower mortality in the univariable analysis ( unadjusted hr of 0.93 ( 95% ci : 0.890.96 , p < 0.01 ) , but not in the multivariable analysis ( adjusted hr 1.01 ( 95% ci : 0.961.07 , p = 0.70 ) ( table 2 ) . association between distance to invasive heart centre and ( a ) achievement of rosc , ( b ) direct admission to invasive heart centre , ( c ) acute cag / pci , and ( d ) 30-day survival . in the univariable analyses , a population density above 2000 per zip code per m was associated with lower mortality ( unadjusted hr of 0.95 ( 95% ci : 0.920.97 , p < population density remained independently associated with lower mortality , corresponding to an adjusted hr of 0.94 ( 95% ci : 0.890.98 , p < 0.001 ) ( table 2 ) . this nationwide study is currently the largest to investigate the association between ohca patients survival and distance to invasive heart centre , performance of acute cag / pci and hospital - level of care . the three main results of the study were , first , that admission to an invasive heart centre and regional use of acute cag / pci were associated with improved 30-day survival ; second , distance from the site of the event to the invasive heart centre was not associated with survival ; third , survival was associated with population density , bystander cpr , witnessed arrest and shockable rhythm . we expected that a longer distance from the site of event to an invasive heart centre would be associated with a lower survival in ohca patients . more importantly , in multivariable analyses the distance from the site of event to the nearest invasive heart centre was not associated with survival . whether admission to an invasive heart centre is a surrogate for a higher level of care , performance of acute cag / pci or both , our results underline the importance of a centralized post - resuscitation care strategy in ohca patients . whether improved survival observed in patients admitted directly to invasive heart centres is caused by increased use of acute cag / pci , therapeutic hypothermia , overall advanced care or selection bias nevertheless , the totality of components available for optimization by high - volume invasive heart centres represents an argument in favour of centralized post - resuscitation care in ohca patients . the strength of this study is the combination of large registries and databases , by far the largest study conducted to date in denmark in ohca patients , providing a detailed description of the association between the distance from the site of the event to an invasive heart centre , acute cag / pci , hospital level - of - care and survival in patients with ohca . the strength of this study is the combination of large registries and databases , by far the largest study conducted to date in denmark in ohca patients , providing a detailed description of the association between the distance from the site of the event to an invasive heart centre , acute cag / pci , hospital level - of - care and survival in patients with ohca . immediate admission to an invasive heart centre and acute cag / pci are associated with improved survival in patients with ohca , whereas distance from site of the event to the invasive heart centre was not .

patients were selected consecutively at the outpatient clinic for dysphagia at the department of otorhinolaryngology , head and neck surgery and at the maastro clinic in the academic hospital , both part of maastricht university medical center ( mumc ) . were recruited by phone after having studied their medical records . to be included in the sample , a patient must have been diagnosed by a laryngologist as having oropharyngeal dysphagia due to oncological disorders . the selected patients received verbal information about the study and were included in the sample only after giving their informed consent . in total , 76 patients were included in the study : 57 ( 75% ) men and 19 ( 25% ) women , ranging in age from 45 to 83 years . the status of the oral feeding restrictions was scored using the functional oral intake scale ( fois ) of crary et al . . two subjects were tube dependent while all other subjects were on a totally oral diet . the latter took various forms : a diet of a single consistency ( n = 7 ) , one of multiple consistencies and requiring special preparation or compensation ( n = 30 ) , one not needing any special preparation but with some food limitations ( n = 28 ) , and a normal oral diet ( n = 9 ) . this study used four questionnaires : three on quality of life related to oropharyngeal dysphagia , namely , the swal - qol , the mdadi , and the dhi ; plus a simple one - item visual analog scale , the dysphagia severity scale . both the mdadi and the dhi were translated into dutch by three independent researchers ; their versions were combined by mutual consensus to form one final translation . the dysphagia severity scale needed no translation , and the swal - qol had already been translated by bogaardt et al . . the first questionnaire , the swal - qol , is considered the gold standard for determining quality of life in persons with oropharyngeal dysphagia . the minimum and maximum scores per subscale are zero and 100 , indicating an extremely impaired quality of life ( 0 ) versus no impairment ( 100 ) as experienced by the individual.table 1descriptive analysis of the md anderson dysphagia inventory ( mdadi ) , the deglutition handicap index ( dhi ) , the dysphagia severity scale , and the swal - qolquality - of - life scalerange of scalemedian ( 25;75 percentiles)nswal qol burden010063 ( 6;75)73 food selection010075 ( 25;88)71 eating duration010025 ( 0;63)71 eating desire010075 ( 27;100)72 fear010088 ( 69;100)71 sleep010075 ( 44;88)73 fatigue010058 ( 33;83)73 communication010063 ( 50;88)71 mental health010065 ( 30;90)71 social functioning010065 ( 25;92)73 symptoms010063 ( 44;77)73dhi total score012036 ( 20;46)42 emotional subscore04010 ( 2;22)46 functional subscore04012 ( 8;19)44 physical subscore04010 ( 6;16)44mdadi total score2010066 ( 51;77)74 global assessment154 ( 2;4)76 emotional subscore63020 ( 15;25)75 functional subscore52517 ( 13;21)75 physical subscore84025 ( 19;29)75dysphagia severity scale010049 ( 34;71)57lower scores indicate more severely impaired quality of life or ability to swallow ( mdadi , dysphagia severity scale , swal - qol)higher scores indicate more severely impaired quality of life ( dhi)according to chen et al . the range of scores is 0 to 100 , while using a scale of 15 . in this study the range of scores has been adjusted descriptive analysis of the md anderson dysphagia inventory ( mdadi ) , the deglutition handicap index ( dhi ) , the dysphagia severity scale , and the swal - qol lower scores indicate more severely impaired quality of life or ability to swallow ( mdadi , dysphagia severity scale , swal - qol ) higher scores indicate more severely impaired quality of life ( dhi ) according to chen et al . the range of scores is 0 to 100 , while using a scale of 15 . in this study the range of scores has been adjusted the dhi is a 30-item questionnaire on deglutition - related aspects of daily life ( 5-point rating scale : 04 ) . the questionnaire is subdivided into three domains of ten items : emotional ( psychosocial consequences ) , functional ( nutritional and respiratory consequences ) , and physical ( symptoms related to swallowing ) . the minimum scores range from zero ( indicating no handicap ) to 120 ( indicating maximum handicap ) . the mdadi consists of 20 items . besides a global assessment ( a single question ) , it comprises three subscales : the emotional subscale ( 8 items ) , the functional subscale ( 5 items ) , and the physical subscale ( 6 items ) . the global assessment refers to the individual s swallowing difficulty as it affects one s overall daily routine . the emotional , functional , and physical subscales refer to the individual s affective response to the swallowing disorder , the impact of the disorder on daily activities , and the self - perception of the swallowing difficulties , respectively . using a five - point scale ( 15 ) , the minimum total score is 20 and the maximum 100 . in the original version of the mdadi , all but two items were scored such that higher scores indicated higher functioning . in the dutch translation , thus , by adjusting the scoring of two items , low scores came to indicate low functioning and high scores high functioning . the dysphagia severity scale is a self - designed evaluation tool consisting of one visual analog scale , quantifying the severity of the swallowing disorder and the extent of impairment experienced by the patient . a score of 100 ( the maximum ) indicates normal swallowing abilities , while a score of zero indicates extreme swallowing impairment or inability to swallow . patients were asked to fill in all four questionnaires , either during their outpatient visit or when recruited by phone at home . within 2 weeks after this first measurement , all patients received by post the mdadi , the dhi , and the dysphagia severity scale for purposes of repeated measurement . the researchers made sure that all repeated measurements were sent back in time for adequate retest interval analysis , reminding patients if necessary by phone . table 2 presents a glossary of the psychometric and statistical terms used in this study . measurement properties of the mdadi and the dhi were determined and compared to the quality criteria as defined by terwee et al . .table 2glossary of psychometric and statistical termstermdefinitionconstruct validitythe extent to which a measurement corresponds to theoretical concepts ( constructs ) concerning the phenomenon under study .convergent validitythe degree to which a measure is correlated with other measures to which it is theoretically predicted to correlate . in contrast , discriminant validity describes the degree to which the measure is not similar to ( diverges from ) other measures to which it theoretically should not be similar . convergent validity and discriminant validity are variants of construct validity .correlation coefficientan index that quantifies the linear relationship between a pair of variables ( range = 1 to 1 ) , with the sign indicating the direction of the relationship and the numerical magnitude its strength . values of 1 or 1 indicate that the sample values fall on a straight line , whereas a value of zero indicates the lack of any linear relationship between the two variables .criterion validitythe extent to which the measurement correlates with an external criterion of the phenomenon under study .cronbach s the estimate of the correlation between the total score across a series of items from a rating scale and the total score that would have been obtained had a comparable series of items been employed . cronbach s is an index of internal consistency of a psychological test ranging from 0 to 1 . ( guidelines for interpretation:<0.60 , unacceptable ; 0.60 - 0.65 , minimally acceptable ; 0.70 - 0.80 , respectable ; 0.80 - 0.90 , very good ; and > 0.90 , consider shortening the scale by reducing the number of items .)factor analysisa set of statistical methods ( e.g. , maximum likelihood estimation ) for analyzing the correlations among several variables in order to estimate the number of fundamental dimensions that underlie the observed data and to describe and measure those dimensions . these underlying , unobservable , latent variables are usually known as the common factors . using exploratory factor analysis , no hypothesis about the number and kind of common factors exists prior to analysis . in the case of confirmatory factor analysis , the number of common factors has been predetermined.floor or ceiling effectthe number of respondents who achieved the lowest or highest possible score [ 10 , 11].goodness of fitthe degree of agreement between an empirically observed distribution and a mathematical or theoretical distribution .internal consistencythe extent to which items in a ( sub)scale are intercorrelated , thus measuring the same construct .intraclass correlationthe proportion of variance of an observation due to between - subject variability in the true scores of a measuring instrument .test - retest reliabilityan index of score consistency over a brief period of time ( typically several weeks ) , usually the correlation coefficient determined between administration of the test twice with a certain amount of time between administrations . glossary of psychometric and statistical terms first , the mdadi and dhi questionnaires were reviewed for possible floor and ceiling effects , noting the number of respondents who obtained the lowest or highest possible scores . next , test - retest reliability was assessed by determining intraclass correlation coefficients ( two - way random effects model , icc ) between repeated measurements on the mdadi , the dhi , and the dysphagia severity scale . confirmatory maximum likelihood ( ml ) factor analyses were performed to determine the number of ( homogeneous ) ( sub)scales in each questionnaire . in addition , by computing cronbach s coefficients , the internal - consistency reliability of the mdadi and the dhi was estimated . the associations among the four administered questionnaires plus the fois and among the subscales per instrument were determined using nonparametric spearman s correlation coefficients . ( sub)scales from the mdadi and the dhi that were supposed to measure the same concept were compared to determine construct validity ( convergent validity ) . finally , criterion validity was determined by computing nonparametric spearman s correlations between the swal - qol ( reference or gold standard ) and both the mdadi and the dhi . patients were selected consecutively at the outpatient clinic for dysphagia at the department of otorhinolaryngology , head and neck surgery and at the maastro clinic in the academic hospital , both part of maastricht university medical center ( mumc ) . were recruited by phone after having studied their medical records . to be included in the sample , a patient must have been diagnosed by a laryngologist as having oropharyngeal dysphagia due to oncological disorders . the selected patients received verbal information about the study and were included in the sample only after giving their informed consent . in total , 76 patients were included in the study : 57 ( 75% ) men and 19 ( 25% ) women , ranging in age from 45 to 83 years . the status of the oral feeding restrictions was scored using the functional oral intake scale ( fois ) of crary et al . . two subjects were tube dependent while all other subjects were on a totally oral diet . the latter took various forms : a diet of a single consistency ( n = 7 ) , one of multiple consistencies and requiring special preparation or compensation ( n = 30 ) , one not needing any special preparation but with some food limitations ( n = 28 ) , and a normal oral diet ( n = 9 ) . this study used four questionnaires : three on quality of life related to oropharyngeal dysphagia , namely , the swal - qol , the mdadi , and the dhi ; plus a simple one - item visual analog scale , the dysphagia severity scale . both the mdadi and the dhi were translated into dutch by three independent researchers ; their versions were combined by mutual consensus to form one final translation . the dysphagia severity scale needed no translation , and the swal - qol had already been translated by bogaardt et al . . the first questionnaire , the swal - qol , is considered the gold standard for determining quality of life in persons with oropharyngeal dysphagia . the minimum and maximum scores per subscale are zero and 100 , indicating an extremely impaired quality of life ( 0 ) versus no impairment ( 100 ) as experienced by the individual.table 1descriptive analysis of the md anderson dysphagia inventory ( mdadi ) , the deglutition handicap index ( dhi ) , the dysphagia severity scale , and the swal - qolquality - of - life scalerange of scalemedian ( 25;75 percentiles)nswal qol burden010063 ( 6;75)73 food selection010075 ( 25;88)71 eating duration010025 ( 0;63)71 eating desire010075 ( 27;100)72 fear010088 ( 69;100)71 sleep010075 ( 44;88)73 fatigue010058 ( 33;83)73 communication010063 ( 50;88)71 mental health010065 ( 30;90)71 social functioning010065 ( 25;92)73 symptoms010063 ( 44;77)73dhi total score012036 ( 20;46)42 emotional subscore04010 ( 2;22)46 functional subscore04012 ( 8;19)44 physical subscore04010 ( 6;16)44mdadi total score2010066 ( 51;77)74 global assessment154 ( 2;4)76 emotional subscore63020 ( 15;25)75 functional subscore52517 ( 13;21)75 physical subscore84025 ( 19;29)75dysphagia severity scale010049 ( 34;71)57lower scores indicate more severely impaired quality of life or ability to swallow ( mdadi , dysphagia severity scale , swal - qol)higher scores indicate more severely impaired quality of life ( dhi)according to chen et al . the range of scores is 0 to 100 , while using a scale of 15 . in this study the range of scores has been adjusted descriptive analysis of the md anderson dysphagia inventory ( mdadi ) , the deglutition handicap index ( dhi ) , the dysphagia severity scale , and the swal - qol lower scores indicate more severely impaired quality of life or ability to swallow ( mdadi , dysphagia severity scale , swal - qol ) higher scores indicate more severely impaired quality of life ( dhi ) according to chen et al . the range of scores is 0 to 100 , while using a scale of 15 . in this study the range of scores has been adjusted the dhi is a 30-item questionnaire on deglutition - related aspects of daily life ( 5-point rating scale : 04 ) . the questionnaire is subdivided into three domains of ten items : emotional ( psychosocial consequences ) , functional ( nutritional and respiratory consequences ) , and physical ( symptoms related to swallowing ) . the minimum scores range from zero ( indicating no handicap ) to 120 ( indicating maximum handicap ) . the mdadi consists of 20 items . besides a global assessment ( a single question ) , it comprises three subscales : the emotional subscale ( 8 items ) , the functional subscale ( 5 items ) , and the physical subscale ( 6 items ) . the global assessment refers to the individual s swallowing difficulty as it affects one s overall daily routine . the emotional , functional , and physical subscales refer to the individual s affective response to the swallowing disorder , the impact of the disorder on daily activities , and the self - perception of the swallowing difficulties , respectively . using a five - point scale ( 15 ) , the minimum total score is 20 and the maximum 100 . in the original version of the mdadi , all but two items were scored such that higher scores indicated higher functioning . in the dutch translation , thus , by adjusting the scoring of two items , low scores came to indicate low functioning and high scores high functioning . the dysphagia severity scale is a self - designed evaluation tool consisting of one visual analog scale , quantifying the severity of the swallowing disorder and the extent of impairment experienced by the patient . a score of 100 ( the maximum ) indicates normal swallowing abilities , while a score of zero indicates extreme swallowing impairment or inability to swallow . patients were asked to fill in all four questionnaires , either during their outpatient visit or when recruited by phone at home . within 2 weeks after this first measurement , all patients received by post the mdadi , the dhi , and the dysphagia severity scale for purposes of repeated measurement . the researchers made sure that all repeated measurements were sent back in time for adequate retest interval analysis , reminding patients if necessary by phone . table 2 presents a glossary of the psychometric and statistical terms used in this study . measurement properties of the mdadi and the dhi were determined and compared to the quality criteria as defined by terwee et al . .table 2glossary of psychometric and statistical termstermdefinitionconstruct validitythe extent to which a measurement corresponds to theoretical concepts ( constructs ) concerning the phenomenon under study .convergent validitythe degree to which a measure is correlated with other measures to which it is theoretically predicted to correlate . in contrast , discriminant validity describes the degree to which the measure is not similar to ( diverges from ) other measures to which it theoretically should not be similar . convergent validity and discriminant validity are variants of construct validity .correlation coefficientan index that quantifies the linear relationship between a pair of variables ( range = 1 to 1 ) , with the sign indicating the direction of the relationship and the numerical magnitude its strength . values of 1 or 1 indicate that the sample values fall on a straight line , whereas a value of zero indicates the lack of any linear relationship between the two variables .criterion validitythe extent to which the measurement correlates with an external criterion of the phenomenon under study .cronbach s the estimate of the correlation between the total score across a series of items from a rating scale and the total score that would have been obtained had a comparable series of items been employed . cronbach s is an index of internal consistency of a psychological test ranging from 0 to 1 . ( guidelines for interpretation:<0.60 , unacceptable ; 0.60 - 0.65 , minimally acceptable ; 0.70 - 0.80 , respectable ; 0.80 - 0.90 , very good ; and > 0.90 , consider shortening the scale by reducing the number of items .)factor analysisa set of statistical methods ( e.g. , maximum likelihood estimation ) for analyzing the correlations among several variables in order to estimate the number of fundamental dimensions that underlie the observed data and to describe and measure those dimensions . these underlying , unobservable , latent variables are usually known as the common factors . using exploratory factor analysis , no hypothesis about the number and kind of common factors exists prior to analysis . in the case of confirmatory factor analysis , the number of common factors has been predetermined.floor or ceiling effectthe number of respondents who achieved the lowest or highest possible score [ 10 , 11].goodness of fitthe degree of agreement between an empirically observed distribution and a mathematical or theoretical distribution .internal consistencythe extent to which items in a ( sub)scale are intercorrelated , thus measuring the same construct .intraclass correlationthe proportion of variance of an observation due to between - subject variability in the true scores of a measuring instrument .test - retest reliabilityan index of score consistency over a brief period of time ( typically several weeks ) , usually the correlation coefficient determined between administration of the test twice with a certain amount of time between administrations . glossary of psychometric and statistical terms first , the mdadi and dhi questionnaires were reviewed for possible floor and ceiling effects , noting the number of respondents who obtained the lowest or highest possible scores . next , test - retest reliability was assessed by determining intraclass correlation coefficients ( two - way random effects model , icc ) between repeated measurements on the mdadi , the dhi , and the dysphagia severity scale . confirmatory maximum likelihood ( ml ) factor analyses were performed to determine the number of ( homogeneous ) ( sub)scales in each questionnaire . in addition , by computing cronbach s coefficients , the internal - consistency reliability of the mdadi and the dhi was estimated . the associations among the four administered questionnaires plus the fois and among the subscales per instrument were determined using nonparametric spearman s correlation coefficients . ( sub)scales from the mdadi and the dhi that were supposed to measure the same concept were compared to determine construct validity ( convergent validity ) . finally , criterion validity was determined by computing nonparametric spearman s correlations between the swal - qol ( reference or gold standard ) and both the mdadi and the dhi . table 1 presents the descriptive statistics for all four questionnaires . to examine a possible floor or ceiling effect , the total score of the mdadi , the total score of the dhi , and the dysphagia severity scale have been visualized by means of histograms ( fig . these figures objectify the number of respondents who obtained the lowest or highest possible scores . as less than 15% of the respondents got the lowest or highest possible score , no floor or ceiling effect was considered to be present [ 10 , 11].fig . the number of patients is displayed as a function of the total score on the mdadi . the number of patients is displayed as a function of the total score on the dhi . the number of patients is displayed as a function of the score on the dysphagia severity scale . the area under the curve equals the total number of patients a data distribution on the mdadi . the number of patients is displayed as a function of the total score on the mdadi . the number of patients is displayed as a function of the total score on the dhi . the number of patients is displayed as a function of the score on the dysphagia severity scale . the area under the curve equals the total number of patients to assess test - retest reliability , intraclass correlation coefficients ( two - way random effects model , icc ) have been determined between repeated measurements on the total scores of the mdadi and the dhi and on the dysphagia severity scale . a positive rating for reliability can be given only when the icc is at least 0.70 in a sample size of at least 50 patients . because of missing values , the actual sample sizes used for icc computation were 64 ( mdadi ) , 35 ( dhi ) , and 49 ( dysphagia severity scale ) . the reliability of the dhi could not be determined appropriately as a consequence of too little data . it describes the extent to which items in a questionnaire ( sub)scale are correlated and thus measure the same concept . for an existing theoretical model or in case the factor structure had been determined previously , confirmatory factor analysis should be applied in order to determine the number of ( homogeneous ) ( sub)scales . to that end , a confirmatory maximum likelihood ( ml ) factor analysis has been performed using all items of the mdadi to test whether three factors could be distinguished ( namely , the three subscales ) . however , this three - factor model was rejected ( goodness - of - fit test , p < 0.000 ) . a four - factor model , referring to the global assessment as a possible fourth factor , was rejected as well ( p = 0.003 ) . a confirmatory ml factor analysis using all items of the dhi and a three - factor model also called for rejection of the possibility of three underlying constructs or subscales ( goodness - of - fit test , p < 0.000 ) . still , as the subject population was rather limited , further analysis was performed to gather more information about the questionnaires psychometric properties . cronbach s was determined because it is considered an adequate measure of internal - consistency reliability . a low cronbach s ( 0.70 ) suggests a lack of correlation , whereas a high cronbach s ( > 0.90 ) indicates redundancy of one or more items [ 9 , 12 ] . cronbach s was calculated separately for each ( sub)scale of the mdadi and the dhi ( table 3 ) . all cronbach s values lie between 0.76 and 0.94 , thus indicating good internal consistency , although some redundancy may be present . considering the outcome of the factor analyses no obvious homogeneous ( sub)scales detected and adequate cronbach s values found per ( sub)scale the internal consistency of both questionnaires seems to remain unclear .table 3cronbach s per ( sub)scale of the md anderson dysphagia inventory ( mdadi ) and the deglutition handicap index ( dhi)quality - of - life scalecronbach s mdadi total score0.94 global assessmentn.a . emotional subscore0.86 functional subscore0.82 physical subscore0.87dhi total score0.93 emotional subscore0.94 functional subscore0.84 physical subscore0.76 cronbach s per ( sub)scale of the md anderson dysphagia inventory ( mdadi ) and the deglutition handicap index ( dhi ) the associations among the four patient - administered questionnaires plus the fois and among the subscales per instrument were determined by nonparametric spearman s correlation coefficients as well ( tables 4 , 5 ) . for the correlation coefficients ( r ) , a minimum value for a strong correlation was set at 0.7 [ 1315 ] . correlation coefficients between 0.3 and 0.7 were considered a substantial correlation , and r values less than 0.3 were considered a weak correlation . negative correlations are expected because all questionnaires except the dhi associate lower scores with more severely impaired quality of life or restricted functional oral intake . correlations between the quality - of - life instruments and the functional feeding status proved low ( 0.013 r 0.53 ) . construct validity could be determined by comparing the ( sub)scales from the mdadi and the dhi that were supposed to measure the same concept . associations between similar subscales from both questionnaires as well as both total scores demonstrated whether they defined the same target construct ( convergent validity ) . correlation coefficients for the emotional , functional , and physical subscales from the mdadi and the dhi were 0.93 , 0.65 , and 0.62 , respectively . the correlations between the dysphagia severity scale and both total scores from the mdadi and the dhi were rather low ( 0.45 and 0.52 , respectively ) , whereas the correlation between both total scores of the mdadi and the dhi was strong ( r = 0.87 ) . the mean correlation coefficients between the subscales of the mdadi and between the subscales of the dhi were 0.80 ( 0.66 r 0.82 ) and 0.60 ( 0.54 r 0.66 ) , respectively.table 4associations among the mdadi , the dhi , the dysphagia severity scale , and the fois ( nonparametric spearman s correlation coefficients)mdadidhidysphagia severity scalefoistotal scoreglobal assessmentemotional subscorefunctional subscorephysical subscoretotal scoreemotional subscorefunctional subscorephysical subscoremdadi total score0.75 * * ( 74)0.94 * * ( 74)0.92 * * ( 74)0.91 * * ( 74)0.87 * * ( 41)0.89 * * ( 44)0.70 * * ( 43)0.57 * * ( 42)0.45 * * ( 57)0.53 * * ( 74 ) global assessment0.68 * * ( 75)0.72 * * ( 75)0.66 * * ( 75)0.72 * * ( 42)0.65 * * ( 46)0.64 * * ( 44)0.65 * * ( 44)0.57 * * ( 57)0.44 * * ( 76 ) emotional subscore0.81 * * ( 74)0.82 * * ( 74)0.85 * * ( 41)0.93 * * ( 45)0.63 * * ( 43)0.52 * * ( 43)0.43 * * ( 57)0.46 * * ( 75 ) functional subscore0.77 * * ( 75)0.82 * * ( 41)0.86 * * ( 45)0.65 * * ( 44)0.54 * * ( 43)0.34 * * ( 57)0.53 * * ( 75 ) physical subscore0.82 * * ( 41)0.75 * * ( 45)0.78 * * ( 44)0.62 * * ( 43)0.44 * * ( 57)0.45 * * ( 75)dhi total score0.89 * * ( 42)0.84 * * ( 42)0.78 * * ( 42)0.52 * * ( 30)0.41 * * ( 42 ) emotional subscore0.60 * * ( 44)0.54 * * ( 44)0.43 * ( 31)0.13 ( 44 ) functional subscore0.66 * * ( 42)0.50 * * ( 31)0.36 * ( 44 ) physical subscore0.45 * * ( 32)0.40 * * ( 46)dysphagia severity scale0.38 * * ( 57)fois * correlation is significant at the 0.01 level ( 2-tailed ) * * correlation is significant at the 0.05 level ( 2-tailed)lower scores indicate more severely impaired oral intaketable 5associations among the swal - qol versus the mdadi , the dhi , the dysphagia severity scale , and the fois ( nonparametric spearman s correlation coefficients)swal - qolmdadidhidysphagia severity scalefoistotal scoreglobal assessmentemotional subscorefunctional subscorephysical subscoretotal scoreemotional subscorefunctional subscorephysical subscoreburden0.84 * * ( 71)0.69 * * ( 73)0.79 * * ( 72)0.79 * * ( 72)0.78 * * ( 72)0.68 * * ( 39)0.77 * * ( 43)0.54 * * ( 41)0.46 * * ( 41)0.54 * * ( 55)0.50 * * ( 73)food selection0.77 * * ( 69)0.67 * * ( 71)0.68 * * ( 70)0.80 * * ( 70)0.78 * * ( 70)0.69 * * ( 38)0.68 * * ( 42)0.69 * * ( 40)0.51 * * ( 40)0.42 * * ( 54)0.40 * * ( 71)eating duration0.70 * * ( 69)0.57 * * ( 71)0.63 * * ( 70)0.66 * * ( 70)0.72 * * ( 70)0.70 * * ( 39)0.63 * * ( 43)0.69 * * ( 41)0.40 * ( 41)0.38 * * ( 55)0.41 * * ( 71)eating desire0.71 * * ( 70)0.56 * * ( 72)0.66 * * ( 71)0.68 * * ( 71)0.73 * * ( 71)0.70 * * ( 39)0.70 * * ( 43)0.64 * * ( 41)0.31 * ( 41)0.32 * ( 55)0.38 * * ( 72)fear0.57 * * ( 69)0.58 * * ( 71)0.52 * * ( 70)0.49 * * ( 70)0.59 * * ( 70)0.38 * ( 37)0.42 * * ( 41)0.32 * ( 39)0.30 ( 39)0.34 * ( 53)0.31 * * ( 71)sleep0.39 * * ( 71)0.36 * * ( 73)0.31 * * ( 72)0.47 * * ( 72)0.42 * * ( 72)0.47 * * ( 39)0.47 * * ( 43)0.40 * * ( 41)0.35 * ( 41)0.12 ( 55)0.26 * ( 73)fatigue0.46 * * ( 71)0.43 * * ( 73)0.36 * * ( 72)0.46 * * ( 72)0.53 * * ( 72)0.42 * * ( 39)0.30 * ( 43)0.58 * * ( 41)0.41 * * ( 41)0.25 ( 55)0.21 ( 73)communication0.63 * * ( 69)0.63 * * ( 71)0.52 * * ( 70)0.61 * * ( 70)0.61 * * ( 70)0.48 * * ( 37)0.46 * * ( 41)0.36 * ( 39)0.47 * * ( 39)0.34 * ( 53)0.42 * * ( 71)mental health0.86 * * ( 69)0.72 * * ( 71)0.82 * * ( 70)0.83 * * ( 70)0.80 * * ( 70)0.80 * * ( 37)0.85 * * ( 41)0.63 * * ( 39)0.49 * * ( 39)0.42 * * ( 53)0.48 * * ( 71)social functioning0.85 * * ( 71)0.73 * * ( 73)0.76 * * ( 72)0.90 * * ( 72)0.75 * * ( 72)0.78 * * ( 39)0.84 * * ( 43)0.62 * * ( 41)0.49 * * ( 41)0.43 * * ( 55)0.61 * * ( 73)symptoms0.62 * * ( 71)0.66 * * ( 73)0.53 * * ( 72)0.58 * * ( 72)0.61 * * ( 72)0.60 * * ( 39)-0.54 * * ( 43)0.51 * * ( 41)0.73 * * ( 41)0.41 * * ( 55)0.33 * * ( 73 ) * correlation is significant at the 0.01 level ( 2-tailed ) * * correlation is significant at the 0.05 level ( 2-tailed)lower scores indicate more severely impaired oral intake associations among the mdadi , the dhi , the dysphagia severity scale , and the fois ( nonparametric spearman s correlation coefficients ) * correlation is significant at the 0.01 level ( 2-tailed ) * * correlation is significant at the 0.05 level ( 2-tailed ) lower scores indicate more severely impaired oral intake associations among the swal - qol versus the mdadi , the dhi , the dysphagia severity scale , and the fois ( nonparametric spearman s correlation coefficients ) * correlation is significant at the 0.01 level ( 2-tailed ) * * correlation is significant at the 0.05 level ( 2-tailed ) lower scores indicate more severely impaired oral intake when considering the swal - qol as the reference standard or gold standard , the extent to which the mdadi and the dhi agreed or correlated with the swal - qol could be defined as the questionnaires criterion validity . table 5 presents the associations among the swal - qol versus the mdadi , the dhi , the dysphagia severity index , and the fois ( nonparametric spearman s correlation coefficients ) . the mean correlation coefficients for the subscales from the swal - qol versus the total score of the mdadi , the total score of the dhi , and the dysphagia severity scale were 0.67 ( 0.39 r 0.86 ) , 0.61 ( 0.38 r 0.80 ) , and 0.36 ( 0.30 r 0.73 ) , respectively . next , based on the authors clinical experience , subscales that were considered to be of lesser importance to oropharyngeal dysphagia were excluded by mutual consensus . thus , when excluding the subscales fear , sleep , fatigue , and communication , the mean correlation coefficients as determined for this restricted group of subscales were 0.76 ( 0.62 r 0.86 ) , 0.71 ( 0.60 r 0.80 ) , and 0.42 ( 0.31 r 0.73 ) , respectively . according to terwee et al . , the correlation with the reference standard needs to be at least 0.70 . only after having excluded the less relevant subscales of the swal - qol did both the mdadi and the dhi show satisfactory associations with the reference standard . in this study , the psychometric characteristics for the mdadi and the dhi have been determined . the dysphagia severity scale was introduced to reveal any advantages or disadvantages of using elaborate questionnaires compared to using a simple visual analog scale , while the swal - qol was considered the reference or gold standard . none of the quality - of - life questionnaires showed any floor or ceiling effect . the test - retest reliability of the mdadi and the dysphagia severity scale proved to be good . however , because too much data were missing for the dhi , its test - retest reliability could not be determined , although the intraclass correlation coefficients were rather high . however , when applying confirmatory factor analysis , the underlying constructs as defined by the subscales per questionnaire could not be distinguished . probably because of unclear constructs , only the two emotional subscales were strongly correlated , whereas the associations between the other corresponding subscales were just moderate . overall , the dysphagia severity scale showed rather low correlations with the other three questionnaires . it seemed that a detailed questionnaire could not be replaced by a single one - item scale quantifying the severity of the swallowing disorder . when considering the criterion validity , the mdadi and the dhi showed satisfactory associations with the swal - qol after having removed its less relevant subscales . considering both the mdadi and the dhi , it is concluded that neither of these two questionnaires will generate perfect psychometric data . while striving to use questionnaires with the most optimal properties , the ultimate choice will be made by future researchers themselves . depending on the purposes of their studies , they may choose the somewhat elaborate swal - qol or one of the other two questionnaires with reasonable ( though not perfect ) psychometric characteristics . in conclusion , when assessing the validity and reliability of the dutch version of the mdadi and the dhi , not all criteria for psychometric properties have been adequately met . in general , the importance of determining these characteristics and of objectifying concepts such as validity and reliability must be stressed when developing a questionnaire . if a questionnaire s quality proves to be poor , the study results can not be interpreted correctly nor can any clinical relevance be determined . therefore , it is recommended that future outcome studies should use only quality - of - life questionnaires that have sufficiently good psychometric characteristics .

quality of life is an important outcome measurement in objectifying the current health status or therapy effects in patients with oropharyngeal dysphagia . in this study , the validity and reliability of the dutch version of the deglutition handicap index ( dhi ) and the md anderson dysphagia inventory ( mdadi ) have been determined for oncological patients with oropharyngeal dysphagia . at maastricht university medical center , 76 consecutive patients were selected and asked to fill in three questionnaires on quality of life related to oropharyngeal dysphagia ( the swal - qol , the mdadi , and the dhi ) as well as a simple one - item visual analog dysphagia severity scale . none of the quality - of - life questionnaires showed any floor or ceiling effect . the test - retest reliability of the mdadi and the dysphagia severity scale proved to be good . the test - retest reliability of the dhi could not be determined because of insufficient data , but the intraclass correlation coefficients were rather high . the internal consistency proved to be good . however , confirmatory factor analysis could not distinguish the underlying constructs as defined by the subscales per questionnaire . when assessing criterion validity , both the mdadi and the dhi showed satisfactory associations with the swal - qol ( reference or gold standard ) after having removed the less relevant subscales of the swal - qol . in conclusion , when assessing the validity and reliability of the dutch version of the dhi or the mdadi , not all psychometric properties have been adequately met . in general , because of difficulties in the interpretation of study results when using questionnaires lacking sufficient psychometric quality , it is recommended that researchers strive to use questionnaires with the most optimal psychometric properties .

Methods Subjects Questionnaires Protocol Statistical Analysis Results Discussion Conclusions

this study used four questionnaires : three on quality of life related to oropharyngeal dysphagia , namely , the swal - qol , the mdadi , and the dhi ; plus a simple one - item visual analog scale , the dysphagia severity scale . the first questionnaire , the swal - qol , is considered the gold standard for determining quality of life in persons with oropharyngeal dysphagia . the minimum and maximum scores per subscale are zero and 100 , indicating an extremely impaired quality of life ( 0 ) versus no impairment ( 100 ) as experienced by the individual.table 1descriptive analysis of the md anderson dysphagia inventory ( mdadi ) , the deglutition handicap index ( dhi ) , the dysphagia severity scale , and the swal - qolquality - of - life scalerange of scalemedian ( 25;75 percentiles)nswal qol burden010063 ( 6;75)73 food selection010075 ( 25;88)71 eating duration010025 ( 0;63)71 eating desire010075 ( 27;100)72 fear010088 ( 69;100)71 sleep010075 ( 44;88)73 fatigue010058 ( 33;83)73 communication010063 ( 50;88)71 mental health010065 ( 30;90)71 social functioning010065 ( 25;92)73 symptoms010063 ( 44;77)73dhi total score012036 ( 20;46)42 emotional subscore04010 ( 2;22)46 functional subscore04012 ( 8;19)44 physical subscore04010 ( 6;16)44mdadi total score2010066 ( 51;77)74 global assessment154 ( 2;4)76 emotional subscore63020 ( 15;25)75 functional subscore52517 ( 13;21)75 physical subscore84025 ( 19;29)75dysphagia severity scale010049 ( 34;71)57lower scores indicate more severely impaired quality of life or ability to swallow ( mdadi , dysphagia severity scale , swal - qol)higher scores indicate more severely impaired quality of life ( dhi)according to chen et al . in this study the range of scores has been adjusted descriptive analysis of the md anderson dysphagia inventory ( mdadi ) , the deglutition handicap index ( dhi ) , the dysphagia severity scale , and the swal - qol lower scores indicate more severely impaired quality of life or ability to swallow ( mdadi , dysphagia severity scale , swal - qol ) higher scores indicate more severely impaired quality of life ( dhi ) according to chen et al . in the case of confirmatory factor analysis , the number of common factors has been predetermined.floor or ceiling effectthe number of respondents who achieved the lowest or highest possible score [ 10 , 11].goodness of fitthe degree of agreement between an empirically observed distribution and a mathematical or theoretical distribution .internal consistencythe extent to which items in a ( sub)scale are intercorrelated , thus measuring the same construct .intraclass correlationthe proportion of variance of an observation due to between - subject variability in the true scores of a measuring instrument .test - retest reliabilityan index of score consistency over a brief period of time ( typically several weeks ) , usually the correlation coefficient determined between administration of the test twice with a certain amount of time between administrations . next , test - retest reliability was assessed by determining intraclass correlation coefficients ( two - way random effects model , icc ) between repeated measurements on the mdadi , the dhi , and the dysphagia severity scale . finally , criterion validity was determined by computing nonparametric spearman s correlations between the swal - qol ( reference or gold standard ) and both the mdadi and the dhi . this study used four questionnaires : three on quality of life related to oropharyngeal dysphagia , namely , the swal - qol , the mdadi , and the dhi ; plus a simple one - item visual analog scale , the dysphagia severity scale . the first questionnaire , the swal - qol , is considered the gold standard for determining quality of life in persons with oropharyngeal dysphagia . the minimum and maximum scores per subscale are zero and 100 , indicating an extremely impaired quality of life ( 0 ) versus no impairment ( 100 ) as experienced by the individual.table 1descriptive analysis of the md anderson dysphagia inventory ( mdadi ) , the deglutition handicap index ( dhi ) , the dysphagia severity scale , and the swal - qolquality - of - life scalerange of scalemedian ( 25;75 percentiles)nswal qol burden010063 ( 6;75)73 food selection010075 ( 25;88)71 eating duration010025 ( 0;63)71 eating desire010075 ( 27;100)72 fear010088 ( 69;100)71 sleep010075 ( 44;88)73 fatigue010058 ( 33;83)73 communication010063 ( 50;88)71 mental health010065 ( 30;90)71 social functioning010065 ( 25;92)73 symptoms010063 ( 44;77)73dhi total score012036 ( 20;46)42 emotional subscore04010 ( 2;22)46 functional subscore04012 ( 8;19)44 physical subscore04010 ( 6;16)44mdadi total score2010066 ( 51;77)74 global assessment154 ( 2;4)76 emotional subscore63020 ( 15;25)75 functional subscore52517 ( 13;21)75 physical subscore84025 ( 19;29)75dysphagia severity scale010049 ( 34;71)57lower scores indicate more severely impaired quality of life or ability to swallow ( mdadi , dysphagia severity scale , swal - qol)higher scores indicate more severely impaired quality of life ( dhi)according to chen et al . in this study the range of scores has been adjusted descriptive analysis of the md anderson dysphagia inventory ( mdadi ) , the deglutition handicap index ( dhi ) , the dysphagia severity scale , and the swal - qol lower scores indicate more severely impaired quality of life or ability to swallow ( mdadi , dysphagia severity scale , swal - qol ) higher scores indicate more severely impaired quality of life ( dhi ) according to chen et al . next , test - retest reliability was assessed by determining intraclass correlation coefficients ( two - way random effects model , icc ) between repeated measurements on the mdadi , the dhi , and the dysphagia severity scale . finally , criterion validity was determined by computing nonparametric spearman s correlations between the swal - qol ( reference or gold standard ) and both the mdadi and the dhi . to examine a possible floor or ceiling effect , the total score of the mdadi , the total score of the dhi , and the dysphagia severity scale have been visualized by means of histograms ( fig . the area under the curve equals the total number of patients to assess test - retest reliability , intraclass correlation coefficients ( two - way random effects model , icc ) have been determined between repeated measurements on the total scores of the mdadi and the dhi and on the dysphagia severity scale . considering the outcome of the factor analyses no obvious homogeneous ( sub)scales detected and adequate cronbach s values found per ( sub)scale the internal consistency of both questionnaires seems to remain unclear .table 3cronbach s per ( sub)scale of the md anderson dysphagia inventory ( mdadi ) and the deglutition handicap index ( dhi)quality - of - life scalecronbach s mdadi total score0.94 global assessmentn.a . emotional subscore0.86 functional subscore0.82 physical subscore0.87dhi total score0.93 emotional subscore0.94 functional subscore0.84 physical subscore0.76 cronbach s per ( sub)scale of the md anderson dysphagia inventory ( mdadi ) and the deglutition handicap index ( dhi ) the associations among the four patient - administered questionnaires plus the fois and among the subscales per instrument were determined by nonparametric spearman s correlation coefficients as well ( tables 4 , 5 ) . the mean correlation coefficients between the subscales of the mdadi and between the subscales of the dhi were 0.80 ( 0.66 r 0.82 ) and 0.60 ( 0.54 r 0.66 ) , respectively.table 4associations among the mdadi , the dhi , the dysphagia severity scale , and the fois ( nonparametric spearman s correlation coefficients)mdadidhidysphagia severity scalefoistotal scoreglobal assessmentemotional subscorefunctional subscorephysical subscoretotal scoreemotional subscorefunctional subscorephysical subscoremdadi total score0.75 * * ( 74)0.94 * * ( 74)0.92 * * ( 74)0.91 * * ( 74)0.87 * * ( 41)0.89 * * ( 44)0.70 * * ( 43)0.57 * * ( 42)0.45 * * ( 57)0.53 * * ( 74 ) global assessment0.68 * * ( 75)0.72 * * ( 75)0.66 * * ( 75)0.72 * * ( 42)0.65 * * ( 46)0.64 * * ( 44)0.65 * * ( 44)0.57 * * ( 57)0.44 * * ( 76 ) emotional subscore0.81 * * ( 74)0.82 * * ( 74)0.85 * * ( 41)0.93 * * ( 45)0.63 * * ( 43)0.52 * * ( 43)0.43 * * ( 57)0.46 * * ( 75 ) functional subscore0.77 * * ( 75)0.82 * * ( 41)0.86 * * ( 45)0.65 * * ( 44)0.54 * * ( 43)0.34 * * ( 57)0.53 * * ( 75 ) physical subscore0.82 * * ( 41)0.75 * * ( 45)0.78 * * ( 44)0.62 * * ( 43)0.44 * * ( 57)0.45 * * ( 75)dhi total score0.89 * * ( 42)0.84 * * ( 42)0.78 * * ( 42)0.52 * * ( 30)0.41 * * ( 42 ) emotional subscore0.60 * * ( 44)0.54 * * ( 44)0.43 * ( 31)0.13 ( 44 ) functional subscore0.66 * * ( 42)0.50 * * ( 31)0.36 * ( 44 ) physical subscore0.45 * * ( 32)0.40 * * ( 46)dysphagia severity scale0.38 * * ( 57)fois * correlation is significant at the 0.01 level ( 2-tailed ) * * correlation is significant at the 0.05 level ( 2-tailed)lower scores indicate more severely impaired oral intaketable 5associations among the swal - qol versus the mdadi , the dhi , the dysphagia severity scale , and the fois ( nonparametric spearman s correlation coefficients)swal - qolmdadidhidysphagia severity scalefoistotal scoreglobal assessmentemotional subscorefunctional subscorephysical subscoretotal scoreemotional subscorefunctional subscorephysical subscoreburden0.84 * * ( 71)0.69 * * ( 73)0.79 * * ( 72)0.79 * * ( 72)0.78 * * ( 72)0.68 * * ( 39)0.77 * * ( 43)0.54 * * ( 41)0.46 * * ( 41)0.54 * * ( 55)0.50 * * ( 73)food selection0.77 * * ( 69)0.67 * * ( 71)0.68 * * ( 70)0.80 * * ( 70)0.78 * * ( 70)0.69 * * ( 38)0.68 * * ( 42)0.69 * * ( 40)0.51 * * ( 40)0.42 * * ( 54)0.40 * * ( 71)eating duration0.70 * * ( 69)0.57 * * ( 71)0.63 * * ( 70)0.66 * * ( 70)0.72 * * ( 70)0.70 * * ( 39)0.63 * * ( 43)0.69 * * ( 41)0.40 * ( 41)0.38 * * ( 55)0.41 * * ( 71)eating desire0.71 * * ( 70)0.56 * * ( 72)0.66 * * ( 71)0.68 * * ( 71)0.73 * * ( 71)0.70 * * ( 39)0.70 * * ( 43)0.64 * * ( 41)0.31 * ( 41)0.32 * ( 55)0.38 * * ( 72)fear0.57 * * ( 69)0.58 * * ( 71)0.52 * * ( 70)0.49 * * ( 70)0.59 * * ( 70)0.38 * ( 37)0.42 * * ( 41)0.32 * ( 39)0.30 ( 39)0.34 * ( 53)0.31 * * ( 71)sleep0.39 * * ( 71)0.36 * * ( 73)0.31 * * ( 72)0.47 * * ( 72)0.42 * * ( 72)0.47 * * ( 39)0.47 * * ( 43)0.40 * * ( 41)0.35 * ( 41)0.12 ( 55)0.26 * ( 73)fatigue0.46 * * ( 71)0.43 * * ( 73)0.36 * * ( 72)0.46 * * ( 72)0.53 * * ( 72)0.42 * * ( 39)0.30 * ( 43)0.58 * * ( 41)0.41 * * ( 41)0.25 ( 55)0.21 ( 73)communication0.63 * * ( 69)0.63 * * ( 71)0.52 * * ( 70)0.61 * * ( 70)0.61 * * ( 70)0.48 * * ( 37)0.46 * * ( 41)0.36 * ( 39)0.47 * * ( 39)0.34 * ( 53)0.42 * * ( 71)mental health0.86 * * ( 69)0.72 * * ( 71)0.82 * * ( 70)0.83 * * ( 70)0.80 * * ( 70)0.80 * * ( 37)0.85 * * ( 41)0.63 * * ( 39)0.49 * * ( 39)0.42 * * ( 53)0.48 * * ( 71)social functioning0.85 * * ( 71)0.73 * * ( 73)0.76 * * ( 72)0.90 * * ( 72)0.75 * * ( 72)0.78 * * ( 39)0.84 * * ( 43)0.62 * * ( 41)0.49 * * ( 41)0.43 * * ( 55)0.61 * * ( 73)symptoms0.62 * * ( 71)0.66 * * ( 73)0.53 * * ( 72)0.58 * * ( 72)0.61 * * ( 72)0.60 * * ( 39)-0.54 * * ( 43)0.51 * * ( 41)0.73 * * ( 41)0.41 * * ( 55)0.33 * * ( 73 ) * correlation is significant at the 0.01 level ( 2-tailed ) * * correlation is significant at the 0.05 level ( 2-tailed)lower scores indicate more severely impaired oral intake associations among the mdadi , the dhi , the dysphagia severity scale , and the fois ( nonparametric spearman s correlation coefficients ) * correlation is significant at the 0.01 level ( 2-tailed ) * * correlation is significant at the 0.05 level ( 2-tailed ) lower scores indicate more severely impaired oral intake associations among the swal - qol versus the mdadi , the dhi , the dysphagia severity scale , and the fois ( nonparametric spearman s correlation coefficients ) * correlation is significant at the 0.01 level ( 2-tailed ) * * correlation is significant at the 0.05 level ( 2-tailed ) lower scores indicate more severely impaired oral intake when considering the swal - qol as the reference standard or gold standard , the extent to which the mdadi and the dhi agreed or correlated with the swal - qol could be defined as the questionnaires criterion validity . table 5 presents the associations among the swal - qol versus the mdadi , the dhi , the dysphagia severity index , and the fois ( nonparametric spearman s correlation coefficients ) . the mean correlation coefficients for the subscales from the swal - qol versus the total score of the mdadi , the total score of the dhi , and the dysphagia severity scale were 0.67 ( 0.39 r 0.86 ) , 0.61 ( 0.38 r 0.80 ) , and 0.36 ( 0.30 r 0.73 ) , respectively . only after having excluded the less relevant subscales of the swal - qol did both the mdadi and the dhi show satisfactory associations with the reference standard . in this study , the psychometric characteristics for the mdadi and the dhi have been determined . the dysphagia severity scale was introduced to reveal any advantages or disadvantages of using elaborate questionnaires compared to using a simple visual analog scale , while the swal - qol was considered the reference or gold standard . none of the quality - of - life questionnaires showed any floor or ceiling effect . the test - retest reliability of the mdadi and the dysphagia severity scale proved to be good . however , because too much data were missing for the dhi , its test - retest reliability could not be determined , although the intraclass correlation coefficients were rather high . however , when applying confirmatory factor analysis , the underlying constructs as defined by the subscales per questionnaire could not be distinguished . when considering the criterion validity , the mdadi and the dhi showed satisfactory associations with the swal - qol after having removed its less relevant subscales . in conclusion , when assessing the validity and reliability of the dutch version of the mdadi and the dhi , not all criteria for psychometric properties have been adequately met .

although anticoagulation with the use of warfarin may effectively reduce risk of cardioembolic stroke in patients with atrial fibrillation ( af ) , warfarin has a narrow therapeutic window . in the re - ly trial with carefully selected and monitored patients , the proportion of time in therapeutic range ( ttr ) , defined by the international normalized ratio ( inr ) of 2 to 3 , varied from 44% to 77% depending on the study or clinical center.1 hemorrhage or thrombosis due to over- or under - dosing may have devastating consequences . poor coagulation control may increase the risk of thromboembolic events2 and warfarin - related bleeding,3 and influence the size of brain infarcts.4 also recent study shows the importance of identification of the patient with atrial fibrillation who has higher risk of stroke , and administration of proper anticoagulation.5 new oral anticoagulants ( noacs ) , i.e. , dabigatran , apixaban , rivaroxaban , and edoxaban,6,7,8,9 are not inferior to warfarin in preventing ischemic stroke and systemic embolism in patients with non - valvular af , and are superior to warfarin in reducing hemorrhagic stroke . for all vascular events , non - hemorrhagic events , and mortality , advantages of noacs may be greater in the case of poor inr control than with good inr control.1 however , no consensus exists regarding the indication for use of these agents in patients with af . the quality of anticoagulation control may depend on genetic factors , notably cyp2c9 and vkorc1 polymorphisms , and on non - genetic patient - related factors , such as gender , race / ethnicity , and paroxysmal vs. permanent af . lip and colleagues recently introduced the same - tt2r2 score , a validated assessment scheme based on clinical variables , to aid in distinguishing patients with af who are likely to do well on warfarin ( having low same - tt2r2 scores ) from those who are likely to have poor anticoagulation control ( predicted by high scores).10 this score appears to provide valuable information relevant to safety and effectiveness of treatment , while avoiding the time and expense of pharmacogenetic study.11 however , the same - tt2r2 score was derived from clinical trial data and did not include all of the consecutive inr measurements . thus , in the present investigation we tested both the same - tt2r2 score and pharmacogenetic data for power to predict anticoagulation control , expressed as the ttr , and the correlation between the two predictors . data for analysis were retrieved retrospectively and included the consecutive inr measurements as well as the cyp2c9 and vkorc1 genotypes for each patient . we collected data from consecutive patients who visited a university medical center , seoul , korea . patients included in this study met the following criteria ( a ) diagnosis of non - valvular af , ( b ) eligibility for warfarin treatment to prevent thromboembolism ( i.e. chads2 score 1 ) , ( c ) genetic testing data available , and ( d ) follow - up greater than 6 months . patients were excluded for ( a ) irregular use of medications ( > 20% missed doses or > 10% extra doses),12 ( b ) active cancer ( c ) chronic alcoholism , and ( d ) chronic consumption of food , beverages , or medications that may interfere with warfarin metabolism , such as green liquor and herbal medications.13,14,15 patients with a target inr range of 1.5 to 2.5 or 2.5 to 3.5 were also excluded . the institutional review board approved this study and all participants gave informed consent before the study began . patients received long - term warfarin treatment with a target inr range of 2.0 to 3.0 . following enrollment of a patient , we obtained a medical history , performed a physical examination , and drew blood for inr measurement and cyp2c9 and vkorc1 genotyping . we contacted each patient as scheduled for an inr test , to assess warfarin dosing , and to ask about the use of any new medications . compliance with warfarin treatment was regularly monitored and patients received dietary instructions for use with warfarin therapy . variables that could potentially affect the quality of anticoagulation control were recorded for each patient . these included age , body surface area , concurrent medications , and risk scores for thrombosis ( cha2ds2-vasc)16 and hemorrhage ( atria)17 . in this study , we used the atria score for bleeding risk instead of the has - bled score because a labile inr is a key component of the has - bled risk scheme.3 the methods used for cyp2c9 and vkorc1 genotyping are explained in our previous publications.18,19 the same - tt2r2 scores were determined for each patient ( sex female , age < 60 years , medical history [ more than 2 comorbidities ] , treatment [ interacting drugs , e.g. , amiodarone for rhythm control ] , tobacco use within 2 years [ doubled ] , race nonwhite [ doubled ] ) . these factors are reportedly associated with poor control of inr in af patients on warfarin therapy ; that is , the factors predict a low value of ttr.10 medical history was defined as having more than two of the following : hypertension , diabetes , coronary artery disease / myocardial infarction , peripheral arterial disease , congestive heart failure , previous stroke , pulmonary disease , and hepatic or renal disease.10 in this study , all participants were korean . to calculate the proportion of time spent within specific inr levels , rosendaal 's method was used.20 to estimate the unknown inr values ( inrx ) between two sequential inr values , linear interpolation of inr was performed . inrx=(inrpost - inrpre)(datepost - datepre)datex - datepre+inrpre the inrpre and inrpost are inr values at datepre or datepost , respectively ( datepre < datex < datepost ) . we evaluated the relationship between cyp2c9 and vkorc1 genotype and ttr ( defined as 2.0 - 3.0 for all patients ) and measured times within specific ranges of inr . these ranges were less than 1.8 , over 4.0 , and between 2.0 and 3.0 . inr levels after one month of initiation of warfarin were used to define quality of anticoagulation control based on detection of labile inrs during warfarin maintenance . in this analysis , we excluded inrs obtained during temporary discontinuation , and during the first month after treatment was restarted . we also evaluated the relationship between the same - tt2r2 scores and ttr , and the relationships of the same - tt2r2 scores with cyp2c9 and vkorc1 genotypes . all numeric data are presented as the meansd or median ( interquartile range [ iqr ] ) . differences in clinical and laboratory parameters were evaluated by one - way anova with post hoc analysis using the fisher 's least - square difference or kruskall - wallis test for continuous variables and pearson 's chi - square , fisher 's exact test or linear by linear association for categorical variables . an independent t test or the mann- whitney u test independent factors for a patient having labile inrs were evaluated using logistic regression . in this study , a ttr of < 65% was taken to indicate a labile inr and 65% as stable inr . the institute for health and care excellence ( nice ) commissioning guide recommended that ttr of over 65% are desirable in the anticoagulation control.21 in the present study , 35% of patients had ttr 65% and 65% had ttr < 65% . among patients with ttr < 66% , the noac are reported to reduce risk of major bleeding more effectively relative to warfarin than in patients with ttr 66%.22 body surface area , warfarin dose and dose assessment interval , concurrent medications , liver disease , cha2ds2-vasc and atria scores , the same - tt2r2 score , and cyp2c9 and vkorc1 genotype all served as predictive variables . all variables with p<0.20 in the univariate analysis and variables of interest in this study ( e.g. , same - tt2r2 score ) were included in the multivariate analysis . in the final models , differences with p values<0.05 all statistical analyses were performed using spss for windows , version 18.0 ( spss inc . , we collected data from consecutive patients who visited a university medical center , seoul , korea . patients included in this study met the following criteria ( a ) diagnosis of non - valvular af , ( b ) eligibility for warfarin treatment to prevent thromboembolism ( i.e. chads2 score 1 ) , ( c ) genetic testing data available , and ( d ) follow - up greater than 6 months . patients were excluded for ( a ) irregular use of medications ( > 20% missed doses or > 10% extra doses),12 ( b ) active cancer ( c ) chronic alcoholism , and ( d ) chronic consumption of food , beverages , or medications that may interfere with warfarin metabolism , such as green liquor and herbal medications.13,14,15 patients with a target inr range of 1.5 to 2.5 or 2.5 to 3.5 were also excluded . the institutional review board approved this study and all participants gave informed consent before the study began . patients received long - term warfarin treatment with a target inr range of 2.0 to 3.0 . following enrollment of a patient , we obtained a medical history , performed a physical examination , and drew blood for inr measurement and cyp2c9 and vkorc1 genotyping . we contacted each patient as scheduled for an inr test , to assess warfarin dosing , and to ask about the use of any new medications . compliance with warfarin treatment was regularly monitored and patients received dietary instructions for use with warfarin therapy . variables that could potentially affect the quality of anticoagulation control were recorded for each patient . these included age , body surface area , concurrent medications , and risk scores for thrombosis ( cha2ds2-vasc)16 and hemorrhage ( atria)17 . in this study , we used the atria score for bleeding risk instead of the has - bled score because a labile inr is a key component of the has - bled risk scheme.3 the methods used for cyp2c9 and vkorc1 genotyping are explained in our previous publications.18,19 the same - tt2r2 scores were determined for each patient ( sex female , age < 60 years , medical history [ more than 2 comorbidities ] , treatment [ interacting drugs , e.g. , amiodarone for rhythm control ] , tobacco use within 2 years [ doubled ] , race nonwhite [ doubled ] ) . these factors are reportedly associated with poor control of inr in af patients on warfarin therapy ; that is , the factors predict a low value of ttr.10 medical history was defined as having more than two of the following : hypertension , diabetes , coronary artery disease / myocardial infarction , peripheral arterial disease , congestive heart failure , previous stroke , pulmonary disease , and hepatic or renal disease.10 in this study , all participants were korean . to calculate the proportion of time spent within specific inr levels , rosendaal 's method was used.20 to estimate the unknown inr values ( inrx ) between two sequential inr values , linear interpolation of inr was performed . inrx=(inrpost - inrpre)(datepost - datepre)datex - datepre+inrpre the inrpre and inrpost are inr values at datepre or datepost , respectively ( datepre < datex < datepost ) . we evaluated the relationship between cyp2c9 and vkorc1 genotype and ttr ( defined as 2.0 - 3.0 for all patients ) and measured times within specific ranges of inr . these ranges were less than 1.8 , over 4.0 , and between 2.0 and 3.0 . inr levels after one month of initiation of warfarin were used to define quality of anticoagulation control based on detection of labile inrs during warfarin maintenance . in this analysis , we excluded inrs obtained during temporary discontinuation , and during the first month after treatment was restarted . we also evaluated the relationship between the same - tt2r2 scores and ttr , and the relationships of the same - tt2r2 scores with cyp2c9 and vkorc1 genotypes . all numeric data are presented as the meansd or median ( interquartile range [ iqr ] ) . differences in clinical and laboratory parameters were evaluated by one - way anova with post hoc analysis using the fisher 's least - square difference or kruskall - wallis test for continuous variables and pearson 's chi - square , fisher 's exact test or linear by linear association for categorical variables . an independent t test or the mann- whitney u test independent factors for a patient having labile inrs were evaluated using logistic regression . in this study , a ttr of < 65% was taken to indicate a labile inr and 65% as stable inr . the institute for health and care excellence ( nice ) commissioning guide recommended that ttr of over 65% are desirable in the anticoagulation control.21 in the present study , 35% of patients had ttr 65% and 65% had ttr < 65% . among patients with ttr < 66% , the noac are reported to reduce risk of major bleeding more effectively relative to warfarin than in patients with ttr 66%.22 body surface area , warfarin dose and dose assessment interval , concurrent medications , liver disease , cha2ds2-vasc and atria scores , the same - tt2r2 score , and cyp2c9 and vkorc1 genotype all served as predictive variables . all variables with p<0.20 in the univariate analysis and variables of interest in this study ( e.g. , same - tt2r2 score ) were included in the multivariate analysis . in the final models , differences with p values<0.05 all statistical analyses were performed using spss for windows , version 18.0 ( spss inc . , clinical and genetic data form 380 korean patients with af were collected and the patients were followed for about 4 years ( meansd , 231.9199.7 weeks ) . mean age was 70.6 ( ranging from 22 to 90 years ) and 153 patients were female . blood sampling for warfarin dose assessment was performed at a meansd interval of 5.52.8 weeks . the same - tt2r2 score was 3.40.9 , range 2 - 7 , and 153 subjects ( 40.2% ) had same - tt2r2 scores of 4 . no scores of 0 or 1 were found in this study because the risk scheme assigns two points for non - white race classification . genotype frequencies for cyp2c9 * 3 ( rs1057910 ) and vkorc1 1173c > t ( rs9934438 ) were 9.5% and 16.3% , respectively . clinical , genetic and demographic characteristics of the patients are summarized in table 1 . for the patient group overall , irrespective of genotype , the percentage of time spent in therapeutic range was 57.5% during the entire follow - up period . carriers of vkorc1 tt spent less time in therapeutic range ( inr 2 - 3 ) than carriers of vkorc1 1173c > t ( p=0.031 ) . on the contrary , the percentages of time in specific inr ranges did not differ according to cyp2c9 isoform or same - tt2r2 score . we expected a negative correlation between the same - tt2r2 score and time in therapeutic range ( inr 2 - 3 ) , but the spearman correlation analysis showed no correlation ( r=0.11 p=0.826 ) . the distribution of the same - tt2r2 scores was similar among the different genotypes related to warfarin dosing ( figure 2 ) . multivariate testing was performed to test further for factors that may independently predict labile inr ( table 3 ) . the vkorc1 tt but none of the other factors , including the same - tt2r2 score , was significantly associated with labile inr . clinical and genetic data form 380 korean patients with af were collected and the patients were followed for about 4 years ( meansd , 231.9199.7 weeks ) . mean age was 70.6 ( ranging from 22 to 90 years ) and 153 patients were female . blood sampling for warfarin dose assessment was performed at a meansd interval of 5.52.8 weeks . the same - tt2r2 score was 3.40.9 , range 2 - 7 , and 153 subjects ( 40.2% ) had same - tt2r2 scores of 4 . no scores of 0 or 1 were found in this study because the risk scheme assigns two points for non - white race classification . genotype frequencies for cyp2c9 * 3 ( rs1057910 ) and vkorc1 1173c > t ( rs9934438 ) were 9.5% and 16.3% , respectively . for the patient group overall , irrespective of genotype , the percentage of time spent in therapeutic range was 57.5% during the entire follow - up period . carriers of vkorc1 tt spent less time in therapeutic range ( inr 2 - 3 ) than carriers of vkorc1 1173c > t ( p=0.031 ) . on the contrary , the percentages of time in specific inr ranges did not differ according to cyp2c9 isoform or same - tt2r2 score . we expected a negative correlation between the same - tt2r2 score and time in therapeutic range ( inr 2 - 3 ) , but the spearman correlation analysis showed no correlation ( r=0.11 p=0.826 ) . the distribution of the same - tt2r2 scores was similar among the different genotypes related to warfarin dosing ( figure 2 ) . multivariate testing was performed to test further for factors that may independently predict labile inr ( table 3 ) . the vkorc1 tt but none of the other factors , the main findings of this study were that ( 1 ) a genetic factor related to warfarin response , but no common clinical or demographic factor , including the same - tt2r2 score , was associated with the quality of anticoagulation control , and ( 2 ) there is poor correlation between clinical and genetic factors . the american heart and stroke association guidelines for oral antithrombotic agents for the prevention of stroke in non - valvular af23 support the use of both warfarin and noacs to prevent first and recurrent strokes in patients with non - valvular af . the guidelines recommend the individualized selection of antithrombotic agent(s ) based on risk calculations , cost , tolerability , patient preference , potential for drug interactions , and other clinical characteristics , including ttr if the patient has been taking warfarin . in determining the selection of warfarin vs. noacs , a dilemma arises in that the ttr can not be known at the time of initial treatment . as a result , it is difficult to identify the newly diagnosed patients with af who would do well on warfarin and who also have high values for time in therapeutic range . this is desirable because the main benefits of noacs compared with warfarin may be only marginal in those with high times in therapeutic range , although the reduction in intracranial hemorrhage is still evident.24 in a recent meta - analysis with data for four phase 3 clinical trials of noacs , the noacs had a favorable risk - benefit profile as compared with warfarin that was consistent across diverse patient groups.22 however , it also showed a greater relative reduction in major bleeding with noacs when ttr was less than 66% than when it was more than 66%.22 high ttr ( > 70% ) are known to be associated with the best efficacy and safety of warfarin.25 genotype - guided dosing of warfarin was recently reported to be associated with a higher percentage of time in the therapeutic inr range than standard dosing during the initiation of warfarin therapy.26 however , the relationship of warfarin pharmacogenetics to long - term ttr during warfarin maintenance is not well studied . if pharmacogenetic analysis could predict ttr at the time of the initial af diagnosis , the selection of antithrombotic agent might be clarified . in particular , those patients who have genetic variability associated with labile inr during the maintenance of warfarin could be selected for treatment with noacs . however , because polymorphism frequency varies between ethnic groups , the results of this study may not apply to other populations with different distributions of cyp2c9 and vkorc1 isoforms.27 further studies are needed to determine the clinical significance of warfarin pharmacogenetics in ethnic groups with higher prevalence of cyp2c9 variants . our findings , that clinical and demographic factors expressed in the same - tt2r2 score were not associated with quality of anticoagulation control , contradict the recent findings of dr . this contradiction may be explained in various ways . in the atrial fibrillation follow - up investigation of rhythm management ( affirm ) , the same patient cohort served in both the derivation and validation phases of the study . although the mean age ( ~70 years - old ) and mean duration of follow - up ( 3.5 - 3.9 years ) were similar between that study and ours , the baseline patient characteristics differed significantly.28 in particular , most subjects ( 89% ) in the affirm study were white , whereas all patients in our study were korean . " non - white race " represents two points from a total of eight points in the same - tt2r2 risk prediction scheme . in addition , only 13% of the affirm study subjects , but 57% of those in our study had experienced stroke or tia . the choice of noacs vs. warfarin would be particularly important in patients who have suffered stroke resulting in severe disability , because the monitoring of inr is often difficult in these patients ; and importantly , the cost of noacs may be prohibitive in developing countries , which include a large proportion of the world 's population . in this study , furthermore , patients with valvular af ( who are not candidates for noac treatment ) and those with a target inr range of 1.5 to 2.5 or 2.5 to 3.5 ( outside the inr target of 2 - 3 ) were excluded , whereas 11%-12% of both the derivation and validation groups in the affirm study had valvular af . , not all of the affirm participants received anticoagulation therapy throughout the study follow - up period and consecutive inrs were not measured . instead , patients with > 12 months of uninterrupted warfarin treatment and more than eight inr values available were included in their analysis.10 our results are in line with recent results from a danish cohort . recently , jane skov et al.29 investigated to determine whether the same - tt2r2 score can predict inr control and the population only included whites , and showed that the same - tt2r2 score was not predictive of ttr . thus , results of our and other studies indicate that further studies are needed for external validation of same tt2r2 score . the strength of the present study resides in the consecutive recruitment of patients with comprehensive evaluation of factors related to the quality of anticoagulation control , such as concurrent medications , scored risk assessments for af - related thromboembolic and hemorrhagic events , and consecutive inr measurements with exclusion of inr values during temporary treatment interruptions and during the first one month after treatment was resumed . limitations of this study include the retrospective design and residence at a single clinical center . although we can not generalize our findings in korean subjects to all other ethnic groups , our analytical approach and findings may inform clinical services for relatively large populations throughout asia , and possibly africa . further prospective studies are needed to identify clinical and genetic factors related to the quality of anticoagulation control during treatment with warfarin in asians . in conclusion , genetic factors , but not the commonly recognized clinical and demographic factors , were associated with quality of anticoagulation control in patients of asian background who had recently survived an af - related stroke . our findings emphasize the need for continuous efforts to find clinical scoring systems to reliably inform therapeutic decisions , such as the choice between warfarin and less widely available noacs . in addition , further studies are needed to test whether noacs may be particularly effective in patients who have genotypes related to inr lability during warfarin maintenance .

background and purposeadvantages of new oral anticoagulations may be greater in atrial fibrillation ( af ) patients of poor anticoagulation control with warfarin . the same - tt2r2 scoring system , based on clinical variables , was recently developed to aid in identifying these patients . in this study , we investigated the association of this clinical composite score with genetic factors related warfarin dosing and the quality of anticoagulation control.methodsclinical and genetic data were collected from 380 consecutive korean patients with af ( cha2ds2-vasc score , 3.51.8 ) who were followed for an average of 4 years . we evaluated factors associated with time in therapeutic range ( ttr , inr 2 - 3 ) , including the cyp2c9 and vkorc1 genotypes and the same - tt2r2 score ( sex female , age < 60 years , medical history [ > two co - morbidities ] , treatment [ interacting drugs , e.g. , amiodarone ] , tobacco use within 2 years [ doubled ] , and race non - white [ doubled]).resultsthe average same - tt2r2 score was 3.40.9 , range 2 - 7 ; and 153 patients ( 40.2% ) had same - tt2r2 scores 4 . time in specific inr ranges varied depending on the vkorc1 genotype but not with the cyp2c9 genotype or the same - tt2r2 score . ttr was higher in patients with the vkorc1 1173c > t than in vkorc1 tt ( 61.716% vs. 56.717.4% , p=0.031 ) . multivariate testing showed that vkorc1 genotype but not the same - tt2r2 score was significantly associated with labile inrs . there was no correlation between the same - tt2r2 scores and pharmacogenetic data.conclusionsa genetic factor , but none of the common clinical and demographic factors , as combined in the same - tt2r2 score , was associated with the quality of anticoagulation control in korean patients with af .

Introduction Methods Patient selection Work-up Quality of anticoagulation control Statistical analysis Results Patients' characteristics Factors associated with quality of anticoagulation control Discussion Conclusion

although anticoagulation with the use of warfarin may effectively reduce risk of cardioembolic stroke in patients with atrial fibrillation ( af ) , warfarin has a narrow therapeutic window . in the re - ly trial with carefully selected and monitored patients , the proportion of time in therapeutic range ( ttr ) , defined by the international normalized ratio ( inr ) of 2 to 3 , varied from 44% to 77% depending on the study or clinical center.1 hemorrhage or thrombosis due to over- or under - dosing may have devastating consequences . poor coagulation control may increase the risk of thromboembolic events2 and warfarin - related bleeding,3 and influence the size of brain infarcts.4 also recent study shows the importance of identification of the patient with atrial fibrillation who has higher risk of stroke , and administration of proper anticoagulation.5 new oral anticoagulants ( noacs ) , i.e. , dabigatran , apixaban , rivaroxaban , and edoxaban,6,7,8,9 are not inferior to warfarin in preventing ischemic stroke and systemic embolism in patients with non - valvular af , and are superior to warfarin in reducing hemorrhagic stroke . for all vascular events , non - hemorrhagic events , and mortality , advantages of noacs may be greater in the case of poor inr control than with good inr control.1 however , no consensus exists regarding the indication for use of these agents in patients with af . the quality of anticoagulation control may depend on genetic factors , notably cyp2c9 and vkorc1 polymorphisms , and on non - genetic patient - related factors , such as gender , race / ethnicity , and paroxysmal vs. permanent af . lip and colleagues recently introduced the same - tt2r2 score , a validated assessment scheme based on clinical variables , to aid in distinguishing patients with af who are likely to do well on warfarin ( having low same - tt2r2 scores ) from those who are likely to have poor anticoagulation control ( predicted by high scores).10 this score appears to provide valuable information relevant to safety and effectiveness of treatment , while avoiding the time and expense of pharmacogenetic study.11 however , the same - tt2r2 score was derived from clinical trial data and did not include all of the consecutive inr measurements . thus , in the present investigation we tested both the same - tt2r2 score and pharmacogenetic data for power to predict anticoagulation control , expressed as the ttr , and the correlation between the two predictors . data for analysis were retrieved retrospectively and included the consecutive inr measurements as well as the cyp2c9 and vkorc1 genotypes for each patient . patients included in this study met the following criteria ( a ) diagnosis of non - valvular af , ( b ) eligibility for warfarin treatment to prevent thromboembolism ( i.e. patients were excluded for ( a ) irregular use of medications ( > 20% missed doses or > 10% extra doses),12 ( b ) active cancer ( c ) chronic alcoholism , and ( d ) chronic consumption of food , beverages , or medications that may interfere with warfarin metabolism , such as green liquor and herbal medications.13,14,15 patients with a target inr range of 1.5 to 2.5 or 2.5 to 3.5 were also excluded . following enrollment of a patient , we obtained a medical history , performed a physical examination , and drew blood for inr measurement and cyp2c9 and vkorc1 genotyping . we contacted each patient as scheduled for an inr test , to assess warfarin dosing , and to ask about the use of any new medications . variables that could potentially affect the quality of anticoagulation control were recorded for each patient . in this study , we used the atria score for bleeding risk instead of the has - bled score because a labile inr is a key component of the has - bled risk scheme.3 the methods used for cyp2c9 and vkorc1 genotyping are explained in our previous publications.18,19 the same - tt2r2 scores were determined for each patient ( sex female , age < 60 years , medical history [ more than 2 comorbidities ] , treatment [ interacting drugs , e.g. , amiodarone for rhythm control ] , tobacco use within 2 years [ doubled ] , race nonwhite [ doubled ] ) . these factors are reportedly associated with poor control of inr in af patients on warfarin therapy ; that is , the factors predict a low value of ttr.10 medical history was defined as having more than two of the following : hypertension , diabetes , coronary artery disease / myocardial infarction , peripheral arterial disease , congestive heart failure , previous stroke , pulmonary disease , and hepatic or renal disease.10 in this study , all participants were korean . we evaluated the relationship between cyp2c9 and vkorc1 genotype and ttr ( defined as 2.0 - 3.0 for all patients ) and measured times within specific ranges of inr . inr levels after one month of initiation of warfarin were used to define quality of anticoagulation control based on detection of labile inrs during warfarin maintenance . in this analysis , we excluded inrs obtained during temporary discontinuation , and during the first month after treatment was restarted . we also evaluated the relationship between the same - tt2r2 scores and ttr , and the relationships of the same - tt2r2 scores with cyp2c9 and vkorc1 genotypes . in this study , a ttr of < 65% was taken to indicate a labile inr and 65% as stable inr . the institute for health and care excellence ( nice ) commissioning guide recommended that ttr of over 65% are desirable in the anticoagulation control.21 in the present study , 35% of patients had ttr 65% and 65% had ttr < 65% . among patients with ttr < 66% , the noac are reported to reduce risk of major bleeding more effectively relative to warfarin than in patients with ttr 66%.22 body surface area , warfarin dose and dose assessment interval , concurrent medications , liver disease , cha2ds2-vasc and atria scores , the same - tt2r2 score , and cyp2c9 and vkorc1 genotype all served as predictive variables . all variables with p<0.20 in the univariate analysis and variables of interest in this study ( e.g. , same - tt2r2 score ) were included in the multivariate analysis . patients included in this study met the following criteria ( a ) diagnosis of non - valvular af , ( b ) eligibility for warfarin treatment to prevent thromboembolism ( i.e. patients were excluded for ( a ) irregular use of medications ( > 20% missed doses or > 10% extra doses),12 ( b ) active cancer ( c ) chronic alcoholism , and ( d ) chronic consumption of food , beverages , or medications that may interfere with warfarin metabolism , such as green liquor and herbal medications.13,14,15 patients with a target inr range of 1.5 to 2.5 or 2.5 to 3.5 were also excluded . following enrollment of a patient , we obtained a medical history , performed a physical examination , and drew blood for inr measurement and cyp2c9 and vkorc1 genotyping . we contacted each patient as scheduled for an inr test , to assess warfarin dosing , and to ask about the use of any new medications . variables that could potentially affect the quality of anticoagulation control were recorded for each patient . in this study , we used the atria score for bleeding risk instead of the has - bled score because a labile inr is a key component of the has - bled risk scheme.3 the methods used for cyp2c9 and vkorc1 genotyping are explained in our previous publications.18,19 the same - tt2r2 scores were determined for each patient ( sex female , age < 60 years , medical history [ more than 2 comorbidities ] , treatment [ interacting drugs , e.g. , amiodarone for rhythm control ] , tobacco use within 2 years [ doubled ] , race nonwhite [ doubled ] ) . these factors are reportedly associated with poor control of inr in af patients on warfarin therapy ; that is , the factors predict a low value of ttr.10 medical history was defined as having more than two of the following : hypertension , diabetes , coronary artery disease / myocardial infarction , peripheral arterial disease , congestive heart failure , previous stroke , pulmonary disease , and hepatic or renal disease.10 in this study , all participants were korean . we evaluated the relationship between cyp2c9 and vkorc1 genotype and ttr ( defined as 2.0 - 3.0 for all patients ) and measured times within specific ranges of inr . inr levels after one month of initiation of warfarin were used to define quality of anticoagulation control based on detection of labile inrs during warfarin maintenance . in this analysis , we excluded inrs obtained during temporary discontinuation , and during the first month after treatment was restarted . we also evaluated the relationship between the same - tt2r2 scores and ttr , and the relationships of the same - tt2r2 scores with cyp2c9 and vkorc1 genotypes . in this study , a ttr of < 65% was taken to indicate a labile inr and 65% as stable inr . the institute for health and care excellence ( nice ) commissioning guide recommended that ttr of over 65% are desirable in the anticoagulation control.21 in the present study , 35% of patients had ttr 65% and 65% had ttr < 65% . among patients with ttr < 66% , the noac are reported to reduce risk of major bleeding more effectively relative to warfarin than in patients with ttr 66%.22 body surface area , warfarin dose and dose assessment interval , concurrent medications , liver disease , cha2ds2-vasc and atria scores , the same - tt2r2 score , and cyp2c9 and vkorc1 genotype all served as predictive variables . all variables with p<0.20 in the univariate analysis and variables of interest in this study ( e.g. , same - tt2r2 score ) were included in the multivariate analysis . , clinical and genetic data form 380 korean patients with af were collected and the patients were followed for about 4 years ( meansd , 231.9199.7 weeks ) . the same - tt2r2 score was 3.40.9 , range 2 - 7 , and 153 subjects ( 40.2% ) had same - tt2r2 scores of 4 . no scores of 0 or 1 were found in this study because the risk scheme assigns two points for non - white race classification . genotype frequencies for cyp2c9 * 3 ( rs1057910 ) and vkorc1 1173c > t ( rs9934438 ) were 9.5% and 16.3% , respectively . for the patient group overall , irrespective of genotype , the percentage of time spent in therapeutic range was 57.5% during the entire follow - up period . carriers of vkorc1 tt spent less time in therapeutic range ( inr 2 - 3 ) than carriers of vkorc1 1173c > t ( p=0.031 ) . on the contrary , the percentages of time in specific inr ranges did not differ according to cyp2c9 isoform or same - tt2r2 score . we expected a negative correlation between the same - tt2r2 score and time in therapeutic range ( inr 2 - 3 ) , but the spearman correlation analysis showed no correlation ( r=0.11 p=0.826 ) . the distribution of the same - tt2r2 scores was similar among the different genotypes related to warfarin dosing ( figure 2 ) . the vkorc1 tt but none of the other factors , including the same - tt2r2 score , was significantly associated with labile inr . clinical and genetic data form 380 korean patients with af were collected and the patients were followed for about 4 years ( meansd , 231.9199.7 weeks ) . the same - tt2r2 score was 3.40.9 , range 2 - 7 , and 153 subjects ( 40.2% ) had same - tt2r2 scores of 4 . no scores of 0 or 1 were found in this study because the risk scheme assigns two points for non - white race classification . genotype frequencies for cyp2c9 * 3 ( rs1057910 ) and vkorc1 1173c > t ( rs9934438 ) were 9.5% and 16.3% , respectively . for the patient group overall , irrespective of genotype , the percentage of time spent in therapeutic range was 57.5% during the entire follow - up period . carriers of vkorc1 tt spent less time in therapeutic range ( inr 2 - 3 ) than carriers of vkorc1 1173c > t ( p=0.031 ) . on the contrary , the percentages of time in specific inr ranges did not differ according to cyp2c9 isoform or same - tt2r2 score . we expected a negative correlation between the same - tt2r2 score and time in therapeutic range ( inr 2 - 3 ) , but the spearman correlation analysis showed no correlation ( r=0.11 p=0.826 ) . the distribution of the same - tt2r2 scores was similar among the different genotypes related to warfarin dosing ( figure 2 ) . the vkorc1 tt but none of the other factors , the main findings of this study were that ( 1 ) a genetic factor related to warfarin response , but no common clinical or demographic factor , including the same - tt2r2 score , was associated with the quality of anticoagulation control , and ( 2 ) there is poor correlation between clinical and genetic factors . the american heart and stroke association guidelines for oral antithrombotic agents for the prevention of stroke in non - valvular af23 support the use of both warfarin and noacs to prevent first and recurrent strokes in patients with non - valvular af . the guidelines recommend the individualized selection of antithrombotic agent(s ) based on risk calculations , cost , tolerability , patient preference , potential for drug interactions , and other clinical characteristics , including ttr if the patient has been taking warfarin . as a result , it is difficult to identify the newly diagnosed patients with af who would do well on warfarin and who also have high values for time in therapeutic range . this is desirable because the main benefits of noacs compared with warfarin may be only marginal in those with high times in therapeutic range , although the reduction in intracranial hemorrhage is still evident.24 in a recent meta - analysis with data for four phase 3 clinical trials of noacs , the noacs had a favorable risk - benefit profile as compared with warfarin that was consistent across diverse patient groups.22 however , it also showed a greater relative reduction in major bleeding with noacs when ttr was less than 66% than when it was more than 66%.22 high ttr ( > 70% ) are known to be associated with the best efficacy and safety of warfarin.25 genotype - guided dosing of warfarin was recently reported to be associated with a higher percentage of time in the therapeutic inr range than standard dosing during the initiation of warfarin therapy.26 however , the relationship of warfarin pharmacogenetics to long - term ttr during warfarin maintenance is not well studied . however , because polymorphism frequency varies between ethnic groups , the results of this study may not apply to other populations with different distributions of cyp2c9 and vkorc1 isoforms.27 further studies are needed to determine the clinical significance of warfarin pharmacogenetics in ethnic groups with higher prevalence of cyp2c9 variants . our findings , that clinical and demographic factors expressed in the same - tt2r2 score were not associated with quality of anticoagulation control , contradict the recent findings of dr . in the atrial fibrillation follow - up investigation of rhythm management ( affirm ) , the same patient cohort served in both the derivation and validation phases of the study . although the mean age ( ~70 years - old ) and mean duration of follow - up ( 3.5 - 3.9 years ) were similar between that study and ours , the baseline patient characteristics differed significantly.28 in particular , most subjects ( 89% ) in the affirm study were white , whereas all patients in our study were korean . " non - white race " represents two points from a total of eight points in the same - tt2r2 risk prediction scheme . the choice of noacs vs. warfarin would be particularly important in patients who have suffered stroke resulting in severe disability , because the monitoring of inr is often difficult in these patients ; and importantly , the cost of noacs may be prohibitive in developing countries , which include a large proportion of the world 's population . in this study , furthermore , patients with valvular af ( who are not candidates for noac treatment ) and those with a target inr range of 1.5 to 2.5 or 2.5 to 3.5 ( outside the inr target of 2 - 3 ) were excluded , whereas 11%-12% of both the derivation and validation groups in the affirm study had valvular af . recently , jane skov et al.29 investigated to determine whether the same - tt2r2 score can predict inr control and the population only included whites , and showed that the same - tt2r2 score was not predictive of ttr . the strength of the present study resides in the consecutive recruitment of patients with comprehensive evaluation of factors related to the quality of anticoagulation control , such as concurrent medications , scored risk assessments for af - related thromboembolic and hemorrhagic events , and consecutive inr measurements with exclusion of inr values during temporary treatment interruptions and during the first one month after treatment was resumed . limitations of this study include the retrospective design and residence at a single clinical center . although we can not generalize our findings in korean subjects to all other ethnic groups , our analytical approach and findings may inform clinical services for relatively large populations throughout asia , and possibly africa . further prospective studies are needed to identify clinical and genetic factors related to the quality of anticoagulation control during treatment with warfarin in asians . in conclusion , genetic factors , but not the commonly recognized clinical and demographic factors , were associated with quality of anticoagulation control in patients of asian background who had recently survived an af - related stroke .

throughout the world , fish stocks are threatened by overharvesting , and there is growing concern that fishing also has negative effects on stock productivity through evolutionary processes ( conover and munch 2002 ; reznick and ghalambor 2005 ) . 2003 ) and select for traits associated with low or unstable productivity , such as reduced age and body size at maturation in otherwise long - lived species ( olsen et al . biocomplexity , including intraspecific diversity in fitness - related traits such as patterns of migration , maturation and spawning , is expected to improve fisheries sustainability ( hilborn et al . for example , biocomplexity in pacific salmon is manifested by a network of local populations , where overall stock productivity is maintained through periods of shifting environmental conditions because certain geographic- and life - history components perform well under some environmental conditions whereas other components perform well under other environmental conditions ( hilborn et al . any given point in time , all geographic- and life - history components are conserved , which ensures the long - term sustainability of the overall stock complex ( hilborn et al . biocomplexity may be defined along three axes : spatial heterogeneity , connectivity and historical contingencies ( cadenasso et al . the spatial component of biocomplexity may involve adaptations to local environmental conditions ( hilborn et al . indeed , such spatially structured evolution depends on the interplay between heritability ( history ) , gene flow ( connectivity ) and local selection regimes ( heterogeneity ) ( lenormand 2002 ; garant et al . the marine environment should offer ample opportunities for homogenizing gene flow through passive dispersal of eggs and larvae with ocean currents and active movement of older individuals ( waples 1998 ) . yet , studies on neutral molecular markers have shown that marine species with highly mobile life stages can be structured into local populations on a surprisingly small scale ( e.g. jorde et al . retention of pelagic early life stages and homing of adult individuals have been identified as important mechanisms influencing the scale of population connectivity in marine systems ( jones et al . evidence for locally adapted marine populations is emerging ( conover and present 1990 ; bricelj et al . 2006 ) , but little is known about the spatial scales of adaptive variation and how this is associated with the spatial scales of gene flow ( but see , hutchings et al . life - history traits are often highly plastic in response to environmental change ( alm 1959 ) . thus , a major challenge is to separate genetic influences from environmental influences on phenotypic variation . for this purpose , reaction norms , defined as the set of phenotypes produced by a genotype over a range of environmental conditions ( schmalhausen 1949 ) , are helpful . a plastic response to environmental change is expected to shift the phenotype along the reaction norm , whereas an evolutionary change in life history is expected to alter the reaction norm itself ( ernande et al . recently , the reaction norm approach has been applied to long - term survey data in order to explore whether fisheries are causing evolutionary changes in fish life histories . 2004 ) , observations from several fish stocks indicate that maturation reaction norms have shifted towards younger age and smaller size ( barot et al . 2004a ; olsen et al . here , we assess the complexity of maturation patterns in atlantic cod ( gadus morhua ) from the norwegian skagerrak coast . earlier work on maturation patterns in fishes focus mainly on quantifying the probability of maturing at a certain age and body length ( e.g. olsen et al . age and body length together determine the growth trajectory of the fish and hence this probabilistic maturation reaction - norm approach ( heino et al . 2002 ; dieckmann and heino 2007 ) describes phenotypic plasticity in maturation linked to any feature of the environment that influences growth ( e.g. temperature and food availability ) . still , age and body length are not expected to explain all variation in maturation ( morita and fukuwaka 2006 ) , and a consistent trend in the environment that is not captured by variation in growth might bias maturation reaction norms estimated from field data ( law 2007 ) . therefore , we have added fish condition as an additional dimension in the reaction norm , realizing that animals should not start reproducing when their physiological state ( condition ) is below a certain critical level ( mcnamara and houston 1996 ) . we acknowledge that maturation reaction norms derived from field data will inevitably be associated with some uncertainty , as compared with common garden studies performed under standardized laboratory conditions , but we will argue that the probabilistic maturation reaction - norm approach is nevertheless valuable for studies on biocomplexity . skagerrak coastal cod are a good candidate for identifying biocomplexity because there is evidence for local populations on a scale of only a few tens of kilometres , revealed as genetic differentiation in presumed selectively neutral loci ( knutsen et al . 2003 ; jorde et al . 2007 ) . using the probabilistic maturation reaction norm approach , we report evidence for spatial structuring of coastal cod life histories associated with patterns in population structure inferred from neutral markers . we argue that management strategies should aim to protect such diversity in fitness - oriented traits that are likely to influence stock productivity : that is , a darwinian approach to fisheries science and management ( conover and munch 2002 ; jrgensen et al . coastal skagerrak cod have a relatively short life cycle , and may spawn already at an age of 2 years and a body length of about 30 cm ( olsen et al . traditional analyses of the proportion of mature fish at a specific age and body length ( maturity ogives ) have identified significant spatial variation in life histories ( olsen et al . 2004b ) , but it is unknown to what extent the observed phenotypic variation merely reflects plastic responses to environmental differences or genetic responses resulting from evolutionary processes . coastal cod from eastern parts of skagerrak have suffered a near collapse during the past 20 years , presumably due to local overfishing ( svedng and bardon 2003 ) . coastal cod were sampled for maturation data during an annual autumn survey ( november december ) with trammel gillnets within five regions of the norwegian skagerrak coast ( table 1 , fig . ( 20012006 ) , the same set of gillnets ( 45 mm mesh size ) was set at the same well - defined sites . the nets were set in the evening and hauled during the morning , fishing for about 15 h in shallow near - shore waters ( 515 m depth ) . gender and maturity stage were determined for each sampled fish via macroscopic inspections of the gonads . liver wet weight was measured to the nearest 0.1 g. somatic weight was not measured . a fish was considered to be age 0 the year of hatching , age 1 the next year , and so on . because the skagerrak coastal cod spawn mainly in late winter , we added 1 year to the age at capture ( in the autumn ) to obtain age during spawning season . skagerrak coastal cod sampled for life history characteristics : sample sizes ( n ) , mean age ( range ) , mean total body length ( range ) and mean liver wet weight ( range ) . coastal cod sampled from the norwegian skagerrak coast : mandal ( 1 ) , hvg ( 2 ) , bjelland ( 3 ) , risr ( 4 ) , krager ( 5 ) , grenland ( 6 ) , vasser ( 7 ) , oslo ( 8) and fredrikstad ( 9 ) . site 1 , 2 , 5 , 7 and 9 were sampled for life history traits , while site 2 , 3 , 4 , 6 , 8 and 9 were sampled for neutral markers . condition is defined as the energy available for life - history decisions such as growth and maturation ( koops et al . 2004 ) . because atlantic cod store most of their lipid energy reserves in the liver ( lambert and dutil 1997 ) , we therefore used liver weight as a measure of the fish s condition . we modelled plasticity in maturation of age 2 cod only the youngest age at which we observed maturing fish ( with the exception of three individuals that may have matured at age 1 ) . older fish with maturing gonads could be either recruit - spawners or repeat - spawners , and including such fish could confound analyses on the actual maturation process . because the age distribution of the coastal cod was dominated by young fish ( table 1 ) these omissions do not seriously reduce the samples available for analyses . note that , in some cases , maturation probability can be estimated even if age at first reproduction is unknown but this method is not robust when fewer than about 100 individuals are sampled from a cohort at a certain age ( barot et al . also , by focussing on age 2 cod , we avoid the potential confounding effect of skipped spawning , referring to the phenomenon that sexually mature fish do not necessarily reproduce every year ( rideout and rose 2006 ) . a mature fish that skips a spawning season might be wrongly classified as a juvenile . the probability of maturing , m , was estimated using a logistic regression model : where s denotes the site , g the gender , lt the total body length and wl the liver wet weight . sample site and gender were modelled as factors , while body length and liver weight were modelled as linear effects . further details about using a logistic regression approach for estimating maturation reaction norms are provided by heino et al . model ( 1 ) was selected based on its aic score ( burnham and anderson 1998 ) when compared with a set of a priory candidate models ( table 2 ) . comparison of logistic regression models for estimating maturation probability ( m ) of 2-year - old skagerrak coastal cod , showing the explanatory variables and interaction effects ( model structure ) , the residual deviance ( dev ) , the residual degrees of freedom ( df ) and the aic value of each model . explanatory variables are site of capture ( s ) , gender ( g ) , total body length ( lt ) and liver wet weight ( wl ) . s and g are modelled as factors while lt and wl are modelled as linear effects . the model selected for inference is shown in bold ( model 1 ) . a global model containing all two - way interaction effects is shown for reference ( model 2 ) . models without an effect of lt on m were considered biologically unrealistic and hence not included among the candidate models . earlier studies on the skagerrak cod have provided evidence for fine - scale neutral genetic population structure ; based on the genetic analysis of mature cod collected during several years ( knutsen et al . our aim is ( i ) to identify potential regions of genetic discontinuity ( barriers ) along this coastline and ( ii ) to compare these regions with the spatial pattern of adaptive structure revealed by the estimated maturation reaction norms . the genetic analyses of population connectivity along the norwegian skagerrak coast were based on data from knutsen et al . briefly , cod from six localities ( table 3 ) were sampled during the spawning season ( january march ) in 2000 and scored for genetic variability at 10 microsatellite loci ( gmo2 , gmo3 , gmo19 , gmo34 , gmo35 , gmo36 , gmo37 , gmo132 , tch12 and tch13 ) . the genetic samples are thus from the same coastline from which the present life - history samples are derived ( fig . , we reanalyse these microsatellite dna data in order to test for potential heterogeneity in the strength of the neutral genetic structure along the coastline . first , we used the exact test in the genepop software ( raymond and rousset 1995 ) to test each locality for genetic difference from the other five sample sites pooled together , one locus at a time and jointly over all 10 loci . second , genetic differences among all pairs of samples were calculated using estimator of weir and cockerham ( 1984 ) , both separately for each locus and averaged over all 10 loci ( table 4 ) . the resulting matrices with pairwise estimates of genetic differences were applied in the barrier software ( manni et al . the relative strength of each barrier is evaluated from the rank of barriers calculated from the matrix of average estimates ( table 4 ) , and we report the three strongest barriers ( labelled a , b and c , in order of rank ) . this number of barriers was chosen because preliminary analyses indicated that only three barriers have any support in the data . the statistical support for the three barriers were evaluated by bootstrapping over loci , i.e. we used 10 single - locus matrices as input for barrier and reported the number of times each of the three barriers were included among the three strongest ones . note that the maturation data and the microsatellite data were not collected from the exact same localities , but rather as independent samples throughout this coastline ( fig . , we do not aim to infer about genetic differences in maturation patterns directly from the neutral genetic population structure . rather , our aim is to compare the spatial scales of adaptive and neutral divergence , because these processes do not necessarily occur on similar temporal and spatial scales ( conover et al . a , number of alleles per locus ; h , gene diversity ( expected heterozygosity ) ; fis , departure from hardy weinberg genotype proportions [ p - values calculated using exact probability test in genepop ( raymond and rousset 1995 ) and summed over loci using fisher s procedure ] . estimated genetic differentiation at 10 microsatellite loci ( : weir and cockerham 1984 ) among pairs of genetic samples , and tests for allele frequency differences [ exact tests for population differentiation in genepop ( raymond and rousset 1995 ) summed over loci using fisher s procedure ] . p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 . coastal skagerrak cod have a relatively short life cycle , and may spawn already at an age of 2 years and a body length of about 30 cm ( olsen et al . traditional analyses of the proportion of mature fish at a specific age and body length ( maturity ogives ) have identified significant spatial variation in life histories ( olsen et al . 2004b ) , but it is unknown to what extent the observed phenotypic variation merely reflects plastic responses to environmental differences or genetic responses resulting from evolutionary processes . coastal cod from eastern parts of skagerrak have suffered a near collapse during the past 20 years , presumably due to local overfishing ( svedng and bardon 2003 ) . coastal cod were sampled for maturation data during an annual autumn survey ( november december ) with trammel gillnets within five regions of the norwegian skagerrak coast ( table 1 , fig . ( 20012006 ) , the same set of gillnets ( 45 mm mesh size ) was set at the same well - defined sites . the nets were set in the evening and hauled during the morning , fishing for about 15 h in shallow near - shore waters ( 515 m depth ) . gender and maturity stage were determined for each sampled fish via macroscopic inspections of the gonads . liver wet weight was measured to the nearest 0.1 g. somatic weight was not measured . a fish was considered to be age 0 the year of hatching , age 1 the next year , and so on . because the skagerrak coastal cod spawn mainly in late winter , we added 1 year to the age at capture ( in the autumn ) to obtain age during spawning season . skagerrak coastal cod sampled for life history characteristics : sample sizes ( n ) , mean age ( range ) , mean total body length ( range ) and mean liver wet weight ( range ) . coastal cod sampled from the norwegian skagerrak coast : mandal ( 1 ) , hvg ( 2 ) , bjelland ( 3 ) , risr ( 4 ) , krager ( 5 ) , grenland ( 6 ) , vasser ( 7 ) , oslo ( 8) and fredrikstad ( 9 ) . site 1 , 2 , 5 , 7 and 9 were sampled for life history traits , while site 2 , 3 , 4 , 6 , 8 and 9 were sampled for neutral markers . condition is defined as the energy available for life - history decisions such as growth and maturation ( koops et al . 2004 ) . because atlantic cod store most of their lipid energy reserves in the liver ( lambert and dutil 1997 ) , we therefore used liver weight as a measure of the fish s condition . we modelled plasticity in maturation of age 2 cod only the youngest age at which we observed maturing fish ( with the exception of three individuals that may have matured at age 1 ) . older fish with maturing gonads could be either recruit - spawners or repeat - spawners , and including such fish could confound analyses on the actual maturation process . because the age distribution of the coastal cod was dominated by young fish ( table 1 ) these omissions do not seriously reduce the samples available for analyses . note that , in some cases , maturation probability can be estimated even if age at first reproduction is unknown but this method is not robust when fewer than about 100 individuals are sampled from a cohort at a certain age ( barot et al . also , by focussing on age 2 cod , we avoid the potential confounding effect of skipped spawning , referring to the phenomenon that sexually mature fish do not necessarily reproduce every year ( rideout and rose 2006 ) . a mature fish that skips a spawning season might be wrongly classified as a juvenile . the probability of maturing , m , was estimated using a logistic regression model : where s denotes the site , g the gender , lt the total body length and wl the liver wet weight . sample site and gender were modelled as factors , while body length and liver weight were modelled as linear effects . further details about using a logistic regression approach for estimating maturation reaction norms are provided by heino et al . model ( 1 ) was selected based on its aic score ( burnham and anderson 1998 ) when compared with a set of a priory candidate models ( table 2 ) . comparison of logistic regression models for estimating maturation probability ( m ) of 2-year - old skagerrak coastal cod , showing the explanatory variables and interaction effects ( model structure ) , the residual deviance ( dev ) , the residual degrees of freedom ( df ) and the aic value of each model . explanatory variables are site of capture ( s ) , gender ( g ) , total body length ( lt ) and liver wet weight ( wl ) . s and g are modelled as factors while lt and wl are modelled as linear effects . the model selected for inference is shown in bold ( model 1 ) . a global model containing all two - way interaction effects is shown for reference ( model 2 ) . models without an effect of lt on m were considered biologically unrealistic and hence not included among the candidate models . earlier studies on the skagerrak cod have provided evidence for fine - scale neutral genetic population structure ; based on the genetic analysis of mature cod collected during several years ( knutsen et al . our aim is ( i ) to identify potential regions of genetic discontinuity ( barriers ) along this coastline and ( ii ) to compare these regions with the spatial pattern of adaptive structure revealed by the estimated maturation reaction norms . the genetic analyses of population connectivity along the norwegian skagerrak coast were based on data from knutsen et al . briefly , cod from six localities ( table 3 ) were sampled during the spawning season ( january march ) in 2000 and scored for genetic variability at 10 microsatellite loci ( gmo2 , gmo3 , gmo19 , gmo34 , gmo35 , gmo36 , gmo37 , gmo132 , tch12 and tch13 ) . the genetic samples are thus from the same coastline from which the present life - history samples are derived ( fig . , we reanalyse these microsatellite dna data in order to test for potential heterogeneity in the strength of the neutral genetic structure along the coastline . first , we used the exact test in the genepop software ( raymond and rousset 1995 ) to test each locality for genetic difference from the other five sample sites pooled together , one locus at a time and jointly over all 10 loci . second , genetic differences among all pairs of samples were calculated using estimator of weir and cockerham ( 1984 ) , both separately for each locus and averaged over all 10 loci ( table 4 ) . the resulting matrices with pairwise estimates of genetic differences were applied in the barrier software ( manni et al . the relative strength of each barrier is evaluated from the rank of barriers calculated from the matrix of average estimates ( table 4 ) , and we report the three strongest barriers ( labelled a , b and c , in order of rank ) . this number of barriers was chosen because preliminary analyses indicated that only three barriers have any support in the data . the statistical support for the three barriers were evaluated by bootstrapping over loci , i.e. we used 10 single - locus matrices as input for barrier and reported the number of times each of the three barriers were included among the three strongest ones . note that the maturation data and the microsatellite data were not collected from the exact same localities , but rather as independent samples throughout this coastline ( fig . , we do not aim to infer about genetic differences in maturation patterns directly from the neutral genetic population structure . rather , our aim is to compare the spatial scales of adaptive and neutral divergence , because these processes do not necessarily occur on similar temporal and spatial scales ( conover et al . a , number of alleles per locus ; h , gene diversity ( expected heterozygosity ) ; fis , departure from hardy weinberg genotype proportions [ p - values calculated using exact probability test in genepop ( raymond and rousset 1995 ) and summed over loci using fisher s procedure ] . data are from knutsen et al . estimated genetic differentiation at 10 microsatellite loci ( : weir and cockerham 1984 ) among pairs of genetic samples , and tests for allele frequency differences [ exact tests for population differentiation in genepop ( raymond and rousset 1995 ) summed over loci using fisher s procedure ] . p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 . considering age 2 cod , the probability of maturing at that age depended on both body length and liver weight ( fig . the effects of body length and liver weight were not additive , as seen in the nonlinearity of the isolines of specific maturation probabilities ( fig . 2 ) . for small - to - intermediate age 2 cod ( up to about 40 cm ) , there was a strong and positive effect of increasing liver weight on the probability of maturing , although the exact shape of this relationship depended on geographic location and gender ( fig . the probability of maturing at age 2 was shifted towards small body size and poor liver weight in northeast skagerrak as compared with southwest skagerrak , while cod from the two sample sites in the central region of this coastline had intermediate and similar maturation schedules ( fig . 2 ) . cod from the two sites in the southwest region also had quite similar maturation schedules , and different from other regions ( fig . 2 ) . condition of cod was quite poor in northeast skagerrak as compared with southwest skagerrak ( fig . the probability of maturing at age 2 was shifted towards small body size and poor liver weight in males as compared with females ( fig . maturation reaction norms estimated for 2-year - old female and male coastal cod , showing the predicted maturation probability ( model 1 , contour lines ) and the observed body length and liver weight ( ) for each site : ( a ) mandal females , ( b ) hvg females , ( c ) krager females , ( d ) vasser females , ( e ) fredrikstad females , ( f ) mandal males , ( g ) hvg males , ( h ) krager males , ( i ) vasser males and ( j ) fredrikstad males . parameter estimates , with se , of the model ( 1 ) selected for inference about coastal cod maturation probability . explanatory variables are site of capture ( s ) , gender ( g ) , total body length ( lt ) and liver wet weight ( wl ) . we also attempted to fit our maturation model ( 1 ) to the data on age 3 cod . as an alternative , we fitted a simper model containing only additive effects of length and liver weight ( pooling males , females and sampling sites ) . this model indicated that there was still an overall positive effect of both length ( 0.052 , se = 0.021 ) and liver weight ( 0.011 , se = 0.004 ) on the probability of spawning ( as differentiated from the probability of maturing , see methods section ) . the analyses of genetic differentiation with the barrier software indicate that the strongest genetic discontinuity separates the western localities ( 1 and 2 ) from the remaining ones , because two barriers ( a and c ) were located in this region ( fig . the third barrier ( b ) appears in the eastern part of the coastline , separating the easternmost locality ( 9 ) from the others . although this barrier has fairly low bootstrap support ( 3 loci ) , the easternmost sample is significantly different from the others , as judged by the low p - value in tests for allele frequency heterogeneity ( p = 0.035 , for the joint test over all 10 loci ) . the allele frequency tests also explain the other barriers and identify site 2 in the southwest and site 6 in the central region as particularly divergent at microsatellite loci ( psite2 = 0.007 , psite6 = 0.014 ) . genetic discontinuity , or barriers to gene flow ( red bars ) in coastal cod , letters indicating relative strength ( a ) and numbers indicating sampling localities ( fig . maturation reaction norm midpoints ( cp50s ) , referring to the site - specific condition ( liver weight ) where a 50% probability of maturing is reached for 2-year - old female ( b ) and male ( c ) cod at mean body length ( 38 cm ) ; barriers to gene flow ( red bars ) superimposed . there is an apparent association between spatial genetic structuring in the microsatellite data and the spatial pattern of maturation reaction norms ( fig . the estimated maturation reaction norms of cod from the two westernmost localities ( 1 and 2 ) are similar to each other and differ significantly from the remaining localities ( fig . the two westernmost localities are also separated from all other localities to the east by two microsatellite barriers ( fig . , the outlying maturation reaction norm estimated for the easternmost site ( 9 ) is in agreement with the estimated barrier to gene flow separating this area from the remaining part of the coast ( fig . we have assessed biocomplexity in coastal atlantic cod , a broadcast - spawning marine fish , and found evidence for a fine - scale spatial diversity in life histories that is associated with patterns of population connectivity based on neutral genetic markers . by estimating probabilistic maturation reaction norms ( heino et al . 2002 ; dieckmann and heino 2007 ) statistically controlling for environmental plasticity in growth ( size at age ) as well as energy reserves ( liver weight 23 months before spawning ) , our finding is indicative of an underlying genetic component to the spatial complexity in maturation patterns . we found an association between spatial structure in life histories and population structure inferred from independent data on microsatellite dna . note that we did not formally correlate gene flow and adaptive divergence ( hendry and taylor 2004 ) . this is because of the complexity of gene flow in this system , where a component of gene flow is probably from oceanic north sea cod and into the coastal populations ( as planktonic larvae ) rather than among coastal populations ( knutsen et al . we presume , however , that observed genetic differences among coastal populations imply some degree of isolation , from both the north sea and from other coastal populations . spawning and retention of eggs and larvae within fjord basins sheltered from coastal currents is a likely mechanism for maintaining genetic differentiation among the local populations ( knutsen et al . 2007 ) , in combination with high site fidelity and natal homing of older cod ( espeland et al . 2007 ; svedng et al . the finding that cod from two pairs of neighbouring localities had very similar maturation reaction norms indicates that we have roughly identified the spatial scale of biocomplexity along this coastline . our data thus suggest that life - history divergence is maintained on a scale of tens of kilometres in coastal cod , in agreement with estimates of local population ranges calculated from microsatellite dna divergence patterns and capture recapture data ( jorde et al . this life - history divergence is maintained despite gene flow suggested to constitute up to nearly 10% of the local age 0 populations ( stenseth et al . earlier studies on spatial patterns of adaptive divergence in marine organisms have focussed on broader spatial patterns ( conover and present 1990 ; bricelj et al . 2006 ) , although in the tropical sea anemone ( condylactis gigantean ) there is evidence for adaptive genetic differentiation among phenotypes on reef habitats only 5 km apart ( stoletzki and schierwater 2005 ) . a recent study on atlantic herring ( clupea harengus ) reported evidence of correlated neutral genetic structure and spawning seasons and spawning location in the north sea ( ruzzante et al . ( 2006 ) by assessing biocomplexity on a smaller spatial scale and by statistically controlling for known environmental influences on life histories , so that the remaining pattern is indicative of a genetic component . also , a recent common garden laboratory study on atlantic cod from the northwest atlantic have revealed population differences in early life - history plasticity on a broader spatial scale ( hutchings et al . cod from northeast skagerrak has suffered a near collapse in abundance during the past 20 years , presumably due to overfishing ( svedng and bardon 2003 ) . a displacement of the maturation reaction norms towards small body size and poor condition in northeast skagerrak induced by high fishing pressure would fit predictions from theory ( stearns and koella 1986 ; ernande et al . still , the relative importance of natural versus anthropogenic causes of life - history divergence in coastal cod remains largely unknown . note also that the heritability of maturation reaction norms in atlantic cod remains to be quantified , but there is some evidence for additive genetic variation in fish phenotypic plasticity ( haugen and vllestad 2000 ; hutchings et al . the shapes of the estimated reaction norms indicate that phenotypic plasticity influences the maturation probability of cod through both somatic growth history and current energetic state . specifically , an increased liver weight is associated with a marked increase in the probability of sexual maturation of small and young cod . this finding that suggests a state - based approach is needed in order to understand how individuals optimize their life histories ( mcnamara and houston 1996 ) . an improvement in the energy available for life - history decisions ( i.e. the condition ) apparently speeds up the life history of the coastal cod through reproduction at a young age . for comparison , marteinsdottir and begg ( 2002 ) found a positive effect of condition on the probability of being mature in atlantic cod . ( 2007 ) modelled the life - history transition from the juvenile to the mature state ( as distinguished from the probability of being mature ) in north sea plaice ( pleuronectes platessa ) , and found a positive effect of condition on maturation probability . plaice condition was estimated from data on body length and total weight sampled during the spawning season , and the apparent condition of spawners will therefore likely be elevated due to the inclusion of gonad weight in total body weight . our study adds to these findings by estimating condition directly from liver weight the main energy store in gadoids measured some months ahead of spawning . we also found that the estimated maturation reaction norms of male cod were shifted towards small body size and poor liver weight , as compared with female reaction norms . hence , male cod apparently have a ( genetic ) tendency to mature relatively early in life even in the face of poor environmental conditions ( see also , olsen et al . 2005 ) . , a large body size will offer a fitness advantage through improved fecundity and offspring quality ( trippel 1998 ) . also , skjraasen et al . ( 2006 ) found a marked positive effect of energy reserves 34 months before spawning on the fecundity of norwegian coastal cod . for male cod , the fitness advantage of a large size appears less clear ( rakitin et al . 2001 ) , although there is evidence that sperm production is positively linked to food availability and condition ( yoneda and wright 2005 ) . the maturation reaction norms could be influenced by other sources of plasticity , in addition to size at age and energy reserves . in fact , the original maturation reaction norms based on body size and age were termed probabilistic , as age and size will typically not explain all variation in maturation ( heino et al . for instance , recent growth history , rather than size at age , could influence the maturation probability ( morita and fukuwaka 2006 ) . however , we consider it likely that energy reserves prior to spawning ( accounted for in the present study ) will capture much of the same environmental influences as recent growth history . also , bias will only become a problem if there is a consistent trend in the environment over time or between localities that is not captured by the reaction norm . common garden studies , where fish are raised under standardized laboratory conditions , are generally regarded as the most powerful technique for inferring about plasticity and evolution ( e.g. conover and present 1990 ; hutchings et al . the probabilistic maturation reaction norm approach will involve added uncertainty about confounding environmental variables , but , on the other hand , this method allows for comparing life histories of long - lived species in a wide range of natural situations that are difficult or impossible to reproduce under controlled conditions . furthermore , the depleted state of many marine fish stocks , including atlantic cod in the north sea region ( cook et al . 1997 ) , have encouraged us to ask how new statistical tools can help to generate knowledge from data that are already at hand . our study highlights the need to manage biocomplexity within harvested marine species such as the coastal cod . as argued by hilborn et al . ( 2006 ) , sustainability may be compromised by a generalized management regime not accounting for intraspecific genetic diversity . in particular , our study supports hutchings et al . ( 2007 ) , pointing out the need to manage adaptive phenotypic plasticity in life histories . selection pressures imposed by humans might affect genetic diversity both directly through selective ( or nonselective ) harvesting and indirectly through environmental change . 2004 ) advocates the need for evolutionary conservation biology where the evolutionary process itself is managed , because this is what will ensure the maintenance of diversity in the long run . for coastal cod , this would mean controlling harvest regimes so as to minimize maladaptive genetic effects of harvesting on the local populations , and also to protect the diversity of coastal environments where the local populations are found . there are probably several ways in which these goals could be reached , but berkley et al . ( 2004 ) concludes that the best way to ensure old - growth age structure ( i.e. selection ) and complex spatial structure in populations of groundfish is through interconnected networks of marine reserves . ( 2005 ) suggests that marine reserves can indeed help to counter maladaptive fisheries - induced evolutionary changes in traits such as age and size at maturation . management approaches that operate on larger spatial scales ( i.e. an overall reduction of fishing pressure ) might help to counter unwanted genetic changes but still involves a risk of depleting local gene pools . in addition , managers could shape the selectivity of fisheries through gear regulations ( e.g. mesh size of gillnets ) . ( 2005 ) argue that a combination of fisher s ecological - knowledge , investigative science and a management system that promotes local community stewardship may help to protect the diversity of coastal cod stocks . as an example , wroblewski et al . ( 2005 ) describe the process towards the establishment of a marine protected area encompassing a local inshore cod stock in gilbert bay , canada . in conclusion , this study presents evidence for small - scale biocomplexity in a harvested marine fish , the atlantic cod , and advocates the need for a darwinian perspective on fisheries management ( conover and munch 2002 ) aimed at protecting such diversity .

harvesting of marine resources raises concerns about how to identify and preserve biocomplexity , including the diversity of life histories found within and among wild populations of a species . in order to fully accomplish this , there is a need to elucidate the underlying causes of phenotypic variation , and how this variation responds to environmental changes . in general , both evolutionary ( genetic ) and nonevolutionary ( plastic ) responses may occur . plastic responses to environmental change are expected to shift the phenotype along a reaction norm , while an evolutionary response is expected to shift the reaction norm itself . here , we assess the maturation patterns of coastal atlantic cod ( gadus morhua ) in skagerrak , where studies using neutral markers have revealed genetically differentiated populations of this harvested fish within tens of kilometres of coastline . our results suggest that physiological state prior to the spawning season , as well as juvenile growth , both influence the probability of completing sexual maturation at a given age . furthermore , our results point towards a spatial structuring of this plasticity ( i.e. the maturation reaction norms ) comparable with population connectivity inferred from neutral markers . we argue that such fine - scale biocomplexity calls for a darwinian approach to fisheries management .

Introduction Methods The Skagerrak coastal cod Sampling of coastal cod maturation data Statistical modelling of cod maturation data Sampling and analyses of cod population genetic data Results Discussion

throughout the world , fish stocks are threatened by overharvesting , and there is growing concern that fishing also has negative effects on stock productivity through evolutionary processes ( conover and munch 2002 ; reznick and ghalambor 2005 ) . 2003 ) and select for traits associated with low or unstable productivity , such as reduced age and body size at maturation in otherwise long - lived species ( olsen et al . biocomplexity , including intraspecific diversity in fitness - related traits such as patterns of migration , maturation and spawning , is expected to improve fisheries sustainability ( hilborn et al . for example , biocomplexity in pacific salmon is manifested by a network of local populations , where overall stock productivity is maintained through periods of shifting environmental conditions because certain geographic- and life - history components perform well under some environmental conditions whereas other components perform well under other environmental conditions ( hilborn et al . indeed , such spatially structured evolution depends on the interplay between heritability ( history ) , gene flow ( connectivity ) and local selection regimes ( heterogeneity ) ( lenormand 2002 ; garant et al . 2006 ) , but little is known about the spatial scales of adaptive variation and how this is associated with the spatial scales of gene flow ( but see , hutchings et al . life - history traits are often highly plastic in response to environmental change ( alm 1959 ) . for this purpose , reaction norms , defined as the set of phenotypes produced by a genotype over a range of environmental conditions ( schmalhausen 1949 ) , are helpful . a plastic response to environmental change is expected to shift the phenotype along the reaction norm , whereas an evolutionary change in life history is expected to alter the reaction norm itself ( ernande et al . recently , the reaction norm approach has been applied to long - term survey data in order to explore whether fisheries are causing evolutionary changes in fish life histories . 2004 ) , observations from several fish stocks indicate that maturation reaction norms have shifted towards younger age and smaller size ( barot et al . here , we assess the complexity of maturation patterns in atlantic cod ( gadus morhua ) from the norwegian skagerrak coast . earlier work on maturation patterns in fishes focus mainly on quantifying the probability of maturing at a certain age and body length ( e.g. still , age and body length are not expected to explain all variation in maturation ( morita and fukuwaka 2006 ) , and a consistent trend in the environment that is not captured by variation in growth might bias maturation reaction norms estimated from field data ( law 2007 ) . therefore , we have added fish condition as an additional dimension in the reaction norm , realizing that animals should not start reproducing when their physiological state ( condition ) is below a certain critical level ( mcnamara and houston 1996 ) . we acknowledge that maturation reaction norms derived from field data will inevitably be associated with some uncertainty , as compared with common garden studies performed under standardized laboratory conditions , but we will argue that the probabilistic maturation reaction - norm approach is nevertheless valuable for studies on biocomplexity . skagerrak coastal cod are a good candidate for identifying biocomplexity because there is evidence for local populations on a scale of only a few tens of kilometres , revealed as genetic differentiation in presumed selectively neutral loci ( knutsen et al . using the probabilistic maturation reaction norm approach , we report evidence for spatial structuring of coastal cod life histories associated with patterns in population structure inferred from neutral markers . we argue that management strategies should aim to protect such diversity in fitness - oriented traits that are likely to influence stock productivity : that is , a darwinian approach to fisheries science and management ( conover and munch 2002 ; jrgensen et al . traditional analyses of the proportion of mature fish at a specific age and body length ( maturity ogives ) have identified significant spatial variation in life histories ( olsen et al . 2004b ) , but it is unknown to what extent the observed phenotypic variation merely reflects plastic responses to environmental differences or genetic responses resulting from evolutionary processes . because the skagerrak coastal cod spawn mainly in late winter , we added 1 year to the age at capture ( in the autumn ) to obtain age during spawning season . site 1 , 2 , 5 , 7 and 9 were sampled for life history traits , while site 2 , 3 , 4 , 6 , 8 and 9 were sampled for neutral markers . because atlantic cod store most of their lipid energy reserves in the liver ( lambert and dutil 1997 ) , we therefore used liver weight as a measure of the fish s condition . also , by focussing on age 2 cod , we avoid the potential confounding effect of skipped spawning , referring to the phenomenon that sexually mature fish do not necessarily reproduce every year ( rideout and rose 2006 ) . the probability of maturing , m , was estimated using a logistic regression model : where s denotes the site , g the gender , lt the total body length and wl the liver wet weight . sample site and gender were modelled as factors , while body length and liver weight were modelled as linear effects . further details about using a logistic regression approach for estimating maturation reaction norms are provided by heino et al . earlier studies on the skagerrak cod have provided evidence for fine - scale neutral genetic population structure ; based on the genetic analysis of mature cod collected during several years ( knutsen et al . our aim is ( i ) to identify potential regions of genetic discontinuity ( barriers ) along this coastline and ( ii ) to compare these regions with the spatial pattern of adaptive structure revealed by the estimated maturation reaction norms . briefly , cod from six localities ( table 3 ) were sampled during the spawning season ( january march ) in 2000 and scored for genetic variability at 10 microsatellite loci ( gmo2 , gmo3 , gmo19 , gmo34 , gmo35 , gmo36 , gmo37 , gmo132 , tch12 and tch13 ) . , we reanalyse these microsatellite dna data in order to test for potential heterogeneity in the strength of the neutral genetic structure along the coastline . first , we used the exact test in the genepop software ( raymond and rousset 1995 ) to test each locality for genetic difference from the other five sample sites pooled together , one locus at a time and jointly over all 10 loci . second , genetic differences among all pairs of samples were calculated using estimator of weir and cockerham ( 1984 ) , both separately for each locus and averaged over all 10 loci ( table 4 ) . the relative strength of each barrier is evaluated from the rank of barriers calculated from the matrix of average estimates ( table 4 ) , and we report the three strongest barriers ( labelled a , b and c , in order of rank ) . , we do not aim to infer about genetic differences in maturation patterns directly from the neutral genetic population structure . a , number of alleles per locus ; h , gene diversity ( expected heterozygosity ) ; fis , departure from hardy weinberg genotype proportions [ p - values calculated using exact probability test in genepop ( raymond and rousset 1995 ) and summed over loci using fisher s procedure ] . coastal skagerrak cod have a relatively short life cycle , and may spawn already at an age of 2 years and a body length of about 30 cm ( olsen et al . traditional analyses of the proportion of mature fish at a specific age and body length ( maturity ogives ) have identified significant spatial variation in life histories ( olsen et al . 2004b ) , but it is unknown to what extent the observed phenotypic variation merely reflects plastic responses to environmental differences or genetic responses resulting from evolutionary processes . because the skagerrak coastal cod spawn mainly in late winter , we added 1 year to the age at capture ( in the autumn ) to obtain age during spawning season . site 1 , 2 , 5 , 7 and 9 were sampled for life history traits , while site 2 , 3 , 4 , 6 , 8 and 9 were sampled for neutral markers . because atlantic cod store most of their lipid energy reserves in the liver ( lambert and dutil 1997 ) , we therefore used liver weight as a measure of the fish s condition . also , by focussing on age 2 cod , we avoid the potential confounding effect of skipped spawning , referring to the phenomenon that sexually mature fish do not necessarily reproduce every year ( rideout and rose 2006 ) . the probability of maturing , m , was estimated using a logistic regression model : where s denotes the site , g the gender , lt the total body length and wl the liver wet weight . further details about using a logistic regression approach for estimating maturation reaction norms are provided by heino et al . earlier studies on the skagerrak cod have provided evidence for fine - scale neutral genetic population structure ; based on the genetic analysis of mature cod collected during several years ( knutsen et al . our aim is ( i ) to identify potential regions of genetic discontinuity ( barriers ) along this coastline and ( ii ) to compare these regions with the spatial pattern of adaptive structure revealed by the estimated maturation reaction norms . briefly , cod from six localities ( table 3 ) were sampled during the spawning season ( january march ) in 2000 and scored for genetic variability at 10 microsatellite loci ( gmo2 , gmo3 , gmo19 , gmo34 , gmo35 , gmo36 , gmo37 , gmo132 , tch12 and tch13 ) . , we reanalyse these microsatellite dna data in order to test for potential heterogeneity in the strength of the neutral genetic structure along the coastline . first , we used the exact test in the genepop software ( raymond and rousset 1995 ) to test each locality for genetic difference from the other five sample sites pooled together , one locus at a time and jointly over all 10 loci . the relative strength of each barrier is evaluated from the rank of barriers calculated from the matrix of average estimates ( table 4 ) , and we report the three strongest barriers ( labelled a , b and c , in order of rank ) . , we do not aim to infer about genetic differences in maturation patterns directly from the neutral genetic population structure . a , number of alleles per locus ; h , gene diversity ( expected heterozygosity ) ; fis , departure from hardy weinberg genotype proportions [ p - values calculated using exact probability test in genepop ( raymond and rousset 1995 ) and summed over loci using fisher s procedure ] . considering age 2 cod , the probability of maturing at that age depended on both body length and liver weight ( fig . for small - to - intermediate age 2 cod ( up to about 40 cm ) , there was a strong and positive effect of increasing liver weight on the probability of maturing , although the exact shape of this relationship depended on geographic location and gender ( fig . the probability of maturing at age 2 was shifted towards small body size and poor liver weight in northeast skagerrak as compared with southwest skagerrak , while cod from the two sample sites in the central region of this coastline had intermediate and similar maturation schedules ( fig . the probability of maturing at age 2 was shifted towards small body size and poor liver weight in males as compared with females ( fig . maturation reaction norms estimated for 2-year - old female and male coastal cod , showing the predicted maturation probability ( model 1 , contour lines ) and the observed body length and liver weight ( ) for each site : ( a ) mandal females , ( b ) hvg females , ( c ) krager females , ( d ) vasser females , ( e ) fredrikstad females , ( f ) mandal males , ( g ) hvg males , ( h ) krager males , ( i ) vasser males and ( j ) fredrikstad males . explanatory variables are site of capture ( s ) , gender ( g ) , total body length ( lt ) and liver wet weight ( wl ) . as an alternative , we fitted a simper model containing only additive effects of length and liver weight ( pooling males , females and sampling sites ) . this model indicated that there was still an overall positive effect of both length ( 0.052 , se = 0.021 ) and liver weight ( 0.011 , se = 0.004 ) on the probability of spawning ( as differentiated from the probability of maturing , see methods section ) . although this barrier has fairly low bootstrap support ( 3 loci ) , the easternmost sample is significantly different from the others , as judged by the low p - value in tests for allele frequency heterogeneity ( p = 0.035 , for the joint test over all 10 loci ) . genetic discontinuity , or barriers to gene flow ( red bars ) in coastal cod , letters indicating relative strength ( a ) and numbers indicating sampling localities ( fig . maturation reaction norm midpoints ( cp50s ) , referring to the site - specific condition ( liver weight ) where a 50% probability of maturing is reached for 2-year - old female ( b ) and male ( c ) cod at mean body length ( 38 cm ) ; barriers to gene flow ( red bars ) superimposed . there is an apparent association between spatial genetic structuring in the microsatellite data and the spatial pattern of maturation reaction norms ( fig . the estimated maturation reaction norms of cod from the two westernmost localities ( 1 and 2 ) are similar to each other and differ significantly from the remaining localities ( fig . the two westernmost localities are also separated from all other localities to the east by two microsatellite barriers ( fig . , the outlying maturation reaction norm estimated for the easternmost site ( 9 ) is in agreement with the estimated barrier to gene flow separating this area from the remaining part of the coast ( fig . we have assessed biocomplexity in coastal atlantic cod , a broadcast - spawning marine fish , and found evidence for a fine - scale spatial diversity in life histories that is associated with patterns of population connectivity based on neutral genetic markers . by estimating probabilistic maturation reaction norms ( heino et al . 2002 ; dieckmann and heino 2007 ) statistically controlling for environmental plasticity in growth ( size at age ) as well as energy reserves ( liver weight 23 months before spawning ) , our finding is indicative of an underlying genetic component to the spatial complexity in maturation patterns . we found an association between spatial structure in life histories and population structure inferred from independent data on microsatellite dna . the finding that cod from two pairs of neighbouring localities had very similar maturation reaction norms indicates that we have roughly identified the spatial scale of biocomplexity along this coastline . our data thus suggest that life - history divergence is maintained on a scale of tens of kilometres in coastal cod , in agreement with estimates of local population ranges calculated from microsatellite dna divergence patterns and capture recapture data ( jorde et al . ( 2006 ) by assessing biocomplexity on a smaller spatial scale and by statistically controlling for known environmental influences on life histories , so that the remaining pattern is indicative of a genetic component . also , a recent common garden laboratory study on atlantic cod from the northwest atlantic have revealed population differences in early life - history plasticity on a broader spatial scale ( hutchings et al . a displacement of the maturation reaction norms towards small body size and poor condition in northeast skagerrak induced by high fishing pressure would fit predictions from theory ( stearns and koella 1986 ; ernande et al . still , the relative importance of natural versus anthropogenic causes of life - history divergence in coastal cod remains largely unknown . note also that the heritability of maturation reaction norms in atlantic cod remains to be quantified , but there is some evidence for additive genetic variation in fish phenotypic plasticity ( haugen and vllestad 2000 ; hutchings et al . the shapes of the estimated reaction norms indicate that phenotypic plasticity influences the maturation probability of cod through both somatic growth history and current energetic state . specifically , an increased liver weight is associated with a marked increase in the probability of sexual maturation of small and young cod . this finding that suggests a state - based approach is needed in order to understand how individuals optimize their life histories ( mcnamara and houston 1996 ) . an improvement in the energy available for life - history decisions ( i.e. for comparison , marteinsdottir and begg ( 2002 ) found a positive effect of condition on the probability of being mature in atlantic cod . ( 2007 ) modelled the life - history transition from the juvenile to the mature state ( as distinguished from the probability of being mature ) in north sea plaice ( pleuronectes platessa ) , and found a positive effect of condition on maturation probability . plaice condition was estimated from data on body length and total weight sampled during the spawning season , and the apparent condition of spawners will therefore likely be elevated due to the inclusion of gonad weight in total body weight . we also found that the estimated maturation reaction norms of male cod were shifted towards small body size and poor liver weight , as compared with female reaction norms . hence , male cod apparently have a ( genetic ) tendency to mature relatively early in life even in the face of poor environmental conditions ( see also , olsen et al . for male cod , the fitness advantage of a large size appears less clear ( rakitin et al . the maturation reaction norms could be influenced by other sources of plasticity , in addition to size at age and energy reserves . in fact , the original maturation reaction norms based on body size and age were termed probabilistic , as age and size will typically not explain all variation in maturation ( heino et al . for instance , recent growth history , rather than size at age , could influence the maturation probability ( morita and fukuwaka 2006 ) . however , we consider it likely that energy reserves prior to spawning ( accounted for in the present study ) will capture much of the same environmental influences as recent growth history . also , bias will only become a problem if there is a consistent trend in the environment over time or between localities that is not captured by the reaction norm . the probabilistic maturation reaction norm approach will involve added uncertainty about confounding environmental variables , but , on the other hand , this method allows for comparing life histories of long - lived species in a wide range of natural situations that are difficult or impossible to reproduce under controlled conditions . furthermore , the depleted state of many marine fish stocks , including atlantic cod in the north sea region ( cook et al . ( 2007 ) , pointing out the need to manage adaptive phenotypic plasticity in life histories . for coastal cod , this would mean controlling harvest regimes so as to minimize maladaptive genetic effects of harvesting on the local populations , and also to protect the diversity of coastal environments where the local populations are found . ( 2004 ) concludes that the best way to ensure old - growth age structure ( i.e. selection ) and complex spatial structure in populations of groundfish is through interconnected networks of marine reserves . ( 2005 ) suggests that marine reserves can indeed help to counter maladaptive fisheries - induced evolutionary changes in traits such as age and size at maturation . management approaches that operate on larger spatial scales ( i.e. ( 2005 ) argue that a combination of fisher s ecological - knowledge , investigative science and a management system that promotes local community stewardship may help to protect the diversity of coastal cod stocks . in conclusion , this study presents evidence for small - scale biocomplexity in a harvested marine fish , the atlantic cod , and advocates the need for a darwinian perspective on fisheries management ( conover and munch 2002 ) aimed at protecting such diversity .

the identification of primary and recurrent cancer diagnoses is critical to clinical and epidemiologic research . despite its importance , however , there is substantial lag between the time of primary cancer diagnoses and complete information capture by cancer registries . additionally , many registries do not track cancer recurrences . consequently , researchers frequently rely on manual chart review and medical claims data , such as international classification of diseases ( icd ) codes , to identify primary and recurrent cancers . chart review , while accurate , is often not feasible in large - scale studies . conversely , claims data are inexpensive and scalable to large studies but are unreliable in terms of accuracy . to address ongoing needs for improved identification of cancer diagnoses , a sas - based coding , extraction , and nomenclature tool ( scent ) was developed at kaiser permanente southern california ( kpsc ) . kpsc is an integrated healthcare organization that provides medical services to a diverse membership of more than 3.5 million people throughout southern california . research conducted at kpsc directly impacts practice guidelines and the medical care that patients receive . given the threat to patients ' health and quality of life , as well as the impact on their families , prevention and treatment of cancer are high priorities for clinicians and researchers . in 1999 , warren et al analyzed the medical claims data of 6784 medicare patients and concluded that such data have limited value in accurately identifying breast cancer cases.1 more recently , lamont et al achieved high levels of sensitivity and specificity using claims data to identify cancer recurrence.2 however , that validation used a small sample of patients ( n=45 ) and the accuracy of claims data in identifying cancer recurrence has yet to be well established . the use of electronic medical record ( emr ) systems among healthcare providers has increased significantly over the past decade . additionally , a provision in the 2009 american recovery and reinvestment act may accelerate the adoption of electronic systems by providing financial incentives for the meaningful use of technology in healthcare delivery.3 according to an annual survey by the centers for disease control and prevention , the use of emrs by office - based physicians increased from 18% to 57% between 2001 and 2011.4 use of emr systems complete with all basic functionality also rose from 11% to 34% between 2006 and 2011 . the transition from paper to emrs not only reduces medical errors and improves communication between providers , but also increases the value and feasibility of medical informatics applications . using text from emrs , natural language processing ( nlp ) has the potential to supplement or replace manual chart review and electronic diagnosis codes in identifying primary and recurrent cancers . a number of studies have already demonstrated the utility of nlp in coding and extracting information from clinical text.57 however , few epidemiologic studies have employed the technology . slow adoption can not be entirely attributed to the technology 's recentness , as working implementations have existed for nearly two decades.8 9 more likely , adoption has been limited by requirements for integration with clinical data systems , technical complexity , and habitual use of medical claims data . in 2011 , chapman et al noted that despite continued improvements in nlp performance , the technology is rarely employed in clinical research settings.10 this is attributed , in part , to a lack of focus on the end - user during development , specifically as it relates to implementation costs and customizability . furthermore , nlp functionality in emr and other clinical systems software currently provides limited value to researchers due to lack of customizability and inability to be readily shared between collaborating institutions . scent was developed to increase the attractiveness of nlp to clinical and epidemiologic researchers by reducing implementation barriers and ensuring accessibility in collaborative multi - site research . scent uses functionality from sas base ( v.9.2 , sas institute ) and does not require the text miner add - on module . components of scent consist primarily of sas macro libraries and collections of excel support files . rules - based nlp approaches have been used effectively in previous studies11 12 and informatics for integrating biology and the bedside challenges.1315 while scent has the flexibility to assign codes to electronic text using a number of different coding systems , snomed 3.x was initially selected due to its use by the copathplus ( v.3.2 ; cerner dht , inc . , copathplus features synoptic reporting , a process by which pathologists provide structured results using predefined cancer checklists.16 results are assigned snomed 3.x codes by copathplus , which can be reviewed and subsequently modified by reporting pathologists . process diagram for a sas - based coding , extraction , and nomenclature tool ( scent ) . for clinical concepts to be matched by scent , their keywords must be found , without negation , in proximate distance to each other . words within reports that contribute to concept matches are tagged with relevant information , such as disease extent and snomed code . scent examines text surrounding concept matches to differentiate clinical suspicions from conclusive findings and to determine disease extent ( eg , non - invasive , invasive , or metastatic ) . scent relies on a dictionary of approximately 1000 clinical concepts and associated snomed 3.x codes related to morphology , anatomic site , and procedural type . the malignancy potential of each morphology concept was classified by up to four physicians with expert pathology or oncology knowledge . dictionary concepts are tokenized into their component words and enhanced with regular expression logic to account for synonyms and plural words . for example , the intraductal papillary adenocarcinoma concept is broken into three distinct words and intraductal is replaced with ( ( intra)?duct(al)?) to match mentions of intraductal , ductal , or duct . in addition to regular expressions that are manually added to individual concepts , some transformations are uniformly applied to the dictionary , including partial reverse lemmatization to account for plural words . exclusionary words are also included for some concepts to prevent matches from occurring in unrelated contexts . , scent performs a number of preprocessing tasks . in the pathology database at kpsc , report text spanning multiple records is often split at locations other than sentence boundaries . to facilitate matching of clinical concepts in addition to recreating sentence boundaries , special characters are removed and common abbreviations replaced . sample pathology report text , in original and preprocessed forms , can be seen in figure 2 . sample pathology report text following preprocessing by a sas - based coding , extraction , and nomenclature tool ( scent ) . using a dictionary of clinical concepts , scent examines preprocessed pathology report text for concept matches . in preparation , a clinical concept dictionary is read into memory and stored as a sas hash object for future use . the text of each report is split by sentence and stored in arrays of character variables . additional hash objects are used to record intermediate information , such as match results of individual concept words , match positioning , and negation status . after preparing the necessary arrays and hash objects the clinical concept dictionary is processed separately for each new sentence and a search for the words of each concept is performed . unmatched words are recorded to prevent unnecessary subsequent searches with concepts containing previously unmatched words . the text surrounding matches is examined for potential negation and the positions of non - negated matches are recorded . dictionary concepts are considered fully matched if all words are found in proximate distance to each other . this distance is a token constant , defined as 10 words in the current study , and ignores prepositions , articles , and certain other words . snomed codes associated with matched concepts is examined to classify disease extent ( eg , non - invasive , invasive , or metastatic ) . as shown in figure 3 , words from reports that contribute to concept matches are tagged with relevant information , such as disease extent and snomed code . scent applies a single tag to multiple match words if they are adjacent or separated by only common prepositions and articles . sample pathology report text following preprocessing and code assignment by a sas - based coding , extraction , and nomenclature tool ( scent ) . using codes from matched clinical concepts and hierarchical decision rules , scent classifies the overall status of reports as either benign , borderline , basaloid , or malignant . the disease extent of assigned morphology codes and anatomic sites are used to differentiate between new primary and recurrent malignancies . to facilitate the process , anatomic site classifications are consolidated into categories . for example , the sternum and clavicle sites are considered part of the bone category . sites relating to regional disease spread , such as the neck and groin , are consolidated into the lymph nodes category . in the case of identified cancer metastases , scent attempts to determine both origin and metastatic sites . this approach is similar to that of the negex algorithm , which has been used successfully in multiple studies.1719 scent contains approximately 40 negation expressions , each assigned values limiting the acceptable distance between themselves and concept matches . free ( of|from) will negate concept matches appearing up to 10 words before or after the expression . without is valid up to only four words before concept matches and zero following them . the maximum number of words permitted between concept matches and negation expressions is modified by the presence of certain words , such as coordinating conjunctions ( eg , and ) . to differentiate clinical suspicions from conclusive findings , scent examines text surrounding matches for uncertainty cues , such as suspicious and uncertain. this process shares some conceptual similarity with the implementation of speculation cues by clark et al in their mitre system.20 the scope of uncertainty by scent , however , is determined using fixed token distances rather than analysis of linguistic structure and statistical classifiers . a validation study was conducted under institutional review board approval using emr records of breast and prostate cancer patients at kpsc . electronic pathology reports were selected for validation due to their availability and significance in diagnosing most cancers . to assess the accuracy of scent , its classifications of pathology reports were compared to those of experienced chart abstractors . breast cancer cases were diagnosed from 2000 to 2007 with american joint committee on cancer ( ajcc ) stage 0iii tumors , and had no prior history of cancer . all prostate cancer cases diagnosed from 2000 to 2005 were included irrespective of stage and previous cancer history . to address needs for improved recurrence identification in ongoing research at kpsc , validation focused on the period following cancer diagnosis and treatment . of the 400 patients randomly selected for each cancer type , 206 breast and 186 prostate cancer patients had one or more pathology reports during the period beginning 6 months after diagnosis and subsequent to primary treatment(s ) through the end of 2008 . in total , 490 breast and 425 prostate cancer patient pathology reports were reviewed by two trained abstractors , one for each cancer group . each report was classified according to malignancy status . in the case of malignant and suspicious findings non - melanoma skin malignancies were considered benign by abstractors , scent , and classifications based on copathplus . independent reviewers made final classifications for the small number of cases about which abstractors were uncertain . reviewers also adjudicated the few cases of discordant classifications between scent and abstractors . to reduce the time and cost of the validation process , we used scent to output pathology text , as can be seen in figure 4 . concepts relating to anatomic site were highlighted in green , malignancies in shades of blue according to disease extent , and suspicious findings in orange . abstractors were instructed to fully review the text of each output report . to investigate the potential for bias stemming from scent highlights , a total of 73 reports were reviewed from the first 30 breast cancer patients in the random sample . performance of scent and classifications based on copathplus were assessed using standard evaluation metrics , including : sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , and cohen 's . sample chart review form used by abstractors to classify the pathology reports of breast and prostate cancer patients . scent uses functionality from sas base ( v.9.2 , sas institute ) and does not require the text miner add - on module . components of scent consist primarily of sas macro libraries and collections of excel support files . rules - based nlp approaches have been used effectively in previous studies11 12 and informatics for integrating biology and the bedside challenges.1315 while scent has the flexibility to assign codes to electronic text using a number of different coding systems , snomed 3.x was initially selected due to its use by the copathplus ( v.3.2 ; cerner dht , inc . , copathplus features synoptic reporting , a process by which pathologists provide structured results using predefined cancer checklists.16 results are assigned snomed 3.x codes by copathplus , which can be reviewed and subsequently modified by reporting pathologists . process diagram for a sas - based coding , extraction , and nomenclature tool ( scent ) . for clinical concepts to be matched by scent , their keywords must be found , without negation , in proximate distance to each other . words within reports that contribute to concept matches are tagged with relevant information , such as disease extent and snomed code . scent examines text surrounding concept matches to differentiate clinical suspicions from conclusive findings and to determine disease extent ( eg , non - invasive , invasive , or metastatic ) . scent relies on a dictionary of approximately 1000 clinical concepts and associated snomed 3.x codes related to morphology , anatomic site , and procedural type . the malignancy potential of each morphology concept was classified by up to four physicians with expert pathology or oncology knowledge . dictionary concepts are tokenized into their component words and enhanced with regular expression logic to account for synonyms and plural words . for example , the intraductal papillary adenocarcinoma concept is broken into three distinct words and intraductal is replaced with ( ( intra)?duct(al)?) to match mentions of intraductal , ductal , or duct . in addition to regular expressions that are manually added to individual concepts , some transformations are uniformly applied to the dictionary , including partial reverse lemmatization to account for plural words . exclusionary words are also included for some concepts to prevent matches from occurring in unrelated contexts . neck anatomic site concept , for instance , includes bladder as an exclusionary word to prevent matching when to prepare electronic pathology report text for analysis , scent performs a number of preprocessing tasks . in the pathology database at kpsc , report text spanning multiple records is often split at locations other than sentence boundaries . to facilitate matching of clinical concepts , in addition to recreating sentence boundaries , special characters are removed and common abbreviations replaced . sample pathology report text , in original and preprocessed forms , can be seen in figure 2 . sample pathology report text following preprocessing by a sas - based coding , extraction , and nomenclature tool ( scent ) . using a dictionary of clinical concepts , scent examines preprocessed pathology report text for concept matches . in preparation , a clinical concept dictionary is read into memory and stored as a sas hash object for future use . the text of each report is split by sentence and stored in arrays of character variables . additional hash objects are used to record intermediate information , such as match results of individual concept words , match positioning , and negation status . after preparing the necessary arrays and hash objects the clinical concept dictionary is processed separately for each new sentence and a search for the words of each concept is performed . unmatched words are recorded to prevent unnecessary subsequent searches with concepts containing previously unmatched words . the text surrounding matches is examined for potential negation and the positions of non - negated matches are recorded . dictionary concepts are considered fully matched if all words are found in proximate distance to each other . this distance is a token constant , defined as 10 words in the current study , and ignores prepositions , articles , and certain other words . snomed codes associated with matched concepts is examined to classify disease extent ( eg , non - invasive , invasive , or metastatic ) . as shown in figure 3 , words from reports that contribute to concept matches are tagged with relevant information , such as disease extent and snomed code . scent applies a single tag to multiple match words if they are adjacent or separated by only common prepositions and articles . sample pathology report text following preprocessing and code assignment by a sas - based coding , extraction , and nomenclature tool ( scent ) . using codes from matched clinical concepts and hierarchical decision rules , scent classifies the overall status of reports as either benign , borderline , basaloid , or malignant . the disease extent of assigned morphology codes and anatomic sites are used to differentiate between new primary and recurrent malignancies . to facilitate the process , anatomic site classifications are consolidated into categories . for example , the sternum and clavicle sites are considered part of the bone category . sites relating to regional disease spread , such as the neck and groin , are consolidated into the lymph nodes category . in the case of identified cancer metastases , scent attempts to determine both origin and metastatic sites . this approach is similar to that of the negex algorithm , which has been used successfully in multiple studies.1719 scent contains approximately 40 negation expressions , each assigned values limiting the acceptable distance between themselves and concept matches . free ( of|from) will negate concept matches appearing up to 10 words before or after the expression . in contrast , the negation word without is valid up to only four words before concept matches and zero following them . the maximum number of words permitted between concept matches and negation expressions is modified by the presence of certain words , such as coordinating conjunctions ( eg , and ) . to differentiate clinical suspicions from conclusive findings , scent examines text surrounding matches for uncertainty cues , such as suspicious and uncertain. this process shares some conceptual similarity with the implementation of speculation cues by clark et al in their mitre system.20 the scope of uncertainty by scent , however , is determined using fixed token distances rather than analysis of linguistic structure and statistical classifiers . a validation study was conducted under institutional review board approval using emr records of breast and prostate cancer patients at kpsc . electronic pathology reports were selected for validation due to their availability and significance in diagnosing most cancers . to assess the accuracy of scent , its classifications of pathology reports were compared to those of experienced chart abstractors . breast cancer cases were diagnosed from 2000 to 2007 with american joint committee on cancer ( ajcc ) stage 0iii tumors , and had no prior history of cancer . all prostate cancer cases diagnosed from 2000 to 2005 were included irrespective of stage and previous cancer history . to address needs for improved recurrence identification in ongoing research at kpsc , validation focused on the period following cancer diagnosis and treatment . of the 400 patients randomly selected for each cancer type , 206 breast and 186 prostate cancer patients had one or more pathology reports during the period beginning 6 months after diagnosis and subsequent to primary treatment(s ) through the end of 2008 . in total , 490 breast and 425 prostate cancer patient pathology reports were reviewed by two trained abstractors , one for each cancer group . each report was classified according to malignancy status . in the case of malignant and suspicious findings non - melanoma skin malignancies were considered benign by abstractors , scent , and classifications based on copathplus . independent reviewers made final classifications for the small number of cases about which abstractors were uncertain . reviewers also adjudicated the few cases of discordant classifications between scent and abstractors . to reduce the time and cost of the validation process , we used scent to output pathology text , as can be seen in figure 4 . concepts relating to anatomic site were highlighted in green , malignancies in shades of blue according to disease extent , and suspicious findings in orange . abstractors were instructed to fully review the text of each output report . to investigate the potential for bias stemming from scent highlights , a total of 73 reports were reviewed from the first 30 breast cancer patients in the random sample . performance of scent and classifications based on copathplus were assessed using standard evaluation metrics , including : sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , and cohen 's . sample chart review form used by abstractors to classify the pathology reports of breast and prostate cancer patients . as shown in table 1 , scent was highly concordant with abstractor classifications , achieving values of 0.96 and 0.95 in the breast and prostate cancer groups , respectively . scent identified 51 of 54 new primary and 60 of 61 recurrent cancer cases across both groups , with only three false positives in 792 true benign cases . agreement was moderate for classifications based on the codes assigned by copathplus , with values of 0.72 and 0.65 in the breast and prostate cancer groups , respectively . to calculate overall performance metrics , results were consolidated into two categories : benign / suspicious and primary / recurrent cancer . within both the breast and prostate cancer groups , scent reached levels of or above 94% for sensitivity , specificity , ppv , and npv ( table 2 ) . ppv was moderately high in both the breast ( 90% ) and prostate ( 88% ) cancer groups . sensitivity for classifications based on copathplus in detecting true positive malignancies was moderate in the breast ( 74% ) and prostate ( 71% ) cancer groups . pathology report classifications of a sas - based coding , extraction , and nomenclature tool ( scent ) and cerner 's copathplus coding , as compared with abstractor review includes the pathology reports of randomly sampled breast and prostate cancer patients from 6 months after diagnosis and subsequent to primary treatment . validation metrics for a sas - based coding , extraction , and nomenclature tool ( scent ) and cerner 's copathplus coding , as compared with abstractor review of pathology reports includes the pathology reports of randomly sampled breast and prostate cancer patients from 6 months after diagnosis and subsequent to primary treatment . a secondary assessment of scent was conducted to evaluate its ability to extract ajcc tumor staging and gleason score information . scent identified and accurately extracted all tumor ( t ) , lymph node ( n ) , and metastasis ( m ) staging information from 19 pathology reports across both cancer groups . within the prostate cancer group , scent identified all gleason scores and accurately extracted the tumor pattern scores from each of the 20 reports . there were no instances in which scent failed to identify reports containing tumor staging or gleason score information . the favorable validation results in this study suggest that sas - based nlp can be used to accurately identify and extract information from electronic clinical text . using the pathology reports of patients previously diagnosed and treated for breast and prostate cancer , scent successfully identified 51 of 54 primary and 60 of 61 recurrent cancers . performance was similar for both the breast and prostate cancer groups , with measures of sensitivity , specificity , ppv , and npv of or above 94% . classifications using codes assigned by copathplus were moderately successful in identifying incident and recurrent cancers within both cancer groups . sensitivity in the breast and prostate cancer groups was 74% and 71% , respectively , while ppv was 90% and 88% . to confirm the accuracy of abstractor classifications , an oncologist reviewed pathology reports associated with discordant copathplus cases . codes assigned to discordant copathplus cases were reviewed to better understand the sources of classification error . primary sources of error for classifications based on copathplus were : coding of historical malignancies , assignment of overly general codes , and lack of code assignment for unknown reasons . additionally , there were three false positives relating to the coding of residual malignancy , which had been excluded a priori from study definitions of primary and recurrent cancer . the widespread adoption of sas in clinical analysis and research settings ensures that scent is highly accessible . integration of sas with relevant data systems has already been established in these settings , allowing electronic text to be readily extracted for analysis . by avoiding the need for additional software installation or systems integration , technical and administrative barriers to nlp implementation additionally , scent does not require annotated training data and can be used by staff without specialized informatics or machine learning knowledge . primary and recurrent cancers are frequently among the main study outcomes in epidemiologic cancer research . despite the importance of accurately identifying these events , electronic diagnosis codes often lack accuracy and cancer registry data are not always available . scent achieved high levels of sensitivity and specificity in identifying pathologically diagnosed malignancies among patients previously diagnosed with breast or prostate cancer . in addition to its utility in retrospective research , scent can be used in prospective research and population care management . cancer registry data , when available , are commonly delayed for months after patient diagnosis . it is often impractical to rely on these data for intervention studies relating to cancer treatment . scent is currently being implemented in one such study aimed at improving adherence to adjuvant hormonal therapy among breast cancer patients . beyond its value in identifying pathologically diagnosed primary and recurrent cancers , scent has the potential for numerous other clinical applications . questions investigated by clinical analysts are often central to medical leadership and decisions relating to patient care . analysts face issues similar to those of prospective research studies , relying on electronic diagnosis codes and available cancer registry data to assess the quality of cancer care . scent has the potential to enhance the availability and depth of data and could be used to identify appropriate patient populations for evaluation . for example , while treatment recommendations for certain precancerous lesions may dictate watchful waiting at present , scent could be used to identify affected patients if those recommendations were to change . extraction of even small numbers of basic data elements can be infeasible in large sample studies due to labor costs and time requirements . as described in the materials and methods section and illustrated in figure 4 , scent can be used to highlight desired clinical concepts within electronic text . this functionality has the potential to dramatically reduce the time and costs associated with chart review . the benefits of scent in expediting chart review will be further explored and quantified in future work . while scent performed well in classifying and extracting information from electronic pathology reports these sources will likely necessitate modifications to the preprocessing and decision rules employed by scent . modifications to preprocessing rules may also be needed prior to implementing scent outside of kpsc . additionally , text sources such clinical progress notes are less structured and have greater linguistic variability . scent does not use formal part - of - speech tagging and is therefore limited in its ability to disambiguate and contextualize identified clinical concepts . the performance of scent relative to that of a general purpose nlp solution will need to be assessed across multiple text sources to identify performance gaps and inform appropriate usage . future development and validation efforts for scent will include identification of incident and recurrent cancer cases that are diagnosed without pathological testing . radiology reports and clinical progress notes are expected to be the primary sources for identifying non - pathologically diagnosed cancer cases . scent may also have generalized utility extracting information outside of the oncology domain , such as standard scoring of cognitive impairment from the mini mental state examination ( mmse ) and depression from the patient health questionnaire ( phq-9 ) . functionality was developed to assign confidence scores to individual scent dictionary concepts according to their false - positive rates from chart review results . these scores can be used in conjunction with specified minimum confidence thresholds to fit the sensitivity requirements and false - positive tolerance of individual studies . this process will be updated to use bayesian methods for incorporating additional evidence from subsequent chart reviews . the feasibility of adopting additional statistical methods to enhance the rules - based classification approach of scent will also be assessed . scent was highly successful in identifying and extracting information on primary and recurrent cancers from electronic pathology reports . this functionality has the potential to provide significant value to clinical and epidemiologic researchers , particularly when statistical nlp is infeasible due to resource or other constraints . scent is proof of concept for sas - based nlp applications that can be easily shared between institutions and used to support clinical and epidemiologic research .

objectivesignificant limitations exist in the timely and complete identification of primary and recurrent cancers for clinical and epidemiologic research . a sas - based coding , extraction , and nomenclature tool ( scent ) was developed to address this problem.materials and methodsscent employs hierarchical classification rules to identify and extract information from electronic pathology reports . reports are analyzed and coded using a dictionary of clinical concepts and associated snomed codes . to assess the accuracy of scent , validation was conducted using manual review of pathology reports from a random sample of 400 breast and 400 prostate cancer patients diagnosed at kaiser permanente southern california . trained abstractors classified the malignancy status of each report.resultsclassifications of scent were highly concordant with those of abstractors , achieving of 0.96 and 0.95 in the breast and prostate cancer groups , respectively . scent identified 51 of 54 new primary and 60 of 61 recurrent cancer cases across both groups , with only three false positives in 792 true benign cases . measures of sensitivity , specificity , positive predictive value , and negative predictive value exceeded 94% in both cancer groups.discussionfavorable validation results suggest that scent can be used to identify , extract , and code information from pathology report text . consequently , scent has wide applicability in research and clinical care . further assessment will be needed to validate performance with other clinical text sources , particularly those with greater linguistic variability.conclusionscent is proof of concept for sas - based natural language processing applications that can be easily shared between institutions and used to support clinical and epidemiologic research .

Introduction Background and significance Materials and methods Overview Clinical concepts Preprocessing Matching Negation and uncertainty Validation Results Discussion Conclusion

the identification of primary and recurrent cancer diagnoses is critical to clinical and epidemiologic research . despite its importance , however , there is substantial lag between the time of primary cancer diagnoses and complete information capture by cancer registries . consequently , researchers frequently rely on manual chart review and medical claims data , such as international classification of diseases ( icd ) codes , to identify primary and recurrent cancers . to address ongoing needs for improved identification of cancer diagnoses , a sas - based coding , extraction , and nomenclature tool ( scent ) was developed at kaiser permanente southern california ( kpsc ) . kpsc is an integrated healthcare organization that provides medical services to a diverse membership of more than 3.5 million people throughout southern california . in 1999 , warren et al analyzed the medical claims data of 6784 medicare patients and concluded that such data have limited value in accurately identifying breast cancer cases.1 more recently , lamont et al achieved high levels of sensitivity and specificity using claims data to identify cancer recurrence.2 however , that validation used a small sample of patients ( n=45 ) and the accuracy of claims data in identifying cancer recurrence has yet to be well established . additionally , a provision in the 2009 american recovery and reinvestment act may accelerate the adoption of electronic systems by providing financial incentives for the meaningful use of technology in healthcare delivery.3 according to an annual survey by the centers for disease control and prevention , the use of emrs by office - based physicians increased from 18% to 57% between 2001 and 2011.4 use of emr systems complete with all basic functionality also rose from 11% to 34% between 2006 and 2011 . using text from emrs , natural language processing ( nlp ) has the potential to supplement or replace manual chart review and electronic diagnosis codes in identifying primary and recurrent cancers . a number of studies have already demonstrated the utility of nlp in coding and extracting information from clinical text.57 however , few epidemiologic studies have employed the technology . slow adoption can not be entirely attributed to the technology 's recentness , as working implementations have existed for nearly two decades.8 9 more likely , adoption has been limited by requirements for integration with clinical data systems , technical complexity , and habitual use of medical claims data . furthermore , nlp functionality in emr and other clinical systems software currently provides limited value to researchers due to lack of customizability and inability to be readily shared between collaborating institutions . scent was developed to increase the attractiveness of nlp to clinical and epidemiologic researchers by reducing implementation barriers and ensuring accessibility in collaborative multi - site research . components of scent consist primarily of sas macro libraries and collections of excel support files . rules - based nlp approaches have been used effectively in previous studies11 12 and informatics for integrating biology and the bedside challenges.1315 while scent has the flexibility to assign codes to electronic text using a number of different coding systems , snomed 3.x was initially selected due to its use by the copathplus ( v.3.2 ; cerner dht , inc . , copathplus features synoptic reporting , a process by which pathologists provide structured results using predefined cancer checklists.16 results are assigned snomed 3.x codes by copathplus , which can be reviewed and subsequently modified by reporting pathologists . process diagram for a sas - based coding , extraction , and nomenclature tool ( scent ) . for clinical concepts to be matched by scent , their keywords must be found , without negation , in proximate distance to each other . scent relies on a dictionary of approximately 1000 clinical concepts and associated snomed 3.x codes related to morphology , anatomic site , and procedural type . the malignancy potential of each morphology concept was classified by up to four physicians with expert pathology or oncology knowledge . , scent performs a number of preprocessing tasks . in the pathology database at kpsc , report text spanning multiple records is often split at locations other than sentence boundaries . to facilitate matching of clinical concepts in addition to recreating sentence boundaries , special characters are removed and common abbreviations replaced . sample pathology report text , in original and preprocessed forms , can be seen in figure 2 . sample pathology report text following preprocessing by a sas - based coding , extraction , and nomenclature tool ( scent ) . using a dictionary of clinical concepts , scent examines preprocessed pathology report text for concept matches . in preparation , a clinical concept dictionary is read into memory and stored as a sas hash object for future use . the text of each report is split by sentence and stored in arrays of character variables . additional hash objects are used to record intermediate information , such as match results of individual concept words , match positioning , and negation status . this distance is a token constant , defined as 10 words in the current study , and ignores prepositions , articles , and certain other words . snomed codes associated with matched concepts is examined to classify disease extent ( eg , non - invasive , invasive , or metastatic ) . sample pathology report text following preprocessing and code assignment by a sas - based coding , extraction , and nomenclature tool ( scent ) . using codes from matched clinical concepts and hierarchical decision rules , scent classifies the overall status of reports as either benign , borderline , basaloid , or malignant . the disease extent of assigned morphology codes and anatomic sites are used to differentiate between new primary and recurrent malignancies . in the case of identified cancer metastases , scent attempts to determine both origin and metastatic sites . to differentiate clinical suspicions from conclusive findings , scent examines text surrounding matches for uncertainty cues , such as suspicious and uncertain. this process shares some conceptual similarity with the implementation of speculation cues by clark et al in their mitre system.20 the scope of uncertainty by scent , however , is determined using fixed token distances rather than analysis of linguistic structure and statistical classifiers . a validation study was conducted under institutional review board approval using emr records of breast and prostate cancer patients at kpsc . electronic pathology reports were selected for validation due to their availability and significance in diagnosing most cancers . to assess the accuracy of scent , its classifications of pathology reports were compared to those of experienced chart abstractors . breast cancer cases were diagnosed from 2000 to 2007 with american joint committee on cancer ( ajcc ) stage 0iii tumors , and had no prior history of cancer . all prostate cancer cases diagnosed from 2000 to 2005 were included irrespective of stage and previous cancer history . to address needs for improved recurrence identification in ongoing research at kpsc , validation focused on the period following cancer diagnosis and treatment . of the 400 patients randomly selected for each cancer type , 206 breast and 186 prostate cancer patients had one or more pathology reports during the period beginning 6 months after diagnosis and subsequent to primary treatment(s ) through the end of 2008 . in total , 490 breast and 425 prostate cancer patient pathology reports were reviewed by two trained abstractors , one for each cancer group . each report was classified according to malignancy status . in the case of malignant and suspicious findings non - melanoma skin malignancies were considered benign by abstractors , scent , and classifications based on copathplus . to reduce the time and cost of the validation process , we used scent to output pathology text , as can be seen in figure 4 . concepts relating to anatomic site were highlighted in green , malignancies in shades of blue according to disease extent , and suspicious findings in orange . abstractors were instructed to fully review the text of each output report . to investigate the potential for bias stemming from scent highlights , a total of 73 reports were reviewed from the first 30 breast cancer patients in the random sample . performance of scent and classifications based on copathplus were assessed using standard evaluation metrics , including : sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , and cohen 's . sample chart review form used by abstractors to classify the pathology reports of breast and prostate cancer patients . components of scent consist primarily of sas macro libraries and collections of excel support files . rules - based nlp approaches have been used effectively in previous studies11 12 and informatics for integrating biology and the bedside challenges.1315 while scent has the flexibility to assign codes to electronic text using a number of different coding systems , snomed 3.x was initially selected due to its use by the copathplus ( v.3.2 ; cerner dht , inc . , copathplus features synoptic reporting , a process by which pathologists provide structured results using predefined cancer checklists.16 results are assigned snomed 3.x codes by copathplus , which can be reviewed and subsequently modified by reporting pathologists . process diagram for a sas - based coding , extraction , and nomenclature tool ( scent ) . for clinical concepts to be matched by scent , their keywords must be found , without negation , in proximate distance to each other . scent relies on a dictionary of approximately 1000 clinical concepts and associated snomed 3.x codes related to morphology , anatomic site , and procedural type . the malignancy potential of each morphology concept was classified by up to four physicians with expert pathology or oncology knowledge . neck anatomic site concept , for instance , includes bladder as an exclusionary word to prevent matching when to prepare electronic pathology report text for analysis , scent performs a number of preprocessing tasks . in the pathology database at kpsc , report text spanning multiple records is often split at locations other than sentence boundaries . to facilitate matching of clinical concepts , in addition to recreating sentence boundaries , special characters are removed and common abbreviations replaced . sample pathology report text , in original and preprocessed forms , can be seen in figure 2 . sample pathology report text following preprocessing by a sas - based coding , extraction , and nomenclature tool ( scent ) . using a dictionary of clinical concepts , scent examines preprocessed pathology report text for concept matches . in preparation , a clinical concept dictionary is read into memory and stored as a sas hash object for future use . the text of each report is split by sentence and stored in arrays of character variables . additional hash objects are used to record intermediate information , such as match results of individual concept words , match positioning , and negation status . after preparing the necessary arrays and hash objects the clinical concept dictionary is processed separately for each new sentence and a search for the words of each concept is performed . this distance is a token constant , defined as 10 words in the current study , and ignores prepositions , articles , and certain other words . sample pathology report text following preprocessing and code assignment by a sas - based coding , extraction , and nomenclature tool ( scent ) . using codes from matched clinical concepts and hierarchical decision rules , scent classifies the overall status of reports as either benign , borderline , basaloid , or malignant . the disease extent of assigned morphology codes and anatomic sites are used to differentiate between new primary and recurrent malignancies . in the case of identified cancer metastases , scent attempts to determine both origin and metastatic sites . this process shares some conceptual similarity with the implementation of speculation cues by clark et al in their mitre system.20 the scope of uncertainty by scent , however , is determined using fixed token distances rather than analysis of linguistic structure and statistical classifiers . a validation study was conducted under institutional review board approval using emr records of breast and prostate cancer patients at kpsc . electronic pathology reports were selected for validation due to their availability and significance in diagnosing most cancers . to assess the accuracy of scent , its classifications of pathology reports were compared to those of experienced chart abstractors . breast cancer cases were diagnosed from 2000 to 2007 with american joint committee on cancer ( ajcc ) stage 0iii tumors , and had no prior history of cancer . all prostate cancer cases diagnosed from 2000 to 2005 were included irrespective of stage and previous cancer history . to address needs for improved recurrence identification in ongoing research at kpsc , validation focused on the period following cancer diagnosis and treatment . of the 400 patients randomly selected for each cancer type , 206 breast and 186 prostate cancer patients had one or more pathology reports during the period beginning 6 months after diagnosis and subsequent to primary treatment(s ) through the end of 2008 . in total , 490 breast and 425 prostate cancer patient pathology reports were reviewed by two trained abstractors , one for each cancer group . each report was classified according to malignancy status . in the case of malignant and suspicious findings non - melanoma skin malignancies were considered benign by abstractors , scent , and classifications based on copathplus . to reduce the time and cost of the validation process , we used scent to output pathology text , as can be seen in figure 4 . concepts relating to anatomic site were highlighted in green , malignancies in shades of blue according to disease extent , and suspicious findings in orange . abstractors were instructed to fully review the text of each output report . to investigate the potential for bias stemming from scent highlights , a total of 73 reports were reviewed from the first 30 breast cancer patients in the random sample . performance of scent and classifications based on copathplus were assessed using standard evaluation metrics , including : sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , and cohen 's . sample chart review form used by abstractors to classify the pathology reports of breast and prostate cancer patients . as shown in table 1 , scent was highly concordant with abstractor classifications , achieving values of 0.96 and 0.95 in the breast and prostate cancer groups , respectively . scent identified 51 of 54 new primary and 60 of 61 recurrent cancer cases across both groups , with only three false positives in 792 true benign cases . agreement was moderate for classifications based on the codes assigned by copathplus , with values of 0.72 and 0.65 in the breast and prostate cancer groups , respectively . to calculate overall performance metrics , results were consolidated into two categories : benign / suspicious and primary / recurrent cancer . within both the breast and prostate cancer groups , scent reached levels of or above 94% for sensitivity , specificity , ppv , and npv ( table 2 ) . ppv was moderately high in both the breast ( 90% ) and prostate ( 88% ) cancer groups . sensitivity for classifications based on copathplus in detecting true positive malignancies was moderate in the breast ( 74% ) and prostate ( 71% ) cancer groups . pathology report classifications of a sas - based coding , extraction , and nomenclature tool ( scent ) and cerner 's copathplus coding , as compared with abstractor review includes the pathology reports of randomly sampled breast and prostate cancer patients from 6 months after diagnosis and subsequent to primary treatment . validation metrics for a sas - based coding , extraction , and nomenclature tool ( scent ) and cerner 's copathplus coding , as compared with abstractor review of pathology reports includes the pathology reports of randomly sampled breast and prostate cancer patients from 6 months after diagnosis and subsequent to primary treatment . a secondary assessment of scent was conducted to evaluate its ability to extract ajcc tumor staging and gleason score information . scent identified and accurately extracted all tumor ( t ) , lymph node ( n ) , and metastasis ( m ) staging information from 19 pathology reports across both cancer groups . within the prostate cancer group , scent identified all gleason scores and accurately extracted the tumor pattern scores from each of the 20 reports . there were no instances in which scent failed to identify reports containing tumor staging or gleason score information . the favorable validation results in this study suggest that sas - based nlp can be used to accurately identify and extract information from electronic clinical text . using the pathology reports of patients previously diagnosed and treated for breast and prostate cancer , scent successfully identified 51 of 54 primary and 60 of 61 recurrent cancers . performance was similar for both the breast and prostate cancer groups , with measures of sensitivity , specificity , ppv , and npv of or above 94% . classifications using codes assigned by copathplus were moderately successful in identifying incident and recurrent cancers within both cancer groups . sensitivity in the breast and prostate cancer groups was 74% and 71% , respectively , while ppv was 90% and 88% . to confirm the accuracy of abstractor classifications , an oncologist reviewed pathology reports associated with discordant copathplus cases . additionally , there were three false positives relating to the coding of residual malignancy , which had been excluded a priori from study definitions of primary and recurrent cancer . by avoiding the need for additional software installation or systems integration , technical and administrative barriers to nlp implementation additionally , scent does not require annotated training data and can be used by staff without specialized informatics or machine learning knowledge . primary and recurrent cancers are frequently among the main study outcomes in epidemiologic cancer research . scent achieved high levels of sensitivity and specificity in identifying pathologically diagnosed malignancies among patients previously diagnosed with breast or prostate cancer . in addition to its utility in retrospective research , scent can be used in prospective research and population care management . scent is currently being implemented in one such study aimed at improving adherence to adjuvant hormonal therapy among breast cancer patients . beyond its value in identifying pathologically diagnosed primary and recurrent cancers , scent has the potential for numerous other clinical applications . analysts face issues similar to those of prospective research studies , relying on electronic diagnosis codes and available cancer registry data to assess the quality of cancer care . scent has the potential to enhance the availability and depth of data and could be used to identify appropriate patient populations for evaluation . for example , while treatment recommendations for certain precancerous lesions may dictate watchful waiting at present , scent could be used to identify affected patients if those recommendations were to change . extraction of even small numbers of basic data elements can be infeasible in large sample studies due to labor costs and time requirements . as described in the materials and methods section and illustrated in figure 4 , scent can be used to highlight desired clinical concepts within electronic text . the benefits of scent in expediting chart review will be further explored and quantified in future work . while scent performed well in classifying and extracting information from electronic pathology reports these sources will likely necessitate modifications to the preprocessing and decision rules employed by scent . additionally , text sources such clinical progress notes are less structured and have greater linguistic variability . scent does not use formal part - of - speech tagging and is therefore limited in its ability to disambiguate and contextualize identified clinical concepts . the performance of scent relative to that of a general purpose nlp solution will need to be assessed across multiple text sources to identify performance gaps and inform appropriate usage . future development and validation efforts for scent will include identification of incident and recurrent cancer cases that are diagnosed without pathological testing . radiology reports and clinical progress notes are expected to be the primary sources for identifying non - pathologically diagnosed cancer cases . functionality was developed to assign confidence scores to individual scent dictionary concepts according to their false - positive rates from chart review results . these scores can be used in conjunction with specified minimum confidence thresholds to fit the sensitivity requirements and false - positive tolerance of individual studies . this process will be updated to use bayesian methods for incorporating additional evidence from subsequent chart reviews . the feasibility of adopting additional statistical methods to enhance the rules - based classification approach of scent will also be assessed . scent was highly successful in identifying and extracting information on primary and recurrent cancers from electronic pathology reports . this functionality has the potential to provide significant value to clinical and epidemiologic researchers , particularly when statistical nlp is infeasible due to resource or other constraints . scent is proof of concept for sas - based nlp applications that can be easily shared between institutions and used to support clinical and epidemiologic research .

the identification of the gene responsible for the primary defect in x - linked muscular dystrophy led to the discovery of its protein product , the large membrane cytoskeletal element dystrophin . the full - length dp427 isoform of dystrophin is almost completely missing from the dystrophic surface membrane , which triggers the characteristic dystrophic phenotype of progressive skeletal muscle weakness and fibre wasting . mutations in the dystrophin gene cause the most prevalent and lethal gender - specific genetic disease of childhood , duchenne muscular dystrophy ( dmd ) , and its more benign and less frequent counterpart becker 's muscular dystrophy , as well as x - linked dilated cardiomyopathy . dystrophin forms a supramolecular complex with various surface glycoproteins that are involved in cellular signaling , receptor clustering and stabilization of the cellular periphery . tissue - specific variations in dystrophin isoforms delegate the composition and subcellular localization of the dystrophin - glycoprotein complex , and hence the function of the specific membrane assembly may vary between the brain , heart , and skeletal muscles . in muscular dystrophy , deficiency in dystrophin causes a significant reduction in the surface - associated glycoprotein complex , which severely impairs the integrity of the sarcolemma and causes a broad spectrum of downstream alterations such as enhanced proteolytic destruction of muscle proteins and abnormal ca - handling . this classifies dmd primarily as a genetic disorder of skeletal muscle , but pathological alterations are also present in cardiac muscle and diaphragm , as well as the peripheral and central nervous system . the majority of dmd patients develop clinical cardiac symptoms during the second decade of life , including arrhythmias , cardiomyopathy , and regional wall abnormalities , leading to fatal cardiac complications in about 20% of cases . on the cellular level , the gradual replacement of cardiac fibres by connective and fatty tissue is a hallmark of the dystrophic heart . while dystrophin - deficient skeletal muscles undergo cycles of fibre degeneration and regeneration , dystrophic heart fibres exhibit only a limited regenerative capacity causing a progressive decline in the cardiomyocyte population of the dystrophic heart . since cardiomyopathy is a frequent occurrence in inherited muscular dystrophies , a number of novel therapeutic approaches are tested to specifically address the cardiac symptoms [ 17 , 18 ] , especially membrane - stabilizing agents such as tri - block poloxamers . in this study , we used the x - linked muscular dystrophy ( mdx ) mouse , a well - established animal model of dmd that is missing dystrophin isoform dp427 . although the dystrophic phenotype of young mdx hearts does not represent a perfect replica of dmd - related cardiomyopathic complications , in aged mdx hearts the pathological changes are of considerable clinical relevance [ 2325 ] and cardiac mdx fibres are frequently used to evaluate new treatment strategies to counteract cardiomyopathic complications [ 2629 ] . the absence of cardiac dystrophin is clearly associated with necrotic changes , extensive infiltration of inflammatory cells , increase in adipose tissue , interstitial fibrosis , tachycardia , and impaired contractile properties in the mdx heart [ 22 , 30 , 31 ] . in analogy to dmd hearts , the expression of dystrophin - associated glycoproteins is also greatly reduced in dystrophin - deficient mdx heart cells . secondary abnormalities in cardiac fibres from the mdx mouse include the drastic reduction in key luminal ca - binding proteins and abnormal stress - induced ca - influx into the cytosol . interestingly , both physical exercise and aging seem to accelerate the dystrophic process in cardiac mdx tissue [ 35 , 36 ] , whereby a recent study by spurney et al . revealed that heart dysfunction was most prominent at 9 months of age . this makes this age group of mdx hearts a suitable dmd model for investigating global changes in the cardiac mdx protein complement and was therefore chosen in this study . the development of mass spectrometry - based proteomics for the swift identification of proteins has decisively enhanced the analytical capability of comparative biomedical studies . in addition , the introduction of fluorescent dyes has drastically increased the total number of identifiable two - dimensional spots , whereby fluorescence difference in - gel electrophoresis ( dige ) can be considered one of the most advanced biomedical tools for studying two different sets of soluble protein complements . dige analysis generates highly reproducible findings due to greatly reduced gel - to - gel variations . we have therefore used this technique here for the proteomic profiling of the dystrophic mdx heart . the usefulness of proteomics for determining the biomarker signature of animal disease models of muscular disorders has recently been reviewed . an important aspect of genetic mouse models is that they show , due to their inbred status , genetically much less interindividual differences than human patients . therefore , considerably fewer experimental repeats are capable of producing meaningful proteomic data . with respect to x - linked muscular dystrophy , proteomic studies have mostly focused on mdx skeletal muscles and have identified numerous biomarker candidates , such as adenylate kinase , calsequestrin , regucalcin , and the small stress protein cvhsp . in general , although the initial pathobiochemical consequence of a primary genetic abnormality in dystrophin is a severe reduction in dystrophin - associated proteins , this seems to result in a variety of downstream alterations in various classes of muscle proteins involved in fibre contraction , chaperone function , ion homeostasis , cytoskeleton formation and metabolism . building on the recent findings of a combined metabolomic and proteomic investigation into dystrophin deficiency in cardiac muscle , this report describes the detailed dige analysis of the expression levels of fluorescently - tagged dystrophic versus normal cardiac proteins using both ph 47 and ph 611 range gels . following the densitometric determination of the expression pattern of 2,509 cardiac protein spots , mass spectrometry identified 26 proteins being decreased , including various myosin light chains , tropomyosin , actin , adenylate kinase , creatine kinase , vimentin , atp synthase , fatty acid binding - protein , isocitrate dehydrogenase , nadh dehydrogenase , myozenin , porin , and peroxiredoxin , and 3 proteins being increased , including lamin - a / c and nucleoside diphosphate kinase . these findings suggest that the loss in dystrophin causes abnormalities in cardiac metabolism , the cellular stress response , the cytoskeleton , and the contractile machinery . in the future , these results will be useful for complementing studies into the molecular mechanisms of muscular dystrophy and for evaluating the effects of novel drugs , genetic modifications , or cell - based therapies on disease progression . for gel electrophoretic analyses , analytical grade chemicals and materials were purchased from amersham biosciences / ge healthcare , little chalfont , buckinghamshire , uk ( cydye dige fluor minimal dyes cy3 and cy5 ; 24 cm ph 47 and 18 cm ph 611 immobilized ph gradient ( ipg ) strips ; ipg buffer ; iodoacetamide ) , biorad laboratories , hemel - hempstead , hertfordshire , uk ( laemmli - type buffer system ; protein molecular mass markers ) , national diagnostics , atlanta , ga , usa ( ultrapure protogel acrylamide stock solution ) , cosmo bio company , tokyo , japan ( 2d silver stain ii kit ) , and perbio science , northumberland , uk ( coomassie brilliant blue g-250 ) . protease inhibitors and chemiluminescence substrate were obtained from roche diagnostics ( mannheim , germany ) . sequencing grade - modified trypsin was purchased from promega ( madison , wi , usa ) . , cambridge , uk ( ab28172 to prohibitin ; ab36329 to isocitrate dehydrogenase ; ab14734 to porin isoform vdac1 ; ab54824 to adenylate kinase isoform ak1 ; ab5432 to atp synthase , ab16915 to cardiac fatty acid binding protein ) , sigma chemical company , dorset , uk ( mab noq7.5.4d to slow / cardiac myosin heavy chain ; pab to laminin ) , visionbiosystems novocastra , newcastle upon tyne , uk ( mab ncl - dys1 against the dp427 isoform of dystrophin ) , thermo fisher scientific inc . , rockford , il , usa ( mab rd301 to desmin ) and santa cruz biotechnology , santa cruz , ca , usa ( sc27992 to succinate dehydrogenase ) . all secondary antibodies were purchased from chemicon international ( temecula , ca , usa ) . for confocal microscopy , superfrost plus positively - charged microscope slides were purchased from menzel glaesser ( braunschweig , germany ) . optimum cutting temperature ( oct ) compound was from sakura finetek europe b.v ( zoeterwoude , netherlands ) and p - phenylenediamine ( ppd)-glycerol was purchased from citifluor ltd . mitotracker red cmxros dye and alexa fluor - conjugated secondary antibodies were from invitrogen molecular probes ( bio sciences ltd . the dna binding dye diamidino - phenylindole ( dapi ) and all other chemicals used were of analytical grade and purchased from sigma chemical company , dorset , uk . hearts from male 9-month old c57 control mice and age - matched dystrophic mdx mice were obtained through the bioresource unit of nui maynooth and the animal house of the university of bielefeld . animals were kept under standard conditions and all procedures were performed in accordance with german and irish guidelines on the use of animals for scientific experiments . as previously demonstrated by immunoblotting , the mdx mouse cohort used in this study lacks the 427 kda isoform of cardiac dystrophin and shows a drastic reduction in dystrophin - associated glycoproteins , such as dystroglycans and sarcoglycans [ 32 , 33 ] . for the proteomic profiling of crude cardiac extracts , three normal and three age - matched dystrophic hearts were separately prepared by washing with distilled water to remove excess blood . ground hearts were solubilized in lysis buffer with the ratio 100 mg wet weight to 1 ml lysis buffer consisting of 9.5 m urea , 2% chaps , 0.8% ipg buffer ph 310 , 1% ( w / v ) dtt and protease inhibitors . samples were gently rocked for 30 minutes and protein concentration was determined using the bradford assay system . for immunoblot analysis samples were placed into homogenization buffer ( 0.5 m hepes ph 7.4 , 200 mm egta , 10% ( w / v ) sucrose , 3 mm mgcl2 , and 0.1% ( w / v ) nan3 ) . extracts were shaken at 4c on a thermomixer from eppendorf ( hamburg , germany ) for 4 hours and then centrifuged at 4c for 20 minutes at 20,000 g in an eppendorf 5417r bench centrifuge . potential differences in the expression pattern of the soluble proteome from normal and mdx cardiac muscle were analysed using the fluorescence difference in - gel electrophoretic dige technique , as recommended by karp and lilley . the fluorescent dyes cy3 and cy5 were reconstituted as a stock solution of 1 mm in fresh dimethylformamide , then diluted to a 0.2 mm solution prior to use . labeling was performed with 200 pmols of cy3 fluor dye per 25 g protein . labelled samples were then vortexed and incubated for 30 minutes on ice in the dark . for isoelectric focusing , ph 611 ipg strips were rehydrated for 12 hours in rehydration buffer ( 8 m urea , 0.5% chaps , 0.2% dtt , and 0.2% ampholytes ) with the addition of 1.2% ( v / v ) destreak rehydration solution ( amersham biosciences / ge healthcare , little chalfont , buckinghamshire , uk ) . sample and equal volumes of reducing lysis buffer ( 9.5 m urea , 2% chaps , 2% dtt , and 1.6% ampholytes ) were added by anodic cup loading . for ph 47 focusing , ipg strips were in - gel rehydrated with sample and equal volumes of reducing lysis buffer made up in rehydration buffer . the strips were run on an amersham ipgphor ief system with the following running conditions : 3500 v for 21.3 hours , a 3500 to 8000 v gradient over 10 minutes in , and 8000 v for 2 hours . for first dimension separation in the ph 611 range the following protocol was employed : 80 v for 4 hours , 100 v for 2 hours , 500 v for 1.5 hours , 1000 v for 1 hour , 2000 v for 1 hour , 4000 v for 1 hour , 6000 v for 2 hours , and finally 8000 v for 2.5 hours . following isoelectric focusing , the ipg strips were equilibrated for 20 minutes firstly in buffer containing 6 m urea , 30% ( w / v ) glycerol , 2% ( w / v ) sds , 100 mm tris - hcl , ph 8.8 with the addition of 100 mm dtt and subsequently for 20 minutes in buffer supplemented with 0.25 m iodoacetamide . strips were briefly washed in sodium dodecyl sulfate - containing running buffer ( 125 mm tris , 0.96 m glycine , 0.1% ( w / v ) sds ) and placed on top of 12.5% ( w / v ) resolving gels . to seal the strip gels , they were overlaid with the above - described running buffer containing 1% ( w / v ) agarose . the separation of the cardiac proteins was carried out in the second dimension by standard sds polyacrylamide gel electrophoresis using the dodecacell system from bio - rad laboratories ( hemel hempstead , herts . , uk ) . electrophoresis was carried out overnight for approximately 18 hours in the dark at 1.5 v per 21 cm - gel until the bromophenol blue tracking dye just ran off the gel . fluorescent cydye - labelled cardiac proteins were visualized using a typhoon trio variable mode imager form amersham biosciences / ge healthcare ( little chalfont , bucks . , uk ) . for image acquisition , cy5- and cy3-labelled proteins were scanned at a wavelength of = 650 nm and = 550 nm , respectively . photomultiplier tube ( pmt ) values were optimised so that the volume of the most abundant spot was between 80,000 and 99,000 when scanned at a resolution of 100 m . this guaranteed that no spot would be saturated on the gel , therefore interfering with accurate analysis . the cy3 images were then analysed using progenesis samespots software version 3.2.3 from nonlinear dynamics ( newcastle upon tyne , uk ) and normalised against their corresponding cy5 image . the gels were placed into groups ( mdx versus normal ) and analysed to determine significant differences in 2d spot abundance . an anova score of 0.5 was required for spots to be included in the subsequent analysis . then principal component analysis ( pca ) was verified with changes displaying power of < 0.8 being removed from the analysis . all remaining changed spots that met the significance criteria were visually checked on the aligned gels to ensure feasibility and were subsequently identified by lc - ms / ms analysis . preparative gels containing 400 g of protein ( 1:1 mdx to normal ) were stained with the dye ruthenium ii bathophenanthroline disulfonate chelate ( rubps ) . rubps - stained gels were scanned at a wavelength of = 550 nm using the criteria outlined above . prior to identification via lc - ms / ms technology , 2d protein spots of interest were excised using the automated ettan spot picker system from amersham biosciences ( little chalfont , bucks . briefly , gel pieces were destained before the addition of 400 ng trypsin per spot , incubated for 30 minutes at 4c before being digested overnight at 37c . 100 l of extraction buffer ( 1:2 v / v of 5% formic acid / acetonitrile ) was added to the peptides and incubated for 15 minutes at 37c . all supernantant was then transferred into fresh tubes and extracts were dried down completely in a vacuum centrifuge . peptides were reconstituted in 12 l of 0.1% formic acid , vortexed , sonicated , and the mixture was centrifuged for 20 minutes in cellulose spin filter tubes to remove any gel particles . samples were analysed on an agilent 6340 ion trap lc mass spectrometer using electrospray ionization ( agilent technologies , santa clara , ca , usa ) on a 10-minute gradient of 5100% acetonitrile/0.1% formic acid with a post run of 1 minute . separation of peptides was performed with a nanoflow agilent 1200 series system , equipped with a zorbax 300 sb c18 m , 4 mm 40 nl pre - column . mobile phases used were a : 0.1% formic acid , b : 90% acetonitrile and 0.1% formic acid . samples ( 8 l ) were loaded onto the enrichment column with capillary flow rate set at 4 l / min with a mix of a : b at a ratio of 19 : 1 . elution was carried out with the nano pump flow rate set at 0.6 l / minute . criterion for each search was set at ( i ) species mus musculus , ( ii ) two missed cleavages by trypsin , ( iii ) variable modification : oxidation of methioine , ( iv ) fixed modification : carboxymethylation of cysteines , and ( v ) mass tolerance of precursor ions 2da and product ions 1da . in order to verify changes in the expression of select cardiac proteins , 1-d gel electrophoresis , transfer unit from invitrogen ( carlsbad , ca , usa ) for semi - dry blotting . blocking of nitrocellulose sheets was achieved with a milk protein solution ( 5% ( w / v ) fat - free milk powder in 0.9% ( w / v ) nacl , 50 mm sodium phosphate , ph 7.4 ) for 1 hour . nitrocellulose sheets were washed and then incubated for 1 hour with secondary peroxidase - conjugated antibodies , diluted in blocking solution . immuno - decorated bands were visualized by the enhanced chemiluminescence method using bm chemiluminescence blotting substrate from roche diagnostics ( mannheim , germany ) . densitometric scanning of immunoblots was performed on a molecular dynamics 300 s computing densitometer ( sunnyvale , ca , usa ) with imagej ( nih , usa ) and graphpad prism ( san diego , ca , usa ) software . for the localization of nuclei , mitochondria and select marker proteins , confocal microscopy was used as previously described in detail . normal and dystrophic mdx hearts were mounted on cyrocassettes at 20c and transverse cryosections of 10 m thickness were cut on a shandon cryotome ( life sciences international , cheshire , uk ) . for labeling of mitochondria , freshly cut tissue sections were incubated with a 1 : 100 diluted solution of mitotracker red chloromethyl - x - rosamine ( cmxros ) dye for 30 minute , briefly washed in phosphate - buffered saline and then immediately examined by microscopy . cmxros is a well - established lipophilic cationic fluorescent dye that is highly specific for mitochondria and is routinely used to measure mitochondrial function and morphology in cell culture and in whole tissues . for immunofluorescence microscopy , cryosections were briefly fixed in ice - cold acetone for 5 minutes , followed by submersion in blocking solution ( 0.2% ( w / v ) bovine serum albumin , 0.2% ( v / v ) triton x-100 , and 2.5% ( v / v ) goat serum in phosphate - buffered saline ) for 30 minute , and then incubated with primary antibodies diluted in 0.2% ( w / v ) bovine serum albumin and 0.2% ( v / v ) triton x-100 in phosphate - buffered saline for 4 hours . tissue sections were washed twice for 30 minute in above solution omitting antibodies and then labelled with secondary antibodies for 1 hour , followed by the above described washing procedure . in order to determine the number of nuclei in normal versus mdx hearts , cardiac tissue sections were labelled with 1 g / ml diamidino - phenylindole ( dapi ) for 30 minute . one drop of p - phenylenediamine ( ppd)-glycerol was applied to the immuno - decorated tissue sections and then coverslips carefully placed over the sections , avoiding the introduction of air bubbles . fluorescent labelling patterns were visualised with an olympus ix81 microscope ( olympus life and material science europe , hamburg , germany ) . in contrast to non - fluorescent protein dye methodology , comparative studies using fluorescent cydyes have an enhanced dynamic range of protein coverage . dige analysis represents a highly accurate quantitative technique that enables multiple protein samples to be separated on the same two - dimensional gel . this reduces the introduction of potential artifacts due to gel - to - gel variations , making comparative dige approaches one of the most powerful analytical tools for conducting comparative biomedical investigations . in order to determine global changes in the heart due to deficiency in the dystrophin isoform dp427 , we have carried out a dige analysis of crude tissue extracts from 9-month old normal versus age - matched dystrophic mdx mice . due to the high cost of breeding and maintaining mice to old age , as well as the considerable cost of fluorescent tagging of large protein populations , the number of biological repeats and analytical dige gels was kept to a minimum . in order to satisfy the statistical requirements for the generation of proper proteomic data sets and perform an optimized dige analysis of normal versus affected proteomes karp and lilley , respectively . in the first dimensional separation step via isoelectric focusing , both a ph 47 and a ph 611 range were employed to cover as many cardiac proteins with differing charges as possible . figure 1 illustrates the neutral and the more acidic range of protein species and figure 2 shows the separation of neutral and more basic heart proteins . shown are representative two - dimensional gels of cy3-labelled normal muscle ( figure 1(a ) ; figure 2(a ) , cy3-labelled dystrophic muscle ( figure 1(c ) ; figure 2(c ) and corresponding cy5-labelled pooled standards ( figures 1(b),1(d ) ; figures 2(b ) , 2(d ) , which were analysed with the help of a typhoon trio variable imager and progenesis 2d analysis software . overall , 2509 distinct protein species were recognized on the lower and higher ph - range gels . the protein spot pattern presented here agrees in large parts with their comprehensive 2d proteomic map of cardiac tissue . out of 2048 detectable protein spots on the ph 47 gels and 487 detectable protein spots on ph 611 gels , 79 protein spots showed a drastic differential expression pattern , with 3 proteins being increased and 26 distinct protein species being decreased . electrospray ionization ms / ms analysis was carried out to unequivocally identify the cardiac proteins with a changed abundance in dystrophic heart fibres . a list of the cardiac protein species with a drastically altered expression level in dystrophic mdx tissue is shown in table 1 . the information on the 79 dige - identified protein spots combine data from both ph 47 and ph 611 gels and contain the spot number , protein name , protein i d , number of matched peptides , percentage sequence coverage , the relative molecular mass , pi - value , mascot score and fold - change of individual proteins affected by deficiency in dystrophin . since the ms - based analysis identified a large number of matched peptide sequences , these data are not shown . the majority of identified cardiac proteins belonged to the main classes of metabolic or contractile proteins . components of the actomyosin apparatus , enzymatic and regulatory elements of mitochondria , glycolytic enzymes , metabolic transporters , cellular stress proteins and cytoskeletal components were clearly identified by ms analysis . cardiac proteins with a dystrophy - related change in abundance ranged in molecular mass from apparent 14 kda to 90 kda and covered a pi - range from approximately 5 to 9 . dige cy5 master gels of both the ph 47 and the ph 611 range are shown in figure 3(a ) , 3(b ) , so that it is possible to correlate ms - identified protein species , listed in table 1 , with distinct two - dimensional spots of altered density in the dystrophic heart . an increased expression level was shown for 3 cardiac proteins , that is , nucleoside diphosphate kinase b ( spot 1 ) , the nuclear lamina matrix protein lamin a / c ( spot 2 ) , and a component of the electron - transferring flavoprotein dehydrogenase ( spot 3 ) . all other identified protein species exhibited a decreased abundance in dystrophin - deficient myocardial tissue . these cardiac proteins are marked and numbered 4 to 79 in the cy5-labelled master gels of figure 3 . the protein species with the highest decrease in concentration was identified as the anti - oxidant enzyme peroxiredoxin-6 ( spot 79 ) . in addition , mitochondrial atp synthase ( spots 50 , 55 , 69 , 72 , 73 , 74 , 77 ) , isocitrate dehydrogenase ( spot 38 , 42 ) , nadh dehydrogenase ( spot 40 ) , pyruvate dehydrogenase ( spots 11 , 14 , 15 , 20 , 25 , 34 ) , isovaleryl - coa dehydrogenae ( spots 8 , 19 ) , dienoyl - coa isomerase ( spot 17 ) , oxoglutarate dehydrogenase ( spot 7 ) , prohibitin ( spot 6 ) , electron - transferring flavoprotein ( spots 5 , 17 ) , myozenin-2 ( spot 4 ) , voltage - dependent anion - selective channel protein vdac1 ( spot 41 ) , the slow / cardiac isoform of myosin light chain mlc2 ( spots 26 , 48 , 62 , 65 , 70 , 78 ) , myosin light chain mlc3 ( spots 53 , 61 , 76 ) , cardiac alpha - actin ( spots 21 , 39 , 45 , 46 , 56 , 60 , 64 , 66 , 67 , 71 , 75 ) , alpha-1 tropomyosin ( spots 35 , 59 ) , adenylate kinase ak1 ( spot 68 ) , creatine kinase ( spot 16 ) , cytochrome c oxidase ( spots 22 , 51 ) , cytochrome b - c complex ( spots 10 , 30 , 57 , 63 ) , desmin ( spot 43 , 47 , 52 , 54 , 58 ) , vimentin ( spots 12 , 23 ) , cardiac fatty acid binding - protein fabp-3 ( spots 27 , 33 , 49 ) , heat shock protein hsp27 ( spot 18 ) , heat shock protein hsp60 ( spots 31 , 37 , 44 ) , enolase ( spots 13 , 28 , 32 ) , dj-1 protein ( spot 29 ) , valsolin - containing protein vcp ( spot 24 ) , and albumin ( spot 9 ) were identified . the results for 2 decreased protein spots have not been included in table 1 , since only 1 matched peptide per cardiac protein could be determined by ms analysis . it is international standard that proteomic studies only list proteins with sequence coverage of less than 10% when at least 2 separate matched peptide sequences are available for an independent identification . the peptide sequences lganslldlvvfgr ( 2% sequence coverage ) and ltfdssfspntgk ( 4% sequence coverage ) identified the nonlisted cardiac proteins as succinate dehydrogenase ( 73.6 kda ; pi 7.1 ) and porin isoform vdac1 ( 32.5 kda ; pi 8.6 ) , respectively . to illustrate representative examples of key cardiac proteins with a drastically decreased expression in dystrophin - deficient fibres , enlarged images of dige - identified protein spots are shown in figure 4 . the direct comparison of fluorescently labeled 2-d protein spots , which have been unequivocally identified by mass spectrometric analysis ( table 1 ) , shows the cytosolic ak1 isoform of adenylate kinase , the mitochondrial proteins atpase synthase , porin isoform vdac1 , isocitrate dehydrogenase , succinate dehydrogenase , and prohibitin , as well as dj1-protein , the stress protein hsp60 and myosin light chain isoform mlc2 . a large proportion of the cardiac proteins identified in this study by dige analysis belong to the contractile apparatus , the cellular stress response and the cytoskeletal network , as well as mitochondrial metabolism including oxidative phosphorylation , the citric acid cycle and fatty acid transportation . previous studies have clearly shown that the deficiency in dystrophin triggers a drastically reduced concentration of the dystrophin - associated /-dystroglycan complex in the mdx heart . immunoblotting has established distinct alterations down - stream of the cardiac dystrophin - glycoprotein complex , such as the reduction in the luminal ca - binding proteins calsequestrin and sarcalumenin . in analogy , we employed western blotting here to verify the results of our dige analysis by immuno - decoration analysis of a select group of cardiac proteins . in this respect , it is important to stress that the mass spectrometry - based proteomic analysis of individual 2d spots identifies changes in distinct subspecies of proteins and not alterations in the total population of a protein isoform . the recent large - scale cataloguing of the heart proteome has shown that cardiac tissues contain thousands of different protein species , whereby many proteins are represented by a large number of distinct 2d - spots in analytical gels [ 5759 ] . it is one of the great advantages of modern proteomics that this analytical approach can differentiate between differently charged or sized subspecies of individual proteins . since 1d immunoblotting or immunofluorescence microscopy often do not recognize all forms of a specific protein due to a lack of antibody specificity , proteomic findings on an individual 2d protein spot sometimes do not correlate fully with the findings from biochemical or cell biological analyses of total protein cohorts . these apparent differences in the fate of individual subspecies of a protein versus the total protein population probably reflect the complexity of disease - related changes in the cardiac proteome . figure 5(a ) shows a coomassie - stained gel of crude preparations from normal and dystrophic hearts and illustrates that no major differences exist in the overall protein expression levels between the two different phenotypes . the immunoblot analysis of laminin , desmin , the slow / cardiac myosin heavy chain and succinate dehydrogenase revealed no major differences in the concentration of these abundant cardiac proteins in normal versus mdx specimens ( figures 5(b)5(d ) and 5(g ) ) . in contrast , immunoblotting of the enzyme adenylate kinase ak1 , the fatty acid transporter fabp3 , isocitrate dehydrogenase , atp synthase and porin isoform vdac1 showed a reduced abundance in mdx preparations ( figures 5(e ) , 5(f ) , 5(h)5(j ) ) . the reduced expression of adenylate kinase isoform ak1 , fatty acid binding protein fabp3 , isocitrate dehydrogenase icdh , porin isoform vdac1 , and atp synthase was found to be statistically significant , while the concentration of desmin , myosin heavy chain and succinate dehydrogenase was shown not to be significantly different between normal and dystrophic preparations . thus the overall isoform population of succinate dehydrogenase and desmin does not appear to be reduced in dystrophinopathy , although the above - described dige analysis showed a lower level of distinct subspecies of these cardiac elements . in general , the immunoblotting results support the main findings from our dige analysis of normal versus dystrophic heart tissue and demonstrate independently that the deficiency in dystrophin has a profound effect on the expression pattern of the cardiac protein complement . since the dige analysis of the normal versus the dystrophic mdx heart has revealed changes in a variety of mitochondrial components , it was important to determine whether this alteration was due to a reduced number of mitochondria in the dystrophic heart or internal abundance changes in the mitochondrial protein complement . figure 7 shows representative results from an immunofluorescence survey of mitochondrial content , nuclei and cardiac marker proteins in dystrophic tissue . cryosections of mdx tissue showed a complete absence of the dystrophin isoform dp427 ( figures 7(c ) , 7(d ) ) , confirming the mutant status of the dystrophic hearts used in this study . dapi staining revealed 229 6 and 270 18 nuclei per examined tissue section in normal versus dystrophic tissue sections , respectively ( figures 7(a ) , 7(b ) ) . the slight increase of nuclei in mdx preparations was not statistically significant ( n = 4 ) . thus the proteomic finding of a dystrophy - dependent increase in lamin - a / c is therefore not directly related to a drastic change in the number of nuclei per cardiac tissue unit . fluorescent labeling of mitochondria with the mitotracker dye cmxros showed a characteristic internal staining pattern in cardiac fibres ( figures 7(e)7(h ) ) . fluorescent intensity values of 1470 36 and 1384 40 for normal versus mdx preparations were found not to be significantly different ( n = 4 ) . hence , lower levels of mitochondrial enzymes do not seem to be a consequence of a drastic decrease in mitochondrial density in the dystrophic mdx heart . the immunofluorescence labeling of desmin ( figures 7(i)7(l ) ) , prohibitin ( figures 7(m)7(p ) ) and succinate dehydrogenase ( figures 7(q)7(t ) ) was shown to be comparable between control and pathological samples . the microscopical localization of desmin and succinate dehydrogenase revealed unexpectedly not a decrease of these proteins in dystrophic tissue sections , as found by dige analysis . hence , although distinct subspecies of certain cardiac proteins are affected in x - linked muscular dystrophy as determined by mass spectrometry - based proteomics , the overall isoform complement of these elements is not drastically altered . this indicates that no major differences exist in the expression levels of desmin - containing intermediate filament structures and the population of cardiac mitochondria in normal versus dystrophin - deficient cells . in order to enhance our knowledge of the molecular pathogenesis of cardiomyopathy linked to x - linked muscular dystrophy , a detailed proteomic profiling of the dystrophic heart was carried out . the biochemical establishment of a comprehensive disease - specific biomarker signature for x - linked muscular dystrophy is crucial for ( i ) the development of superior diagnostic methods , ( ii ) the identification of new therapeutic targets to address cellular alterations down - stream of the primary abnormality in dystrophin , and ( iii ) the evaluation of new treatment approaches to counter - act the various pathological aspects of dystrophinopathy . the proteomic profiling of mdx skeletal muscle has recently established altered expression levels of soluble proteins involved in nucleotide metabolism , ca - handling and the cellular stress response [ 4245 ] . the mass spectrometric analysis of experimental exon skipping therapy revealed a partial reversal of dystrophic changes in these new signature molecules , illustrating the potential of proteomic technology to decisively enhance the capabilities of biomedical research into the molecular mechanisms that underlie neuromuscular disorders . in contrast to skeletal muscle , very little is known about the global pathobiochemical mechanisms that lead to cardiac complications in the dystrophin - deficient organism . here , we have successfully applied the fluorescent dige method to the large - scale analysis of the dystrophic heart . ms - based proteomics clearly revealed a drastic decrease in key metabolic , regulatory and contractile proteins , as well as components of the cytoskeleton and the cellular stress response . the pie chart of figure 8 summarizes the apparent functions of dige - identified proteins with a differential expression in normal versus dystrophin - deficient heart muscle . the dige analysis presented here showed a moderate increase in lamin - a / c , which might improve nuclear stability , chromatin structure and gene expression in the dystrophic heart . since comparative dapi staining indicates comparable numbers of nuclei in normal versus dystrophic fibres , tissue scarring is probably not responsible for an increase in the number of nuclei per tissue unit . therefore , the elevated lamin concentration does not appear to be associated with increased numbers of nuclei . cardiac lamins make up the matrix that is located close to the inner nuclear membrane . the only drastic increase in a cardiac protein was the 4-fold change in the expression of nucleoside diphosphate kinase b. nucleoside diphosphate kinases are key regulatory enzymes involved in intracellular di- and tri - phosphonucleoside homeostasis . their cellular functions are critical for signaling , proliferation , differentiation and bioenergetics , which suggests that the up - regulation of one of their isoforms could be considered a counter measure of the dystrophin - deficient heart to stabilize essential cellular functions in severely stressed muscle cells . in contrast to the 4-fold increase in nucleoside diphosphate kinase , our proteomic screening demonstrated a massive decrease in a variety of cardiac proteins in the mdx heart . the dystrophinopathy - associated loss in numerous key proteins , which belong to different cellular pathways and functions in the heart , agrees with the severity of the cardiomyopathic complications observed in aged mdx mice and dmd patients [ 1315 ] . key proteins involved in cardiac contraction , cytoskeletal integrity , nucleotide metabolism , cellular stress response , mitochondrial metabolism , and fatty acid transportation were shown to be affected by a deficiency in cardiac dystrophin . interestingly , braun et al . could show an altered integration of mitochondria and atpases in slow - twitching mdx muscles . the coupling between mitochondrial creatine kinase and the adenine nucleotide translocase system appears to be weakened in dystrophin - deficient fibres . the 6-fold decrease in peroxiredoxin-6 may have drastic consequences for the capability of the dystrophic heart to counter - act the detrimental effects of stressors . peroxiredoxin-6 is a crucial enzyme that protects cells from oxidative stress . in analogy to the reduced concentration of this antioxidant enzyme in cardiac mdx tissue , the decrease in the small heat shock protein hsp27 , as well as hsp60 and dj-1 protein , also agrees with an impaired cellular stress response in muscular dystrophy - associated cardiomyopathy . small heat shock proteins provide crucial chaperone function in striated muscles and are upregulated in the heart following exercise training . an impaired stress response could result in the detrimental aggregation of misfolded proteins causing cellular dysfunction in the dystrophic heart . the dystrophinopathy - associated decrease in valsolin - containing protein vcp might also impair cardiac function . vcp belongs to the aaa - atpase superfamily and is involved in various cellular functions such as membrane fusion and cell - cycle control , as well as protein degradation through the ubiquitin - proteasome pathway . the dige analysis of the mdx heart presented here has clearly shown a severely altered concentration of numerous key players involved in cardiac contraction and cytoskeletal organization . in contrast to comparable levels of myosin heavy chain , other critical contractile proteins are affected by the lack of dystrophin . the biomolecules that form the thick and thin filaments of the basic contractile units exist in a complex arrangement of myosin heavy chains , myosin light chains , and actins . muscle myosin consists of a hexameric arrangement that is formed by two heavy chains and two pairs of myosin light chains . changes in myosin light chains have been implicated in severe cardiac impairments , such as familial hypertrophic cardiomyopathy . hence , the reduction in the cardiac isoform of myosin light chain mlc2 , myosin light chain mlc3 , and cardiac alpha - actin might have a severe impact on the contractile apparatus in the dystrophic heart . contractile weakness observed in dystrophinopathy could be directly related to the altered concentration of myosin light chains and actin . cardiac muscle cells are activated by ca - binding to tnc - subunit of troponin and is regulated by the interactions of tropomyosin and troponin in the thin filament . thus , the observed reduction of alpha-1 tropomyosin in the mdx heart might also be of pathophysiological significance . since the molecular coupling between the myosin head structure and actin filaments , in the presence of atp , represents the major step that underlies the sliding of thin filaments past thick filaments , abnormal concentrations of regulatory proteins may result in impaired patterns of sarcomeric shortening . in addition , a close connection exists between the contractile apparatus , the membrane - associated actin network and the cytoskeleton . intermediate filaments are of crucial importance for the mechanical integrity and elasticity of contractile fibres . although proteomics showed that the expression levels of distinct subspecies of desmin were reduced in dystrophic preparations , confocal microscopy and immunoblotting revealed that the overall population of desmin isoforms is comparable between normal and mdx heart . however , the deficiency in cardiac dystrophin appears to trigger a concomitant reduction in the density of myozenin-2 and vimentin . myozenin-2 belongs to the calsarcin family of proteins , a new type of sarcomeric calcineurin - binding protein . vimentin is a crucial intermediary filament protein and plays a key role in cytoskeletal network formation . the abnormal expression levels of these proteins might therefore cause disturbed interfilament interactions and impaired cystoskeletal arrays , thereby causing cellular instability in the dystrophic heart . one of the most important findings of this mass spectrometry - based proteomic study is the drastically reduced concentration of numerous mitochondrial proteins and metabolic transporters . since mitochondria represent the primary site for energy generation via oxidative phosphorylation , a reduction in elements belonging to the oxidative phosphorylation complexes , the citric acid cycle and ion homeostasis will have a profound influence on the bioenergetic status of the dystrophic heart . cardiac mitochondria are involved in intermediary metabolism , cell cycle progression , calcium signaling and the regulation of apoptosis , as well as the production of heme and iron - sulfur clusters . previous studies have shown that altered expression levels within the mitochondrial proteome influence many aspects of normal development , diseases and the aging process . with respect to the heart , mitochondrial dysfunction appears to be associated with numerous pathologies such as congestive heart failure , ischaemia reperfusion injury and cardiomyopathy . therefore , the reduced expression of many mitochondrial proteins associated with the matrix , outer membrane and inner membrane system suggests an impaired metabolism in the dystrophic heart . reduced expression levels within the mitochondrial proteome appear to affect the functioning of the mitochondrial atp synthase , isocitrate dehydrogenase , nadh dehydrogenase , oxoglutarate dehydrogenase , isovaleryl - coa dehydrogenase , cytochrome c oxidase and the cytochrome b - c complex , all critical elements involved in mitochondrial function in the heart . this agrees with a previous study by zhang et al . , who reported a decreased citrate synthase activity in the mdx heart . although a certain degree of cellular scarring and the infiltration of fatty and connective tissue may reduce the number of contractile fibres in the dystrophic heart , as previously shown by histochemical analysis , this does not appear to drastically alter the density of muscle - associated mitochondria . the confocal microscopy analysis shown in this report suggests no major changes in the number of mitochondria between normal and dystrophic heart muscle tissue , as judged by labeling with the established chloromethyl - x - rosamine mitotracker dye which is concentrated inside mitochondria by their negative membrane potential [ 5355 ] . our dige analysis revealed that individual protein spots representing electron - transferring flavoprotein showed both increased and decreased abundance . electron transfer flavoprotein ubiquinone oxidoreductase provides a crucial link between electrons derived from fatty acid oxidation and certain amino acids to the main respiratory chain system of mitochondria . the differential effect on different isoforms of this protein is difficult to interpret , but dystrophin deficiency must affect distinct subspecies of this metabolic element with differing post - translational modifications in a dissimilar way . the observed reduction in the rate - limiting enzyme pyruvate dehydrogenase could have severe impact on the utilization of energy carriers and thereby impair the overall bioenergetics of the mdx heart . isoforms of cardiac prohibitin are present in the inner mitochondrial membrane and are responsible for the efficient assembly of mitochondrial respiratory chain enzymes and perform chaperone activity . interestingly , a proteomic survey of mitochondrial proteins in cardiomyocytes from chronic stressed rats has shown a drastic increase in prohibitin . although the overall complement of prohibitin isoforms is not drastically affected in muscular dystrophy , a reduced expression of distinct subspecies of prohibitin in the dystrophic heart the only affected glycolytic enzyme appears to be enolase , indicating that mitochondrial metabolism is more severely affected than glycolysis in dystrophy - related cardiomyopathy . interestingly , the abundance of the extracellular and intracellular fatty acid transporters albumin and fabp-3 , which are considered limiting factors of oxidative metabolism in striated muscles , is decreased in the dystrophin - deficient heart . thus , dystrophic heart cells would be starved of essential fuel supplies , which might trigger a decline in contractile strength . a mitochondrial receptor present in the outer mitochondrial membrane was identified as a porin isoform that forms voltage - dependent anion channels . these ion channels belong to a family of pore - forming proteins that provide large membrane - spanning aqueous channels . the decreased concentration in the porin transport protein would indicate a dystrophy - associated decrease in mitochondrial function and abnormal ion homeostasis . in contrast to a recent combined metabolomic and proteomic study of the mdx heart , and in agreement with two comprehensive proteomic studies on mdx skeletal muscle [ 42 , 45 ] , we could show here a decreased abundance of the ak1 isoform of adenylate kinase . nucleotide metabolism , involving adenylate kinase and creatine kinase appears to be disturbed in the dystrophic mdx heart , and this finding might be useful for the future development of a comprehensive biomarker signature of cardiomyopathy associated with muscular dystrophy . overall , our dige - based screening of the soluble proteome from dystrophic mdx hearts has revealed a severely perturbed protein expression pattern due to deficiency in dystrophin . the observed changes in essential proteins involved in cardiac contraction , mitochondrial metabolism , the cellular stress response and nucleotide metabolism might be useful for the future improvement of differential diagnostic procedures , the assessment of cardiac disease progression in x - linked muscular dystrophies , the identification of novel therapeutic targets to treat cardiomopathic complications in duchenne muscular dystrophy , and the evaluation of novel treatment strategies to counter - act the loss in cardiac dystrophin .

although duchenne muscular dystrophy is primarily classified as a neuromuscular disease , cardiac complications play an important role in the course of this x - linked inherited disorder . the pathobiochemical steps causing a progressive decline in the dystrophic heart are not well understood . we therefore carried out a fluorescence difference in - gel electrophoretic analysis of 9-month - old dystrophin - deficient versus age - matched normal heart , using the established mdx mouse model of muscular dystrophy - related cardiomyopathy . out of 2,509 detectable protein spots , 79 2d - spots showed a drastic differential expression pattern , with the concentration of 3 proteins being increased , including nucleoside diphosphate kinase and lamin - a / c , and of 26 protein species being decreased , including atp synthase , fatty acid binding - protein , isocitrate dehydrogenase , nadh dehydrogenase , porin , peroxiredoxin , adenylate kinase , tropomyosin , actin , and myosin light chains . hence , the lack of cardiac dystrophin appears to trigger a generally perturbed protein expression pattern in the mdx heart , affecting especially energy metabolism and contractile proteins .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

the identification of the gene responsible for the primary defect in x - linked muscular dystrophy led to the discovery of its protein product , the large membrane cytoskeletal element dystrophin . mutations in the dystrophin gene cause the most prevalent and lethal gender - specific genetic disease of childhood , duchenne muscular dystrophy ( dmd ) , and its more benign and less frequent counterpart becker 's muscular dystrophy , as well as x - linked dilated cardiomyopathy . tissue - specific variations in dystrophin isoforms delegate the composition and subcellular localization of the dystrophin - glycoprotein complex , and hence the function of the specific membrane assembly may vary between the brain , heart , and skeletal muscles . the majority of dmd patients develop clinical cardiac symptoms during the second decade of life , including arrhythmias , cardiomyopathy , and regional wall abnormalities , leading to fatal cardiac complications in about 20% of cases . on the cellular level , the gradual replacement of cardiac fibres by connective and fatty tissue is a hallmark of the dystrophic heart . while dystrophin - deficient skeletal muscles undergo cycles of fibre degeneration and regeneration , dystrophic heart fibres exhibit only a limited regenerative capacity causing a progressive decline in the cardiomyocyte population of the dystrophic heart . in this study , we used the x - linked muscular dystrophy ( mdx ) mouse , a well - established animal model of dmd that is missing dystrophin isoform dp427 . the absence of cardiac dystrophin is clearly associated with necrotic changes , extensive infiltration of inflammatory cells , increase in adipose tissue , interstitial fibrosis , tachycardia , and impaired contractile properties in the mdx heart [ 22 , 30 , 31 ] . in analogy to dmd hearts , the expression of dystrophin - associated glycoproteins is also greatly reduced in dystrophin - deficient mdx heart cells . in addition , the introduction of fluorescent dyes has drastically increased the total number of identifiable two - dimensional spots , whereby fluorescence difference in - gel electrophoresis ( dige ) can be considered one of the most advanced biomedical tools for studying two different sets of soluble protein complements . with respect to x - linked muscular dystrophy , proteomic studies have mostly focused on mdx skeletal muscles and have identified numerous biomarker candidates , such as adenylate kinase , calsequestrin , regucalcin , and the small stress protein cvhsp . following the densitometric determination of the expression pattern of 2,509 cardiac protein spots , mass spectrometry identified 26 proteins being decreased , including various myosin light chains , tropomyosin , actin , adenylate kinase , creatine kinase , vimentin , atp synthase , fatty acid binding - protein , isocitrate dehydrogenase , nadh dehydrogenase , myozenin , porin , and peroxiredoxin , and 3 proteins being increased , including lamin - a / c and nucleoside diphosphate kinase . in the future , these results will be useful for complementing studies into the molecular mechanisms of muscular dystrophy and for evaluating the effects of novel drugs , genetic modifications , or cell - based therapies on disease progression . , cambridge , uk ( ab28172 to prohibitin ; ab36329 to isocitrate dehydrogenase ; ab14734 to porin isoform vdac1 ; ab54824 to adenylate kinase isoform ak1 ; ab5432 to atp synthase , ab16915 to cardiac fatty acid binding protein ) , sigma chemical company , dorset , uk ( mab noq7.5.4d to slow / cardiac myosin heavy chain ; pab to laminin ) , visionbiosystems novocastra , newcastle upon tyne , uk ( mab ncl - dys1 against the dp427 isoform of dystrophin ) , thermo fisher scientific inc . as previously demonstrated by immunoblotting , the mdx mouse cohort used in this study lacks the 427 kda isoform of cardiac dystrophin and shows a drastic reduction in dystrophin - associated glycoproteins , such as dystroglycans and sarcoglycans [ 32 , 33 ] . potential differences in the expression pattern of the soluble proteome from normal and mdx cardiac muscle were analysed using the fluorescence difference in - gel electrophoretic dige technique , as recommended by karp and lilley . the separation of the cardiac proteins was carried out in the second dimension by standard sds polyacrylamide gel electrophoresis using the dodecacell system from bio - rad laboratories ( hemel hempstead , herts . in order to determine global changes in the heart due to deficiency in the dystrophin isoform dp427 , we have carried out a dige analysis of crude tissue extracts from 9-month old normal versus age - matched dystrophic mdx mice . out of 2048 detectable protein spots on the ph 47 gels and 487 detectable protein spots on ph 611 gels , 79 protein spots showed a drastic differential expression pattern , with 3 proteins being increased and 26 distinct protein species being decreased . dige cy5 master gels of both the ph 47 and the ph 611 range are shown in figure 3(a ) , 3(b ) , so that it is possible to correlate ms - identified protein species , listed in table 1 , with distinct two - dimensional spots of altered density in the dystrophic heart . an increased expression level was shown for 3 cardiac proteins , that is , nucleoside diphosphate kinase b ( spot 1 ) , the nuclear lamina matrix protein lamin a / c ( spot 2 ) , and a component of the electron - transferring flavoprotein dehydrogenase ( spot 3 ) . all other identified protein species exhibited a decreased abundance in dystrophin - deficient myocardial tissue . in addition , mitochondrial atp synthase ( spots 50 , 55 , 69 , 72 , 73 , 74 , 77 ) , isocitrate dehydrogenase ( spot 38 , 42 ) , nadh dehydrogenase ( spot 40 ) , pyruvate dehydrogenase ( spots 11 , 14 , 15 , 20 , 25 , 34 ) , isovaleryl - coa dehydrogenae ( spots 8 , 19 ) , dienoyl - coa isomerase ( spot 17 ) , oxoglutarate dehydrogenase ( spot 7 ) , prohibitin ( spot 6 ) , electron - transferring flavoprotein ( spots 5 , 17 ) , myozenin-2 ( spot 4 ) , voltage - dependent anion - selective channel protein vdac1 ( spot 41 ) , the slow / cardiac isoform of myosin light chain mlc2 ( spots 26 , 48 , 62 , 65 , 70 , 78 ) , myosin light chain mlc3 ( spots 53 , 61 , 76 ) , cardiac alpha - actin ( spots 21 , 39 , 45 , 46 , 56 , 60 , 64 , 66 , 67 , 71 , 75 ) , alpha-1 tropomyosin ( spots 35 , 59 ) , adenylate kinase ak1 ( spot 68 ) , creatine kinase ( spot 16 ) , cytochrome c oxidase ( spots 22 , 51 ) , cytochrome b - c complex ( spots 10 , 30 , 57 , 63 ) , desmin ( spot 43 , 47 , 52 , 54 , 58 ) , vimentin ( spots 12 , 23 ) , cardiac fatty acid binding - protein fabp-3 ( spots 27 , 33 , 49 ) , heat shock protein hsp27 ( spot 18 ) , heat shock protein hsp60 ( spots 31 , 37 , 44 ) , enolase ( spots 13 , 28 , 32 ) , dj-1 protein ( spot 29 ) , valsolin - containing protein vcp ( spot 24 ) , and albumin ( spot 9 ) were identified . to illustrate representative examples of key cardiac proteins with a drastically decreased expression in dystrophin - deficient fibres , enlarged images of dige - identified protein spots are shown in figure 4 . the direct comparison of fluorescently labeled 2-d protein spots , which have been unequivocally identified by mass spectrometric analysis ( table 1 ) , shows the cytosolic ak1 isoform of adenylate kinase , the mitochondrial proteins atpase synthase , porin isoform vdac1 , isocitrate dehydrogenase , succinate dehydrogenase , and prohibitin , as well as dj1-protein , the stress protein hsp60 and myosin light chain isoform mlc2 . previous studies have clearly shown that the deficiency in dystrophin triggers a drastically reduced concentration of the dystrophin - associated /-dystroglycan complex in the mdx heart . the recent large - scale cataloguing of the heart proteome has shown that cardiac tissues contain thousands of different protein species , whereby many proteins are represented by a large number of distinct 2d - spots in analytical gels [ 5759 ] . the immunoblot analysis of laminin , desmin , the slow / cardiac myosin heavy chain and succinate dehydrogenase revealed no major differences in the concentration of these abundant cardiac proteins in normal versus mdx specimens ( figures 5(b)5(d ) and 5(g ) ) . in contrast , immunoblotting of the enzyme adenylate kinase ak1 , the fatty acid transporter fabp3 , isocitrate dehydrogenase , atp synthase and porin isoform vdac1 showed a reduced abundance in mdx preparations ( figures 5(e ) , 5(f ) , 5(h)5(j ) ) . the reduced expression of adenylate kinase isoform ak1 , fatty acid binding protein fabp3 , isocitrate dehydrogenase icdh , porin isoform vdac1 , and atp synthase was found to be statistically significant , while the concentration of desmin , myosin heavy chain and succinate dehydrogenase was shown not to be significantly different between normal and dystrophic preparations . in general , the immunoblotting results support the main findings from our dige analysis of normal versus dystrophic heart tissue and demonstrate independently that the deficiency in dystrophin has a profound effect on the expression pattern of the cardiac protein complement . since the dige analysis of the normal versus the dystrophic mdx heart has revealed changes in a variety of mitochondrial components , it was important to determine whether this alteration was due to a reduced number of mitochondria in the dystrophic heart or internal abundance changes in the mitochondrial protein complement . thus the proteomic finding of a dystrophy - dependent increase in lamin - a / c is therefore not directly related to a drastic change in the number of nuclei per cardiac tissue unit . fluorescent labeling of mitochondria with the mitotracker dye cmxros showed a characteristic internal staining pattern in cardiac fibres ( figures 7(e)7(h ) ) . hence , lower levels of mitochondrial enzymes do not seem to be a consequence of a drastic decrease in mitochondrial density in the dystrophic mdx heart . hence , although distinct subspecies of certain cardiac proteins are affected in x - linked muscular dystrophy as determined by mass spectrometry - based proteomics , the overall isoform complement of these elements is not drastically altered . this indicates that no major differences exist in the expression levels of desmin - containing intermediate filament structures and the population of cardiac mitochondria in normal versus dystrophin - deficient cells . in order to enhance our knowledge of the molecular pathogenesis of cardiomyopathy linked to x - linked muscular dystrophy , a detailed proteomic profiling of the dystrophic heart was carried out . the biochemical establishment of a comprehensive disease - specific biomarker signature for x - linked muscular dystrophy is crucial for ( i ) the development of superior diagnostic methods , ( ii ) the identification of new therapeutic targets to address cellular alterations down - stream of the primary abnormality in dystrophin , and ( iii ) the evaluation of new treatment approaches to counter - act the various pathological aspects of dystrophinopathy . in contrast to skeletal muscle , very little is known about the global pathobiochemical mechanisms that lead to cardiac complications in the dystrophin - deficient organism . here , we have successfully applied the fluorescent dige method to the large - scale analysis of the dystrophic heart . ms - based proteomics clearly revealed a drastic decrease in key metabolic , regulatory and contractile proteins , as well as components of the cytoskeleton and the cellular stress response . the pie chart of figure 8 summarizes the apparent functions of dige - identified proteins with a differential expression in normal versus dystrophin - deficient heart muscle . the dige analysis presented here showed a moderate increase in lamin - a / c , which might improve nuclear stability , chromatin structure and gene expression in the dystrophic heart . the only drastic increase in a cardiac protein was the 4-fold change in the expression of nucleoside diphosphate kinase b. nucleoside diphosphate kinases are key regulatory enzymes involved in intracellular di- and tri - phosphonucleoside homeostasis . in contrast to the 4-fold increase in nucleoside diphosphate kinase , our proteomic screening demonstrated a massive decrease in a variety of cardiac proteins in the mdx heart . the dystrophinopathy - associated loss in numerous key proteins , which belong to different cellular pathways and functions in the heart , agrees with the severity of the cardiomyopathic complications observed in aged mdx mice and dmd patients [ 1315 ] . key proteins involved in cardiac contraction , cytoskeletal integrity , nucleotide metabolism , cellular stress response , mitochondrial metabolism , and fatty acid transportation were shown to be affected by a deficiency in cardiac dystrophin . the coupling between mitochondrial creatine kinase and the adenine nucleotide translocase system appears to be weakened in dystrophin - deficient fibres . in analogy to the reduced concentration of this antioxidant enzyme in cardiac mdx tissue , the decrease in the small heat shock protein hsp27 , as well as hsp60 and dj-1 protein , also agrees with an impaired cellular stress response in muscular dystrophy - associated cardiomyopathy . an impaired stress response could result in the detrimental aggregation of misfolded proteins causing cellular dysfunction in the dystrophic heart . the dige analysis of the mdx heart presented here has clearly shown a severely altered concentration of numerous key players involved in cardiac contraction and cytoskeletal organization . in contrast to comparable levels of myosin heavy chain , other critical contractile proteins are affected by the lack of dystrophin . the biomolecules that form the thick and thin filaments of the basic contractile units exist in a complex arrangement of myosin heavy chains , myosin light chains , and actins . hence , the reduction in the cardiac isoform of myosin light chain mlc2 , myosin light chain mlc3 , and cardiac alpha - actin might have a severe impact on the contractile apparatus in the dystrophic heart . contractile weakness observed in dystrophinopathy could be directly related to the altered concentration of myosin light chains and actin . thus , the observed reduction of alpha-1 tropomyosin in the mdx heart might also be of pathophysiological significance . however , the deficiency in cardiac dystrophin appears to trigger a concomitant reduction in the density of myozenin-2 and vimentin . the abnormal expression levels of these proteins might therefore cause disturbed interfilament interactions and impaired cystoskeletal arrays , thereby causing cellular instability in the dystrophic heart . since mitochondria represent the primary site for energy generation via oxidative phosphorylation , a reduction in elements belonging to the oxidative phosphorylation complexes , the citric acid cycle and ion homeostasis will have a profound influence on the bioenergetic status of the dystrophic heart . therefore , the reduced expression of many mitochondrial proteins associated with the matrix , outer membrane and inner membrane system suggests an impaired metabolism in the dystrophic heart . reduced expression levels within the mitochondrial proteome appear to affect the functioning of the mitochondrial atp synthase , isocitrate dehydrogenase , nadh dehydrogenase , oxoglutarate dehydrogenase , isovaleryl - coa dehydrogenase , cytochrome c oxidase and the cytochrome b - c complex , all critical elements involved in mitochondrial function in the heart . , who reported a decreased citrate synthase activity in the mdx heart . although a certain degree of cellular scarring and the infiltration of fatty and connective tissue may reduce the number of contractile fibres in the dystrophic heart , as previously shown by histochemical analysis , this does not appear to drastically alter the density of muscle - associated mitochondria . the confocal microscopy analysis shown in this report suggests no major changes in the number of mitochondria between normal and dystrophic heart muscle tissue , as judged by labeling with the established chloromethyl - x - rosamine mitotracker dye which is concentrated inside mitochondria by their negative membrane potential [ 5355 ] . the observed reduction in the rate - limiting enzyme pyruvate dehydrogenase could have severe impact on the utilization of energy carriers and thereby impair the overall bioenergetics of the mdx heart . although the overall complement of prohibitin isoforms is not drastically affected in muscular dystrophy , a reduced expression of distinct subspecies of prohibitin in the dystrophic heart the only affected glycolytic enzyme appears to be enolase , indicating that mitochondrial metabolism is more severely affected than glycolysis in dystrophy - related cardiomyopathy . interestingly , the abundance of the extracellular and intracellular fatty acid transporters albumin and fabp-3 , which are considered limiting factors of oxidative metabolism in striated muscles , is decreased in the dystrophin - deficient heart . thus , dystrophic heart cells would be starved of essential fuel supplies , which might trigger a decline in contractile strength . in contrast to a recent combined metabolomic and proteomic study of the mdx heart , and in agreement with two comprehensive proteomic studies on mdx skeletal muscle [ 42 , 45 ] , we could show here a decreased abundance of the ak1 isoform of adenylate kinase . nucleotide metabolism , involving adenylate kinase and creatine kinase appears to be disturbed in the dystrophic mdx heart , and this finding might be useful for the future development of a comprehensive biomarker signature of cardiomyopathy associated with muscular dystrophy . overall , our dige - based screening of the soluble proteome from dystrophic mdx hearts has revealed a severely perturbed protein expression pattern due to deficiency in dystrophin . the observed changes in essential proteins involved in cardiac contraction , mitochondrial metabolism , the cellular stress response and nucleotide metabolism might be useful for the future improvement of differential diagnostic procedures , the assessment of cardiac disease progression in x - linked muscular dystrophies , the identification of novel therapeutic targets to treat cardiomopathic complications in duchenne muscular dystrophy , and the evaluation of novel treatment strategies to counter - act the loss in cardiac dystrophin .

as part of baseline measurements in the 2-year cardiovascular , diabetes , and ethanol ( cascade ) randomized controlled trial , a cross - sectional analysis was performed in a subgroup of 73 men and women , aged 4173 years , with type 2 diabetes ( defined as fasting plasma glucose [ fpg ] > 126 , hba1c > 6.5 , physician diagnosis , or evidence of purchase of oral hypoglycemic medications ) who underwent magnetic resonance imaging ( mri ) of the abdomen . persons were excluded if they were smokers , pregnant , lactating , or using an insulin pump or injecting insulin more than twice per day ; had evidence of severe diabetes complications ( e.g. , proliferative retinopathy or advanced renal disease ) ; had autonomic neuropathy manifested as postural hypertension or hypoglycemia unawareness ; had a fasting serum triglyceride level > 400 mg / dl or a serum creatinine level of 2 mg / dl ( 177 mol / l ) ; had liver dysfunction ( greater than twice the upper limit of normal of alanine aminotransferase and aspartate aminotransferase levels ) ; had active cancer or received chemotherapy in the last 3 years ; or were participating in another trial . the study protocol was approved by the soroka university medical center medical ethics board and helsinki committee . mri scans of the abdomen were performed using a 1.5 tesla machine ( intera , philips medical systems , best , the netherlands ) using a body coil . subjects were examined in the supine position with arms positioned parallel along the lateral sides of the body . mri scans demonstrating fat in the different compartments were assessed using a matlab - based in - house program ( fig . the mri scan allows visualizing the fascia superficialis as a fine black line . to divide ssat from dsat after quantification , fat tissues were divided into color - coded groups : superficial subcutaneous fat = dark blue ; deep subcutaneous fat = light blue ; vat = green ; perimuscular fat ( fat surrounding and within the latissimus dorsi and diaphragm ) = purple ; and nonclassified fat ( fat surrounding the vertebrae and fat depots unrelated to any of the groups listed above ) = red . selecting the specified fat mass area was performed using a semiautomatic method ( i.e. , connected pixels ) or various manual tools , such as rectangle / circle / polygon or free hand for fine adjustments and corrections if needed . quantification of the fat mass regions included the area of each fat type and the proportion ( percentage ) of the total area of all fat types . to obtain absolute measurements in metric units , a scaling procedure was applied before the segmentation to determine real pixel dimensions . finally , in accordance with other studies ( 15 ) , we calculated the fat distribution using the mean of the three slices : s1l5 , l5l4 , and l3l2 . perimuscular and the nonclassified fat tissues , totaling a negligible fraction of total abdominal fat , were omitted from our analysis . all multivariate analyses were performed twice , using the absolute abdominal fat tissue distribution ( ssat , dsat , vat ) or the relative ( in percent ) of each depot from total abdominal adipose tissue ( taat ) ( ssat% , dsat% , vat%).figure 1mri imaging of abdominal fat tissues compartments . the subcutaneous fascial plane was delineated using the computer interface semiautomatic method , where initially an intensity - based automatic segmentation was generated and presented followed by semimanual fine tuning . fat tissues of specific anatomical landmarks were quantified and divided into color - coded groups as follows : dark blue , superficial subcutaneous fat ; light blue , deep subcutaneous fat ; green , visceral adipose tissue ; and red , nonclassified fat fat surrounding the vertebrae and fat depots that were unrelated to each of the groups listed above . ( a high - quality digital representation of this figure is available in the online issue . ) mri imaging of abdominal fat tissues compartments . the subcutaneous fascial plane was delineated using the computer interface semiautomatic method , where initially an intensity - based automatic segmentation was generated and presented followed by semimanual fine tuning . fat tissues of specific anatomical landmarks were quantified and divided into color - coded groups as follows : dark blue , superficial subcutaneous fat ; light blue , deep subcutaneous fat ; green , visceral adipose tissue ; and red , nonclassified fat fat surrounding the vertebrae and fat depots that were unrelated to each of the groups listed above . ( a high - quality digital representation of this figure is available in the online issue . ) height was measured using a wall - mounted stadiometer to the nearest millimeter for determination of bmi . waist circumference was measured halfway between the last rib and the iliac crest by the qualified study nurse , with the same type of measuring tape . mean blood pressure from two measures was recorded after resting , with the use of an automated system ( datascop acutorr 4 , soma technology , inc . , blood samples were drawn after at least an 8-h fast , and current use of all medication was recorded . to assess blood pressure and heart rate variability , 24-h ambulatory blood pressure monitoring ( oscar 2 oscillometric suntech medical model 222 , morrisville , nc ) and 24-h ambulatory electrocardiography ( ecg ) ( lifecard - cf , delmar reynolds medical ltd . , hertfordshire , uk ) were performed in a substudy group of our population ( n = 31 and n = 37 , respectively ) . heart rate variability parameters included time domain variables ( 16 ) : the mean duration of the time interval between two r waves ( rr ) , graphically presented in the form of an rr interval tachogram ; the sd of all normal rr ( sdnn ) ; the mean of all the 5-min sds of nn ( normal rr ) intervals during the 24-h period ; the root mean square successive difference , calculates the square root of the mean of the squared differences between successive nn intervals over 24 h ; and the 24-h triangular index , the integral of the density distribution ( i.e. , the number of all nn intervals ) divided by the maximum of the density distribution , which is more influenced by the lower than the higher frequencies . from the 24-h blood pressure monitor , we calculated the average systolic and diastolic blood pressure during the day ( average of records between 6 a.m. and 10 p.m. ) and night ( average of records between 10 p.m. and 6 a.m. ) . we evaluated dietary assessment by a validated food - frequency questionnaire ( 17 ) that included 127 food items and 3 portion - size pictures for 17 items . electronic questionnaires ensured completeness of the data by prompting the participant when a question was not answered . we divided our study population across tertiles of ssat ( range 5,05226,836 mm ) and used both absolute and proportional fat distribution for the analysis to account for both subdepot adiposity and interdepot distribution , respectively . fat distribution was calculated by dividing each fat depot by taat , creating three new variables : ssat% , dsat% , and vat% . anova linear test was used to evaluate the characteristics of the study population across ssat tertiles . we performed multivariate linear regression models , adjusted for age and waist circumference , to evaluate associations among ssat , anthropometric measures , diet , blood biomarkers , 24-h ambulatory blood pressure monitoring , and 24-h ambulatory ecg recordings . models including cardiovascular outcomes ( 24-h ambulatory blood pressure monitoring and 24-h ambulatory ecg recordings ) were further adjusted , one at a time , for fpg or hba1c . we further performed similar models adjusted one at a time to the various classes of medical treatments used in this patient population ( insulin , oral hypoglycemic medications , antihypertensive medications , and lipid - lowering medications ) and performed the same models , stratified by sex . multiple linear regression results are reported with the parameter estimate and p value for each variable . as part of baseline measurements in the 2-year cardiovascular , diabetes , and ethanol ( cascade ) randomized controlled trial , a cross - sectional analysis was performed in a subgroup of 73 men and women , aged 4173 years , with type 2 diabetes ( defined as fasting plasma glucose [ fpg ] > 126 , hba1c > 6.5 , physician diagnosis , or evidence of purchase of oral hypoglycemic medications ) who underwent magnetic resonance imaging ( mri ) of the abdomen . persons were excluded if they were smokers , pregnant , lactating , or using an insulin pump or injecting insulin more than twice per day ; had evidence of severe diabetes complications ( e.g. , proliferative retinopathy or advanced renal disease ) ; had autonomic neuropathy manifested as postural hypertension or hypoglycemia unawareness ; had a fasting serum triglyceride level > 400 mg / dl or a serum creatinine level of 2 mg / dl ( 177 mol / l ) ; had liver dysfunction ( greater than twice the upper limit of normal of alanine aminotransferase and aspartate aminotransferase levels ) ; had active cancer or received chemotherapy in the last 3 years ; or were participating in another trial . the study protocol was approved by the soroka university medical center medical ethics board and helsinki committee . mri scans of the abdomen were performed using a 1.5 tesla machine ( intera , philips medical systems , best , the netherlands ) using a body coil . subjects were examined in the supine position with arms positioned parallel along the lateral sides of the body . mri scans demonstrating fat in the different compartments were assessed using a matlab - based in - house program ( fig . the mri scan allows visualizing the fascia superficialis as a fine black line . to divide ssat from dsat after quantification , fat tissues were divided into color - coded groups : superficial subcutaneous fat = dark blue ; deep subcutaneous fat = light blue ; vat = green ; perimuscular fat ( fat surrounding and within the latissimus dorsi and diaphragm ) = purple ; and nonclassified fat ( fat surrounding the vertebrae and fat depots unrelated to any of the groups listed above ) = red . selecting the specified fat mass area was performed using a semiautomatic method ( i.e. , connected pixels ) or various manual tools , such as rectangle / circle / polygon or free hand for fine adjustments and corrections if needed . quantification of the fat mass regions included the area of each fat type and the proportion ( percentage ) of the total area of all fat types . to obtain absolute measurements in metric units , a scaling procedure was applied before the segmentation to determine real pixel dimensions . finally , in accordance with other studies ( 15 ) , we calculated the fat distribution using the mean of the three slices : s1l5 , l5l4 , and l3l2 . perimuscular and the nonclassified fat tissues , totaling a negligible fraction of total abdominal fat , were omitted from our analysis . all multivariate analyses were performed twice , using the absolute abdominal fat tissue distribution ( ssat , dsat , vat ) or the relative ( in percent ) of each depot from total abdominal adipose tissue ( taat ) ( ssat% , dsat% , vat%).figure 1mri imaging of abdominal fat tissues compartments . the subcutaneous fascial plane was delineated using the computer interface semiautomatic method , where initially an intensity - based automatic segmentation was generated and presented followed by semimanual fine tuning . fat tissues of specific anatomical landmarks were quantified and divided into color - coded groups as follows : dark blue , superficial subcutaneous fat ; light blue , deep subcutaneous fat ; green , visceral adipose tissue ; and red , nonclassified fat fat surrounding the vertebrae and fat depots that were unrelated to each of the groups listed above . ( a high - quality digital representation of this figure is available in the online issue . ) the subcutaneous fascial plane was delineated using the computer interface semiautomatic method , where initially an intensity - based automatic segmentation was generated and presented followed by semimanual fine tuning . fat tissues of specific anatomical landmarks were quantified and divided into color - coded groups as follows : dark blue , superficial subcutaneous fat ; light blue , deep subcutaneous fat ; green , visceral adipose tissue ; and red , nonclassified fat fat surrounding the vertebrae and fat depots that were unrelated to each of the groups listed above . ( a high - quality digital representation of this figure is available in the online issue . ) height was measured using a wall - mounted stadiometer to the nearest millimeter for determination of bmi . waist circumference was measured halfway between the last rib and the iliac crest by the qualified study nurse , with the same type of measuring tape . mean blood pressure from two measures was recorded after resting , with the use of an automated system ( datascop acutorr 4 , soma technology , inc . , blood samples were drawn after at least an 8-h fast , and current use of all medication was recorded . to assess blood pressure and heart rate variability , 24-h ambulatory blood pressure monitoring ( oscar 2 oscillometric suntech medical model 222 , morrisville , nc ) and 24-h ambulatory electrocardiography ( ecg ) ( lifecard - cf , delmar reynolds medical ltd . , hertfordshire , uk ) were performed in a substudy group of our population ( n = 31 and n = 37 , respectively ) . heart rate variability parameters included time domain variables ( 16 ) : the mean duration of the time interval between two r waves ( rr ) , graphically presented in the form of an rr interval tachogram ; the sd of all normal rr ( sdnn ) ; the mean of all the 5-min sds of nn ( normal rr ) intervals during the 24-h period ; the root mean square successive difference , calculates the square root of the mean of the squared differences between successive nn intervals over 24 h ; and the 24-h triangular index , the integral of the density distribution ( i.e. , the number of all nn intervals ) divided by the maximum of the density distribution , which is more influenced by the lower than the higher frequencies . from the 24-h blood pressure monitor , we calculated the average systolic and diastolic blood pressure during the day ( average of records between 6 a.m. and 10 p.m. ) and night ( average of records between 10 p.m. and 6 a.m. ) . we evaluated dietary assessment by a validated food - frequency questionnaire ( 17 ) that included 127 food items and 3 portion - size pictures for 17 items . electronic questionnaires ensured completeness of the data by prompting the participant when a question was not answered . we divided our study population across tertiles of ssat ( range 5,05226,836 mm ) and used both absolute and proportional fat distribution for the analysis to account for both subdepot adiposity and interdepot distribution , respectively . fat distribution was calculated by dividing each fat depot by taat , creating three new variables : ssat% , dsat% , and vat% . anova linear test was used to evaluate the characteristics of the study population across ssat tertiles . we performed multivariate linear regression models , adjusted for age and waist circumference , to evaluate associations among ssat , anthropometric measures , diet , blood biomarkers , 24-h ambulatory blood pressure monitoring , and 24-h ambulatory ecg recordings . models including cardiovascular outcomes ( 24-h ambulatory blood pressure monitoring and 24-h ambulatory ecg recordings ) were further adjusted , one at a time , for fpg or hba1c . we further performed similar models adjusted one at a time to the various classes of medical treatments used in this patient population ( insulin , oral hypoglycemic medications , antihypertensive medications , and lipid - lowering medications ) and performed the same models , stratified by sex . multiple linear regression results are reported with the parameter estimate and p value for each variable . the baseline characteristics of the participants in the entire study group and acrosstertiles of ssat are shown in table 1 . mean hba1c was 7.5 1.1% , and 10 ( 14% ) patients were taking one daily dose of insulin . the oral hypoglycemic medications used by the patients included sulfonylureas ( 11 patients,15% ) , dipeptidyl peptidase-4 inhibitor ( 9 patients , 12% ) , and metformin ( 31 patients , 42% ) . the mean fat tissues distribution was as follows : ssat 26% , dsat 23% , and vat 51% . ssat positively correlated with dsat ( r = 0.389 , p < 0.001 ) and taat ( r = 0.461 , p < 0.001 ) . ssat , dsat , and taat correlated positively and significantly with waist circumference ( ssat : r = 0.313 , p = 0.009 , dsat : r = 0.276 , p = 0.023 , taat : r = 0.449 , p < 0.001 ) and bmi ( ssat : r = 0.490 , p < 0.001 , dsat : r = 0.327 , p = 0.005 , taat r = 0.508 , p < 0.001 ) . vat correlated positively and significantly with waist circumference and weight ( r = 0.330 , p = 0.006 ; r = 0.346 , p = 0.003 , respectively ) . medical treatment , including insulin therapy and antihypertensive , lipid - lowering , antiplatelet , and oral hypoglycemic medications , was similarly distributed across ssat tertiles ( table 1 ) . distribution of fat depots , biomarkers , and clinical parameters across tertiles of absolute abdominal ssat among patients with type 2 diabetes fpg ( p of trend = 0.046 , p = 0.073 between extreme tertiles ) and hba1c ( p of trend = 0.006 , p = 0.011 between extreme tertiles ) were both lower with increased tertile of ssat . ssat was inversely correlated with hba1c ( r = 0.262 , p = 0.027 ) , whereas vat ( r = 0.240 , p = 0.042 ) and taat positively correlated with fasting triglycerides ( r = 0.278 , p = 0.018 ) . in a regression model ( fig . 2a ) , adjusted for age and waist circumference , increased absolute ssat was associated with decreased hba1c ( = 0.289 , p = 0.017 ) and tended to be associated with decreased fasting glucose ( = 0.208 , 0.084 ) . further adjustment of the model to medical treatment classes ( one at a time : insulin therapy and antihypertensive , lipid - lowering , antiplatelet , and oral hypoglycemic medications ) did not significantly attenuate these associations ( data not shown ) . total energy intake , total dietary fat , saturated fat , and unsaturated fat content were not associated with fat tissue mass ( data not shown ) . however , increased total dietary intake of trans fatty acids tended to be positively correlated with total sat ( r = 0.228 , p = 0.054 ) and dsat ( r = 0.200 , p = 0.093 ) . collectively , the absolute area ( mass ) of abdominal ssat seemed to be associated with more favorable glycemic and cardiovascular parameters , unlike dsat or vat . association of metabolic parameters and cardiovascular parameters with absolute abdominal fat tissues in patients with type 2 diabetes , stratified by sex . numbers represent standardized coefficient : the amount and direction by which absolute abdominal fat tissues change ( mm ) for each unit change in the metabolic parameters , while accounting for the other variables in the model . we next assessed whether the relative adipose tissue distribution , i.e. , the percentage of fat in a certain abdominal ( sub)-depot , was associated with clinical parameters . the percentage of ssat from taat ( % ssat ) was associated with lower hba1c in regression models adjusted for age and waist circumference ( = 0.304 , p = 0.017 ) , consistent with the finding using the absolute ssat area mentioned above . when we added medical treatment to the model ( insulin therapy and antihypertensive , lipid - lowering , antiplatelet , and oral hypoglycemic medications ) , the association remained significant . conversely , increased dsat was associated with increased levels of hba1c ( = 0.266 , p = 0.039 ) and fasting glucose ( = 0.246 , p = 0.054 ) . increased % ssat was associated with increased hdl cholesterol ( = 0.251 , p = 0.047 ) ( fig . association of metabolic and cardiovascular parameters with abdominal fat tissues distribution in patients with type 2 diabetes , stratified by sex . numbers represent standardized coefficient : the amount and direction by which proportional abdominal fat tissues change ( % ) for each unit change in the metabolic parameters , while accounting for the other variables in the model . tg , triglycerides ( mg / day ) . * p < 0.05 . of the 12 women recruited , 11 were postmenopausal , none of whom were taking hormone replacement therapy . although men and women had similar waist circumferences ( 104 vs. 103 cm , p = 0.902 ) , taat ( 37,095 vs. 37,108 mm , p = 0.975 ) , and medical treatment , women had higher % ssat than did men and twice the ratio of ssat to vat ( table 2 ) . they had lower fasting glucose ( p < 0.001 ) and lower hba1c ( p = 0.002 ) , and tended to have higher hdl cholesterol ( p = 0.076 ) . men had a higher vat to sat ratio , whereas % dsat was similar between men and women . to verify that the association between ssat and favorable cardiometabolic parameters did not simply reflect intrinsic differences between genders , we stratified the regression models by sex ( figs . 2b and 3b ) . in a subgroup of the men only , similar opposite associations among % ssat , % dsat , and cardiometabolic parameters were observed ( fig . 3b ) . in addition , in the men - only subgroup , % dsat was significantly associated with higher glucose ( = 0.292 , p = 0.040 ) and hba1c ( = 0.298 , p = 0.038 ) . abdominal fat tissue distribution , biomarkers , and clinical parameters in men and women with type 2 diabetes we further found associations among the absolute abdominal subdepot fat ( mass ) , proportional abdominal subdepot fat area , blood pressure , and heart rate variability parameters : both absolute ( r = 0.381 , p = 0.050 ) and proportional dsat ( r = 0.428 , p = 0.026 ) negatively correlated with sdnn . in regression models , adjusted for age and waist circumference , higher absolute dsat ( = 0.425 , p = 0.034 ) and higher proportional dsat ( = 0.431 p = 0.029 ) were associated with lower sdnn . proportional dsat was significantly associated with decreased 24-h triangular index ( = 0.417 , p = 0.030 ) , suggesting decreased heart rate variability with increased fat distribution to the dsat depot . however , further adjustment to oral hypoglycemic medications , but not to insulin therapy , antihypertensive , lipid - lowering , or antiplatelet drugs , attenuated the association between dsat and markers of heart rate variability ( 24-h triangular index : = 0.249 , p = 0.228 , sdnn : = 0.294 , p = 0.186 ) . because hyperglycemia is a key risk factor for autonomic neuropathy , we further adjusted the models , one at a time , for hba1c or fpg . the association between both absolute and proportional dsat and heart rate variability remained negative , but the significance was attenuated . when adjusted for hba1c , higher absolute ( = 0.355 , p = 0.093 ) and proportional ( = 0.401 p = 0.073 ) dsat were associated with lower sdnn . proportional dsat was negatively associated with decreased 24-h triangular index ( = 0.406 , p = 0.063 ) . when adjusted for glucose , higher absolute ( = 0.278 p = 0.144 ) and proportional ( = 0.322 p = 0.122 ) dsat were not associated with lower sdnn . in contrast , opposite trends were observed with absolute and proportional abdominal ssat : higher absolute ( = 0.329 , p = 0.043 ) and proportional ssat ( = 0.425 , p = 0.008 ) were associated with lower mean daytime diastolic blood pressure . when medical treatment was added to the model ( insulin therapy , antihypertensive , lipid - lowering , antiplatelet , or oral hypoglycemic medications ) , the association remained significant . increased proportional ssat tended to positively correlate with increased square root of the mean of the squared differences between successive nn intervals over 24 h ( r = 0.367 , p = 0.060 ) . when hba1c was added to the model , the association between absolute ssat and mean daytime diastolic blood pressure was attenuated ( = 0.284 , p = 0.072 ) , although the association between proportional ssat and mean daytime diastolic blood pressure remained significant ( = 0.379 , p = 0.008 ) . when adjusted for glucose , the association between both absolute ( = 0.329 , p = 0.043 ) and proportional ( = 0.444 , p = 0.006 ) ssat and mean daytime diastolic blood pressure remained significant . when the models were stratified by sex , we found that the men - only subgroup also exhibited similar associations among abdominal subdepot fat ( mass and proportion ) , blood pressure , and heart rate variability parameters . higher absolute dsat ( = 0.452 , p = 0.014 ) and proportional dsat ( = 0.535 p = 0.009 ) were associated with lower sdnn . higher absolute dsat ( = 0.486 , p = 0.007 ) and proportional dsat ( = 0.557 , p = 0.005 ) were associated with decreased 24-h triangular index . further adjustment to oral hypoglycemic medications , but not to insulin therapy , antihypertensive , lipid - lowering , or antiplatelet drugs , attenuated the association between proportional dsat and markers of heart rate variability ( 24-h triangular index : = 0.439 , p = 0.075 , sdnn : = 0.443 , p = 0.074 ) and between absolute markers of heart rate variability ( 24-h triangular index : = 0.408 , p = 0.073 , sdnn : = 0.408 , p = 0.072 ) . the baseline characteristics of the participants in the entire study group and acrosstertiles of ssat are shown in table 1 . mean hba1c was 7.5 1.1% , and 10 ( 14% ) patients were taking one daily dose of insulin . the oral hypoglycemic medications used by the patients included sulfonylureas ( 11 patients,15% ) , dipeptidyl peptidase-4 inhibitor ( 9 patients , 12% ) , and metformin ( 31 patients , 42% ) . the mean fat tissues distribution was as follows : ssat 26% , dsat 23% , and vat 51% . ssat positively correlated with dsat ( r = 0.389 , p < 0.001 ) and taat ( r = 0.461 , p < 0.001 ) . ssat , dsat , and taat correlated positively and significantly with waist circumference ( ssat : r = 0.313 , p = 0.009 , dsat : r = 0.276 , p = 0.023 , taat : r = 0.449 , p < 0.001 ) and bmi ( ssat : r = 0.490 , p < 0.001 , dsat : r = 0.327 , p = 0.005 , taat r = 0.508 , p < 0.001 ) . vat correlated positively and significantly with waist circumference and weight ( r = 0.330 , p = 0.006 ; r = 0.346 , p = 0.003 , respectively ) . medical treatment , including insulin therapy and antihypertensive , lipid - lowering , antiplatelet , and oral hypoglycemic medications , was similarly distributed across ssat tertiles ( table 1 ) . distribution of fat depots , biomarkers , and clinical parameters across tertiles of absolute abdominal ssat among patients with type 2 diabetes fpg ( p of trend = 0.046 , p = 0.073 between extreme tertiles ) and hba1c ( p of trend = 0.006 , p = 0.011 between extreme tertiles ) were both lower with increased tertile of ssat . ssat was inversely correlated with hba1c ( r = 0.262 , p = 0.027 ) , whereas vat ( r = 0.240 , p = 0.042 ) and taat positively correlated with fasting triglycerides ( r = 0.278 , p = 0.018 ) . in a regression model ( fig . 2a ) , adjusted for age and waist circumference , increased absolute ssat was associated with decreased hba1c ( = 0.289 , p = 0.017 ) and tended to be associated with decreased fasting glucose ( = 0.208 , 0.084 ) . further adjustment of the model to medical treatment classes ( one at a time : insulin therapy and antihypertensive , lipid - lowering , antiplatelet , and oral hypoglycemic medications ) did not significantly attenuate these associations ( data not shown ) . total energy intake , total dietary fat , saturated fat , and unsaturated fat content were not associated with fat tissue mass ( data not shown ) . however , increased total dietary intake of trans fatty acids tended to be positively correlated with total sat ( r = 0.228 , p = 0.054 ) and dsat ( r = 0.200 , p = 0.093 ) . collectively , the absolute area ( mass ) of abdominal ssat seemed to be associated with more favorable glycemic and cardiovascular parameters , unlike dsat or vat . association of metabolic parameters and cardiovascular parameters with absolute abdominal fat tissues in patients with type 2 diabetes , stratified by sex . numbers represent standardized coefficient : the amount and direction by which absolute abdominal fat tissues change ( mm ) for each unit change in the metabolic parameters , while accounting for the other variables in the model . we next assessed whether the relative adipose tissue distribution , i.e. , the percentage of fat in a certain abdominal ( sub)-depot , was associated with clinical parameters . the percentage of ssat from taat ( % ssat ) was associated with lower hba1c in regression models adjusted for age and waist circumference ( = 0.304 , p = 0.017 ) , consistent with the finding using the absolute ssat area mentioned above . when we added medical treatment to the model ( insulin therapy and antihypertensive , lipid - lowering , antiplatelet , and oral hypoglycemic medications ) , the association remained significant . conversely , increased dsat was associated with increased levels of hba1c ( = 0.266 , p = 0.039 ) and fasting glucose ( = 0.246 , p = 0.054 ) . increased % ssat was associated with increased hdl cholesterol ( = 0.251 , p = 0.047 ) ( fig . association of metabolic and cardiovascular parameters with abdominal fat tissues distribution in patients with type 2 diabetes , stratified by sex . a : entire group . numbers represent standardized coefficient : the amount and direction by which proportional abdominal fat tissues change ( % ) for each unit change in the metabolic parameters , while accounting for the other variables in the model . tg , triglycerides ( mg / day ) . * p < 0.05 . of the 12 women recruited , 11 were postmenopausal , none of whom were taking hormone replacement therapy . although men and women had similar waist circumferences ( 104 vs. 103 cm , p = 0.902 ) , taat ( 37,095 vs. 37,108 mm , p = 0.975 ) , and medical treatment , women had higher % ssat than did men and twice the ratio of ssat to vat ( table 2 ) . they had lower fasting glucose ( p < 0.001 ) and lower hba1c ( p = 0.002 ) , and tended to have higher hdl cholesterol ( p = 0.076 ) . men had a higher vat to sat ratio , whereas % dsat was similar between men and women . to verify that the association between ssat and favorable cardiometabolic parameters did not simply reflect intrinsic differences between genders , we stratified the regression models by sex ( figs . only , similar opposite associations among % ssat , % dsat , and cardiometabolic parameters were observed ( fig . 3b ) . in addition , in the men - only subgroup , % dsat was significantly associated with higher glucose ( = 0.292 , p = 0.040 ) and hba1c ( = 0.298 , p = 0.038 ) . abdominal fat tissue distribution , biomarkers , and clinical parameters in men and women with type 2 diabetes we further found associations among the absolute abdominal subdepot fat ( mass ) , proportional abdominal subdepot fat area , blood pressure , and heart rate variability parameters : both absolute ( r = 0.381 , p = 0.050 ) and proportional dsat ( r = 0.428 , p = 0.026 ) negatively correlated with sdnn . in regression models , adjusted for age and waist circumference , higher absolute dsat ( = 0.425 , p = 0.034 ) and higher proportional dsat ( = 0.431 p = 0.029 ) proportional dsat was significantly associated with decreased 24-h triangular index ( = 0.417 , p = 0.030 ) , suggesting decreased heart rate variability with increased fat distribution to the dsat depot . however , further adjustment to oral hypoglycemic medications , but not to insulin therapy , antihypertensive , lipid - lowering , or antiplatelet drugs , attenuated the association between dsat and markers of heart rate variability ( 24-h triangular index : = 0.249 , p = 0.228 , sdnn : = 0.294 , p = 0.186 ) . because hyperglycemia is a key risk factor for autonomic neuropathy , we further adjusted the models , one at a time , for hba1c or fpg . the association between both absolute and proportional dsat and heart rate variability remained negative , but the significance was attenuated . when adjusted for hba1c , higher absolute ( = 0.355 , p = 0.093 ) and proportional ( = 0.401 p = 0.073 ) dsat were associated with lower sdnn . proportional dsat was negatively associated with decreased 24-h triangular index ( = 0.406 , p = 0.063 ) . when adjusted for glucose , higher absolute ( = 0.278 p = 0.144 ) and proportional ( = 0.322 p = 0.122 ) dsat were not associated with lower sdnn . in contrast , opposite trends were observed with absolute and proportional abdominal ssat : higher absolute ( = 0.329 , p = 0.043 ) and proportional ssat ( = 0.425 , p = 0.008 ) were associated with lower mean daytime diastolic blood pressure . when medical treatment was added to the model ( insulin therapy , antihypertensive , lipid - lowering , antiplatelet , or oral hypoglycemic medications ) , the association remained significant . increased proportional ssat tended to positively correlate with increased square root of the mean of the squared differences between successive nn intervals over 24 h ( r = 0.367 , p = 0.060 ) . when hba1c was added to the model , the association between absolute ssat and mean daytime diastolic blood pressure was attenuated ( = 0.284 , p = 0.072 ) , although the association between proportional ssat and mean daytime diastolic blood pressure remained significant ( = 0.379 , p = 0.008 ) . when adjusted for glucose , the association between both absolute ( = 0.329 , p = 0.043 ) and proportional ( = 0.444 , p = 0.006 ) ssat and mean daytime diastolic blood pressure remained significant . when the models were stratified by sex , we found that the men - only subgroup also exhibited similar associations among abdominal subdepot fat ( mass and proportion ) , blood pressure , and heart rate variability parameters . higher absolute dsat ( = 0.452 , p = 0.014 ) and proportional dsat ( = 0.535 p = 0.009 ) were associated with lower sdnn . higher absolute dsat ( = 0.486 , p = 0.007 ) and proportional dsat ( = 0.557 , p = 0.005 ) were associated with decreased 24-h triangular index . further adjustment to oral hypoglycemic medications , but not to insulin therapy , antihypertensive , lipid - lowering , or antiplatelet drugs , attenuated the association between proportional dsat and markers of heart rate variability ( 24-h triangular index : = 0.439 , p = 0.075 , sdnn : = 0.443 , p = 0.074 ) and between absolute markers of heart rate variability ( 24-h triangular index : = 0.408 , p = 0.073 , sdnn : = 0.408 , p = 0.072 ) . in this study of patients with type 2 diabetes , we observed a distinct association between both the absolute ( representing sub - depot adiposity ) and the relative amounts ( representing interdepot distribution ) of abdominal ssat and markers of more favorable glycemic control and cardiovascular function as determined by higher heart rate variability and lower blood pressure . after adjusting for age and waist circumference , higher relative distribution of abdominal fat in ssat was correlated with improved glycemic control ( hba1c and fasting glucose ) and better indicators of cardiovascular health ( lower blood pressure and higher heart rate variability ) . conversely , dsat correlated with higher heart rate and lower heart rate variability , both indicators of autonomic neuropathy ( 18 ) , and therefore indicators for increased cardiovascular risk . of note , controlling for markers of glycemic control attenuated the negative association between dsat and cardiovascular end point , but not the favorable association with ssat . in terms of diet , increased intake of trans fat tended to be associated with total abdominal sat and dsat , but not with ssat . because most studies support the more adverse metabolic role of intraabdominal / vat , whether sat is simply less pathogenic than vat or exerts direct or indirect protective effects on cardiovascular and metabolic morbidity is still controversial ( 6,9 ) . furthermore , even more uncertainty exists as to the functional differences and risk associated with the dsat or ssat subdepots in persons with type 2 diabetes ( 19 ) . our results suggest a favorable distinct association between abdominal ssat subdepot and cardiometabolic health in type 2 diabetes . this is a cross - sectional design that may suggest associations but not clear cause effect protective relationships between abdominal ssat and cardiometabolic parameters . because we did not measure peripheral ( lower body ) our sample size limits the statistical power , although we still could identify significant differences between parameters . a significant proportion of our patients received treatment with medications that may modify the levels of risk factors and directly affect glycemic control parameters . although we have made an attempt to adjust our results to the use of these drugs , doses were not assessed . finally , we had a lower proportion of women , but nevertheless we could identify significant differences between the sexes . the strengths of our study include the specific group with type 2 diabetes , the high mri quality imaging , the comprehensive 24-h ambulatory blood pressure and 24-h ambulatory ecg measurements as cardiometabolic measurements , the medication follow - up , and the dietary assessment . we found that abdominal ssat correlated with improved glycemic control and indicators of cardiovascular risk . the ssat depot may be less lipolytic than vat , or even dsat , and so improved insulin sensitivity of ssat may favor accumulation of excess energy in this depot . in this regard , higher deposition of excess calories in the ssat is a consequence , not the cause , of improved metabolic function . conversely , it is plausible that the abdominal ssat fat mass may be a unique abdominal fat subdepot that has protective effects on glycemic control and cardiovascular function . this is reminiscent of a finding by some , but not all , studies that suggest peripheral sat might be less pathogenic than vat . currently , two hypotheses have been put forward to explain the difference between peripheral sat and vat : the portal theory ( 6,20 ) implicates a direct mechanism whereby vat is more pathogenic because its venous blood drainage is directly via the portal vein to the liver . the ectopic fat hypothesis ( 6 ) suggests an indirect mechanism whereby increased energy storage in peripheral sat exerts a protective effect by decreasing fat deposition in the liver , muscle , and heart . because abdominal dsat exhibits an intermediate phenotype between vat and abdominal ssat in various functions tested ( lipolysis , adipocytokine profile ) ( 4 ) , it is possible that these theories can underlie the unique positive association between abdominal ssat and cardiovascular and metabolic health . although it may be possible that peripheral sat differs significantly from abdominal sat , and some studies have indicated the potential pathogenic role of increased abdominal ( total ) sat as opposed to peripheral sat ( 911 ) , further studies are required to fully understand the distinct role of the ssat subdepot . it is tempting to speculate that discrepancies in the literature among studies assessing associations between abdominal sat and morbidity were confined by differences in the ssat or ssat / dsat distribution . nutritional habits may also affect fat distribution . in our study , increased trans fatty acids consumption tended to be associated with increased total abdominal sat and dsat , but not ssat . limited evidence has suggested that increased dietary intake of trans fatty acids may increase fat accumulation and abdominal circumference ( 21,22 ) . although these studies emphasize the increase in vat , our findings suggest that dsat also may be increased by excessive intake of trans fatty acids . women in our study had a higher proportion of abdominal ssat , whereas men had a higher intra - abdominal fat to subcutaneous fat ratio . fat is distributed in a sexual dimorphic manner , beyond the differences between upper body ( more in men ) versus lower body locations ( more in women ) . for a given amount of intra - abdominal fat , women , who are relatively protected from cardiometabolic morbidity , at least before menopause ( 6 ) , possess up to twice as much subcutaneous fat as men ( 25,26 ) . of note , although sexual dimorphism in fat distribution usually tends to diminish with postmenopausal state , we observed such changes in our cohort despite the fact that most ( 11/12 ) women were postmenopausal . this finding may complement a prior report that postmenopausal women who possess a higher proportion of adipose tissue located in the total midthigh depot had a more favorable metabolic profile ( 25 ) . although studies have reported an association between hormone replacement therapy and cardiovascular health ( 27,28 ) , none of the women in our study were on such therapy . future studies will unravel whether increased abdominal ssat and its seemingly favorable metabolic and cardiovascular correlates diminish with years into menopause , or have other determinants , possibly unique to persons with diabetes . finally , it has been proposed that antidiabetic medications , particularly of the thiazolidinedione family , may exert their therapeutic effect at least partially by inducing redistribution of fat from pathogenic to less - pathogenic depots ( e.g. , from vat to sat ) ( 29,30 ) . other hypoglycemic medications were distributed similarly among tertiles of ssat and controlled for in the regression models . in summary , the current study adds to the understanding of the pattern of abdominal fat distribution , including sub - depots , and its relation to glycemic control and cardiovascular risk in diabetic patients . effective interventions that can alter abdominal fat distribution may help sort out whether fat distribution phenotypes are merely a reflection of obesity subphenotypes or can causally affect risk and severity of obesity - associated morbidities .

objectiveunlike visceral adipose tissue ( vat ) , the association between subcutaneous adipose tissue ( sat ) and obesity - related morbidity is controversial . in patients with type 2 diabetes , we assessed whether this variability can be explained by a putative favorable , distinct association between abdominal superficial sat ( ssat ) ( absolute amount or its proportion ) and cardiometabolic parameters.research design and methodswe performed abdominal magnetic resonance imaging ( mri ) in 73 patients with diabetes ( mean age 58 years , 83% were men ) and cross - sectionally analyzed fat distribution at s1-l5 , l5-l4 , and l3-l2 levels . patients completed food frequency questionnaires , and subgroups had 24-h ambulatory blood pressure monitoring and 24-h ambulatory electrocardiography.resultswomen had higher % ssat ( 37 vs. 23% in men ; p < 0.001 ) despite a similar mean waist circumference . fasting plasma glucose ( p = 0.046 ) and hba1c ( p = 0.006 ) were both lower with increased tertile of absolute ssat . in regression models adjusted for age , waist circumference , and classes of medical treatments used in this patient population , increased % ssat was significantly associated with decreased hba1c ( = 0.317 ; p = 0.013 ) , decreased daytime ambulatory blood pressure ( = 0.426 ; p = 0.008 ) , and increased hdl cholesterol ( = 0.257 ; p = 0.042 ) . in contrast , increased percent of deep sat ( dsat ) was associated with increased hba1c ( = 0.266 ; p = 0.040 ) and poorer heart rate variability parameters ( p = 0.030 ) . although total fat and energy intake were not correlated with fat tissue distribution , increased intake of trans fat tended to be associated with total sat ( r = 0.228 ; p = 0.05 ) and dsat ( r = 0.20 ; p = 0.093 ) , but not with ssat.conclusionsabdominal sat is composed of two subdepots that associate differently with cardiometabolic parameters . higher absolute and relative distribution of fat in abdominal ssat may signify beneficial cardiometabolic effects in patients with type 2 diabetes .

RESEARCH DESIGN AND METHODS Study population MRI acquisition and image analysis Clinical parameters Dietary assessment Statistical analysis RESULTS Fat distribution, blood biomarkers, and diet Proportional fat tissue distribution Fat distribution, 24-h ambulatory blood pressure monitoring, and 24-h ambulatory ECG CONCLUSIONS

as part of baseline measurements in the 2-year cardiovascular , diabetes , and ethanol ( cascade ) randomized controlled trial , a cross - sectional analysis was performed in a subgroup of 73 men and women , aged 4173 years , with type 2 diabetes ( defined as fasting plasma glucose [ fpg ] > 126 , hba1c > 6.5 , physician diagnosis , or evidence of purchase of oral hypoglycemic medications ) who underwent magnetic resonance imaging ( mri ) of the abdomen . all multivariate analyses were performed twice , using the absolute abdominal fat tissue distribution ( ssat , dsat , vat ) or the relative ( in percent ) of each depot from total abdominal adipose tissue ( taat ) ( ssat% , dsat% , vat%).figure 1mri imaging of abdominal fat tissues compartments . to assess blood pressure and heart rate variability , 24-h ambulatory blood pressure monitoring ( oscar 2 oscillometric suntech medical model 222 , morrisville , nc ) and 24-h ambulatory electrocardiography ( ecg ) ( lifecard - cf , delmar reynolds medical ltd . we performed multivariate linear regression models , adjusted for age and waist circumference , to evaluate associations among ssat , anthropometric measures , diet , blood biomarkers , 24-h ambulatory blood pressure monitoring , and 24-h ambulatory ecg recordings . models including cardiovascular outcomes ( 24-h ambulatory blood pressure monitoring and 24-h ambulatory ecg recordings ) were further adjusted , one at a time , for fpg or hba1c . we further performed similar models adjusted one at a time to the various classes of medical treatments used in this patient population ( insulin , oral hypoglycemic medications , antihypertensive medications , and lipid - lowering medications ) and performed the same models , stratified by sex . as part of baseline measurements in the 2-year cardiovascular , diabetes , and ethanol ( cascade ) randomized controlled trial , a cross - sectional analysis was performed in a subgroup of 73 men and women , aged 4173 years , with type 2 diabetes ( defined as fasting plasma glucose [ fpg ] > 126 , hba1c > 6.5 , physician diagnosis , or evidence of purchase of oral hypoglycemic medications ) who underwent magnetic resonance imaging ( mri ) of the abdomen . all multivariate analyses were performed twice , using the absolute abdominal fat tissue distribution ( ssat , dsat , vat ) or the relative ( in percent ) of each depot from total abdominal adipose tissue ( taat ) ( ssat% , dsat% , vat%).figure 1mri imaging of abdominal fat tissues compartments . to assess blood pressure and heart rate variability , 24-h ambulatory blood pressure monitoring ( oscar 2 oscillometric suntech medical model 222 , morrisville , nc ) and 24-h ambulatory electrocardiography ( ecg ) ( lifecard - cf , delmar reynolds medical ltd . we performed multivariate linear regression models , adjusted for age and waist circumference , to evaluate associations among ssat , anthropometric measures , diet , blood biomarkers , 24-h ambulatory blood pressure monitoring , and 24-h ambulatory ecg recordings . models including cardiovascular outcomes ( 24-h ambulatory blood pressure monitoring and 24-h ambulatory ecg recordings ) were further adjusted , one at a time , for fpg or hba1c . we further performed similar models adjusted one at a time to the various classes of medical treatments used in this patient population ( insulin , oral hypoglycemic medications , antihypertensive medications , and lipid - lowering medications ) and performed the same models , stratified by sex . ssat positively correlated with dsat ( r = 0.389 , p < 0.001 ) and taat ( r = 0.461 , p < 0.001 ) . ssat , dsat , and taat correlated positively and significantly with waist circumference ( ssat : r = 0.313 , p = 0.009 , dsat : r = 0.276 , p = 0.023 , taat : r = 0.449 , p < 0.001 ) and bmi ( ssat : r = 0.490 , p < 0.001 , dsat : r = 0.327 , p = 0.005 , taat r = 0.508 , p < 0.001 ) . distribution of fat depots , biomarkers , and clinical parameters across tertiles of absolute abdominal ssat among patients with type 2 diabetes fpg ( p of trend = 0.046 , p = 0.073 between extreme tertiles ) and hba1c ( p of trend = 0.006 , p = 0.011 between extreme tertiles ) were both lower with increased tertile of ssat . ssat was inversely correlated with hba1c ( r = 0.262 , p = 0.027 ) , whereas vat ( r = 0.240 , p = 0.042 ) and taat positively correlated with fasting triglycerides ( r = 0.278 , p = 0.018 ) . 2a ) , adjusted for age and waist circumference , increased absolute ssat was associated with decreased hba1c ( = 0.289 , p = 0.017 ) and tended to be associated with decreased fasting glucose ( = 0.208 , 0.084 ) . however , increased total dietary intake of trans fatty acids tended to be positively correlated with total sat ( r = 0.228 , p = 0.054 ) and dsat ( r = 0.200 , p = 0.093 ) . the percentage of ssat from taat ( % ssat ) was associated with lower hba1c in regression models adjusted for age and waist circumference ( = 0.304 , p = 0.017 ) , consistent with the finding using the absolute ssat area mentioned above . conversely , increased dsat was associated with increased levels of hba1c ( = 0.266 , p = 0.039 ) and fasting glucose ( = 0.246 , p = 0.054 ) . increased % ssat was associated with increased hdl cholesterol ( = 0.251 , p = 0.047 ) ( fig . although men and women had similar waist circumferences ( 104 vs. 103 cm , p = 0.902 ) , taat ( 37,095 vs. 37,108 mm , p = 0.975 ) , and medical treatment , women had higher % ssat than did men and twice the ratio of ssat to vat ( table 2 ) . they had lower fasting glucose ( p < 0.001 ) and lower hba1c ( p = 0.002 ) , and tended to have higher hdl cholesterol ( p = 0.076 ) . in addition , in the men - only subgroup , % dsat was significantly associated with higher glucose ( = 0.292 , p = 0.040 ) and hba1c ( = 0.298 , p = 0.038 ) . abdominal fat tissue distribution , biomarkers , and clinical parameters in men and women with type 2 diabetes we further found associations among the absolute abdominal subdepot fat ( mass ) , proportional abdominal subdepot fat area , blood pressure , and heart rate variability parameters : both absolute ( r = 0.381 , p = 0.050 ) and proportional dsat ( r = 0.428 , p = 0.026 ) negatively correlated with sdnn . in regression models , adjusted for age and waist circumference , higher absolute dsat ( = 0.425 , p = 0.034 ) and higher proportional dsat ( = 0.431 p = 0.029 ) were associated with lower sdnn . proportional dsat was significantly associated with decreased 24-h triangular index ( = 0.417 , p = 0.030 ) , suggesting decreased heart rate variability with increased fat distribution to the dsat depot . however , further adjustment to oral hypoglycemic medications , but not to insulin therapy , antihypertensive , lipid - lowering , or antiplatelet drugs , attenuated the association between dsat and markers of heart rate variability ( 24-h triangular index : = 0.249 , p = 0.228 , sdnn : = 0.294 , p = 0.186 ) . when adjusted for hba1c , higher absolute ( = 0.355 , p = 0.093 ) and proportional ( = 0.401 p = 0.073 ) dsat were associated with lower sdnn . when adjusted for glucose , higher absolute ( = 0.278 p = 0.144 ) and proportional ( = 0.322 p = 0.122 ) dsat were not associated with lower sdnn . in contrast , opposite trends were observed with absolute and proportional abdominal ssat : higher absolute ( = 0.329 , p = 0.043 ) and proportional ssat ( = 0.425 , p = 0.008 ) were associated with lower mean daytime diastolic blood pressure . increased proportional ssat tended to positively correlate with increased square root of the mean of the squared differences between successive nn intervals over 24 h ( r = 0.367 , p = 0.060 ) . when hba1c was added to the model , the association between absolute ssat and mean daytime diastolic blood pressure was attenuated ( = 0.284 , p = 0.072 ) , although the association between proportional ssat and mean daytime diastolic blood pressure remained significant ( = 0.379 , p = 0.008 ) . when adjusted for glucose , the association between both absolute ( = 0.329 , p = 0.043 ) and proportional ( = 0.444 , p = 0.006 ) ssat and mean daytime diastolic blood pressure remained significant . when the models were stratified by sex , we found that the men - only subgroup also exhibited similar associations among abdominal subdepot fat ( mass and proportion ) , blood pressure , and heart rate variability parameters . higher absolute dsat ( = 0.452 , p = 0.014 ) and proportional dsat ( = 0.535 p = 0.009 ) were associated with lower sdnn . higher absolute dsat ( = 0.486 , p = 0.007 ) and proportional dsat ( = 0.557 , p = 0.005 ) were associated with decreased 24-h triangular index . further adjustment to oral hypoglycemic medications , but not to insulin therapy , antihypertensive , lipid - lowering , or antiplatelet drugs , attenuated the association between proportional dsat and markers of heart rate variability ( 24-h triangular index : = 0.439 , p = 0.075 , sdnn : = 0.443 , p = 0.074 ) and between absolute markers of heart rate variability ( 24-h triangular index : = 0.408 , p = 0.073 , sdnn : = 0.408 , p = 0.072 ) . ssat positively correlated with dsat ( r = 0.389 , p < 0.001 ) and taat ( r = 0.461 , p < 0.001 ) . ssat , dsat , and taat correlated positively and significantly with waist circumference ( ssat : r = 0.313 , p = 0.009 , dsat : r = 0.276 , p = 0.023 , taat : r = 0.449 , p < 0.001 ) and bmi ( ssat : r = 0.490 , p < 0.001 , dsat : r = 0.327 , p = 0.005 , taat r = 0.508 , p < 0.001 ) . distribution of fat depots , biomarkers , and clinical parameters across tertiles of absolute abdominal ssat among patients with type 2 diabetes fpg ( p of trend = 0.046 , p = 0.073 between extreme tertiles ) and hba1c ( p of trend = 0.006 , p = 0.011 between extreme tertiles ) were both lower with increased tertile of ssat . ssat was inversely correlated with hba1c ( r = 0.262 , p = 0.027 ) , whereas vat ( r = 0.240 , p = 0.042 ) and taat positively correlated with fasting triglycerides ( r = 0.278 , p = 0.018 ) . 2a ) , adjusted for age and waist circumference , increased absolute ssat was associated with decreased hba1c ( = 0.289 , p = 0.017 ) and tended to be associated with decreased fasting glucose ( = 0.208 , 0.084 ) . total energy intake , total dietary fat , saturated fat , and unsaturated fat content were not associated with fat tissue mass ( data not shown ) . however , increased total dietary intake of trans fatty acids tended to be positively correlated with total sat ( r = 0.228 , p = 0.054 ) and dsat ( r = 0.200 , p = 0.093 ) . the percentage of ssat from taat ( % ssat ) was associated with lower hba1c in regression models adjusted for age and waist circumference ( = 0.304 , p = 0.017 ) , consistent with the finding using the absolute ssat area mentioned above . conversely , increased dsat was associated with increased levels of hba1c ( = 0.266 , p = 0.039 ) and fasting glucose ( = 0.246 , p = 0.054 ) . increased % ssat was associated with increased hdl cholesterol ( = 0.251 , p = 0.047 ) ( fig . although men and women had similar waist circumferences ( 104 vs. 103 cm , p = 0.902 ) , taat ( 37,095 vs. 37,108 mm , p = 0.975 ) , and medical treatment , women had higher % ssat than did men and twice the ratio of ssat to vat ( table 2 ) . they had lower fasting glucose ( p < 0.001 ) and lower hba1c ( p = 0.002 ) , and tended to have higher hdl cholesterol ( p = 0.076 ) . in addition , in the men - only subgroup , % dsat was significantly associated with higher glucose ( = 0.292 , p = 0.040 ) and hba1c ( = 0.298 , p = 0.038 ) . abdominal fat tissue distribution , biomarkers , and clinical parameters in men and women with type 2 diabetes we further found associations among the absolute abdominal subdepot fat ( mass ) , proportional abdominal subdepot fat area , blood pressure , and heart rate variability parameters : both absolute ( r = 0.381 , p = 0.050 ) and proportional dsat ( r = 0.428 , p = 0.026 ) negatively correlated with sdnn . in regression models , adjusted for age and waist circumference , higher absolute dsat ( = 0.425 , p = 0.034 ) and higher proportional dsat ( = 0.431 p = 0.029 ) proportional dsat was significantly associated with decreased 24-h triangular index ( = 0.417 , p = 0.030 ) , suggesting decreased heart rate variability with increased fat distribution to the dsat depot . however , further adjustment to oral hypoglycemic medications , but not to insulin therapy , antihypertensive , lipid - lowering , or antiplatelet drugs , attenuated the association between dsat and markers of heart rate variability ( 24-h triangular index : = 0.249 , p = 0.228 , sdnn : = 0.294 , p = 0.186 ) . when adjusted for hba1c , higher absolute ( = 0.355 , p = 0.093 ) and proportional ( = 0.401 p = 0.073 ) dsat were associated with lower sdnn . when adjusted for glucose , higher absolute ( = 0.278 p = 0.144 ) and proportional ( = 0.322 p = 0.122 ) dsat were not associated with lower sdnn . in contrast , opposite trends were observed with absolute and proportional abdominal ssat : higher absolute ( = 0.329 , p = 0.043 ) and proportional ssat ( = 0.425 , p = 0.008 ) were associated with lower mean daytime diastolic blood pressure . when hba1c was added to the model , the association between absolute ssat and mean daytime diastolic blood pressure was attenuated ( = 0.284 , p = 0.072 ) , although the association between proportional ssat and mean daytime diastolic blood pressure remained significant ( = 0.379 , p = 0.008 ) . when adjusted for glucose , the association between both absolute ( = 0.329 , p = 0.043 ) and proportional ( = 0.444 , p = 0.006 ) ssat and mean daytime diastolic blood pressure remained significant . when the models were stratified by sex , we found that the men - only subgroup also exhibited similar associations among abdominal subdepot fat ( mass and proportion ) , blood pressure , and heart rate variability parameters . higher absolute dsat ( = 0.452 , p = 0.014 ) and proportional dsat ( = 0.535 p = 0.009 ) were associated with lower sdnn . higher absolute dsat ( = 0.486 , p = 0.007 ) and proportional dsat ( = 0.557 , p = 0.005 ) were associated with decreased 24-h triangular index . further adjustment to oral hypoglycemic medications , but not to insulin therapy , antihypertensive , lipid - lowering , or antiplatelet drugs , attenuated the association between proportional dsat and markers of heart rate variability ( 24-h triangular index : = 0.439 , p = 0.075 , sdnn : = 0.443 , p = 0.074 ) and between absolute markers of heart rate variability ( 24-h triangular index : = 0.408 , p = 0.073 , sdnn : = 0.408 , p = 0.072 ) . in this study of patients with type 2 diabetes , we observed a distinct association between both the absolute ( representing sub - depot adiposity ) and the relative amounts ( representing interdepot distribution ) of abdominal ssat and markers of more favorable glycemic control and cardiovascular function as determined by higher heart rate variability and lower blood pressure . after adjusting for age and waist circumference , higher relative distribution of abdominal fat in ssat was correlated with improved glycemic control ( hba1c and fasting glucose ) and better indicators of cardiovascular health ( lower blood pressure and higher heart rate variability ) . in terms of diet , increased intake of trans fat tended to be associated with total abdominal sat and dsat , but not with ssat . our results suggest a favorable distinct association between abdominal ssat subdepot and cardiometabolic health in type 2 diabetes . this is a cross - sectional design that may suggest associations but not clear cause effect protective relationships between abdominal ssat and cardiometabolic parameters . the strengths of our study include the specific group with type 2 diabetes , the high mri quality imaging , the comprehensive 24-h ambulatory blood pressure and 24-h ambulatory ecg measurements as cardiometabolic measurements , the medication follow - up , and the dietary assessment . in our study , increased trans fatty acids consumption tended to be associated with increased total abdominal sat and dsat , but not ssat .

studies in the caribbean , and jamaica in particular , on male issues have concentrated on marginalisation[14 ] , educational underachievement , fatherhood , masculinity , reproductive health and survivability[912 ] , and crime and violence[1316 ] . in the english - speaking caribbean , even among the new and emerging themes , studies have avoided men 's health status , men 's health conditions , and issues affecting men 's health except statistics on mortality and morbidity . in a recently published text titled health issues in the caribbean , the coverage of topics included child health , reproductive health , the elderly , chronic non - communicable diseases , disability , health care - delivery and health issues in the caribbean , which reinforces the claim of the lack of research on men 's health . another text entitled gender in the 21 century : caribbean perspectives , visions and possibilities had articles on new and emerging themes , redefining masculinities and femininities , but none that examined gender and health in the region , particularly men 's health in the 21 century and the challenges of ill - health of the sexes . men 's health is therefore in need of research as it remains relatively unexamined in the region . a comprehensive search of health literature in the english - speaking caribbean revealed a lack of study regarding men 's health , men 's health care - seeking behaviour , correlates of good health status of men , and the epidemiological shifts in health conditions of men . in jamaica , statistics for 2007 estimated that 49% of the population was male , yet no study exists on this cohort . this denotes neglect of men 's health although they have had a greater mortality than their female counterparts . concurrently , since 1988 when the planning institute of jamaica and the statistical institute of jamaica began collecting data on jamaicans living conditions , women have been outnumbering men in health care utilisation . life expectancy for the sexes in jamaica revealed that women continue to outlive men , and this is as much as 6 years in 2004 . it can be extrapolated from the aforementioned statistics that the lack of health care - seeking behaviour is accounting for 1 ) premature mortality of men , 2 ) men spending more time getting care when becoming ill , and 3 ) men having poorer health status than women . there is a paradox in the health statistics for the sexes as males in jamaica report fewer illnesses than females , yet they tend to live 6 fewer years than women . is it that 1 ) males are underreporting their illnesses , and 2 ) that there are issues surrounding subjective health data ? self - reported health conditions have been widely used to measure the health of people or a population[2022 ] . using self - reported illness to measure health status in jamaica , is there a validity issue with self - reported health to assess health ? there are some fundamental challenges associated with the utilisation of subjective measures in the evaluation of health and these include 1 ) subjectivity , 2 ) response bias , 3 ) recall bias , and 4 ) validity . studies have revealed that current illness is strongly correlated with health status , self - reported illness and life expectancy , self - reported health and mortality , and self - reported health and functional ability , indicating some validity in subjective measures in assessing objective health status . one study found that self - rated health status is a highly reliable measure to proxy health and that this successfully crosses cultural lines . concurrently , odonnell & tait found that self - reported health status can be used to assess health status as they found that all respondents who had chronic diseases reported very poor health . the fact that people are asked to recall and state their health status opens the issue of subjectivity and biasness . subjective indexes as a measure of any phenomenon introduce some element of biasness . if people are asked to recall and provide their assessment ( i.e. , perception ) of a matter , this subjectivity denotes not only people 's perceptions , but it also includes their biases . people 's perceptions are highly biased as they can provide an inflated or deflated account of their state in an interview or on a self - administered questionnaire . it is for this reason that empirical researchers have avoided and decried its utilisation in the measurement of health . even though subjective indexes are in keeping with the who 's widened definition of health , their biasness must be understood as challenges . it can not be denied that the discourse on subjective wellbeing , using survey data , is based on person 's judgement , and therefore must be prone to systematic and non - systematic biases . diener argued that the subjective measure seemed to contain substantial amounts of valid variance , suggesting that this indicated the validity of subjective indexes . kahneman devised a procedure of integrating and reducing the subjective biases when he found that instantaneous subjective evaluations are more reliable than assessments of recall of experiences . this highlights the biasness , therefore , that remains in cross - sectional surveys asking people to remember over a long time . embedded in the aforementioned findings are whether particular subjective indexes that comprised recall over 2 - 4 weeks is a good measure for objective indexes of health . embodied in the literature is the need to carry out empirical research on subjective and objective indexes with emphasis on subjective indexes that are not on instantaneous assessment . the literature has provided some empiricism to the use of self - reported health conditions or self - reported health in assessing health status of people . within the context that subjective indexes are good in measuring health status , this approach will be used to examine men 's health in jamaica . the objectives of the current study are to 1 ) evaluate the changing epidemiological patterns of diseases affecting men in jamaica ; 2 ) ascertain factors that correlate with good health status of men ; 3 ) compare and contrast the differences in health status of men , in particular marital status ; and 4 ) determine which marital status group has the greatest health status . population this study utilised secondary cross - sectional datasets for 2002 and 2007 taken from the jamaica survey of living conditions ( jslc ) . the jslc is a joint publication from the planning institute of jamaica ( pioj ) and the statistical institute of jamaica ( statin ) for analysis[3234 ] . the jslc began in 1988 to collect data on the living conditions of jamaicans in order to measure government policies . these cross - sectional surveys were conducted between may and october of each year across the 14 parishes of jamaica . the current study extracted 8,078 respondents who were 15 years and older from the 2002 sample ( n=25,018 respondents ) and 2,224 from the 2007 sample of 6,783 respondents . the jslc used stratified random probability sampling technique to draw the original sample of respondents . the non - response rates for 2002 and 2007 lie between 23 and 27% respectively . the jslc survey uses a complex design with multiple stratifications to ensure that it represents the population , marital status , area of residence , and social class . instrument the jslc used an administered questionnaire where respondents are asked to recall detailed information on particular activities . the questionnaire was modelled from the world bank 's living standards measurement study ( lsms ) household survey . there are some modifications to the lsms , as the jslc is more focused on policy impacts . the questionnaire covers demographic variables , health , immunisation of children 059 months , education , daily expenses , non - food consumption expenditure , housing conditions , inventory of durable goods and social assistance . statistical methods descriptive statistics were used to analyse the socio - demographic characteristics of the samples . chi - square analyses were used to examine the association between non - metric variables for area of residence , and gender of respondents . t - test statistic and analysis of variance were used to evaluate metric and either a dichotomous or non - dichotomous variable respectively . logistic regression analyses examined the 1 ) association between good health status and some socio - demographic , economic and biological variables ; as well as 2 ) a correlation between self - reported health conditions ( illnesses , dysfunctions or ailments ) and some socio - demographic , economic and biological variables . spss for windows 16.0 ( spss inc ; chicago , il , usa ) was used to store , retrieve and analyse the data . a 95% confidence interval was used for the analysis , and the final models ( i.e. , equations ) were based on a p - value less than 5% . the only selection criterion for this study was based on the respondents being 15 years and older and male . for the model , the selection criteria were based on the 1 ) literature review , 2 ) low correlations , and 3 ) non - response rate . the correlation matrix was examined in order to ascertain if autocorrelation and/or multicollinearity existed between variables . according to cohen & holliday , correlation can be low ( weak ) from 0 to 0.39 ; moderate any correlation that had at least moderate was excluded from the model in order to reduce multicollinearity and/or autocorrelation between or among the independent variables[3945 ] . another approach in addressing and/or reducing autocorrelation is to include in the model all variables that were identified from the literature review with the exception of those with which the percentage of missing cases were in excess of 30% . forward stepwise logistic regression technique was used to determine the magnitude ( or contribution ) of each statistically significant variable in comparison with the others , and the odds ratios ( or ) aided the interpretation of each significant variable . models the current study will employ multivariate analyses in the study of self - reported health status and self - reported health conditions . the use of this approach is better than bivariate analyses as many variables can be tested simultaneously for their impact ( if any ) on a dependent variable[21263845 ] . the final model for the current study is shown below : ht = f(ai , gi , hhi , ari , inc , edi , mri , si , mct , srii , i ) where ht ( i.e. , self - rated health conditions in time t ) is a function of age of respondents ai ; sex of individual i , gi ; household head of individual i , hhi ; area of residence , ari ; logged consumption per person per household member , lnc ; education level of individual i , ed i ; marital status of person i , mr i ; social class of person i , s i ; summation of medical expenditure of individual i in time period t , mc t ; self - reported illness , sri i , and an error term ( i.e. , residual error ) . measures self - reported illness status is a dummy variable , where 1 = reporting an ailment or dysfunction or illness in the last 4 weeks , which was the survey period ; 0 if there were no self - reported ailments , injuries or illnesses . while self - reported ill - health is not an ideal indicator of actual health conditions because people may underreport , it is still an accurate proxy of ill - health and mortality . health status is a binary measure where 1 = good to excellent health , 0 = otherwise[242646 ] , which is determined from generally , how do you feel about your health ? answers for this question are in a likert scale matter ranging from excellent to poor . medical care - seeking behaviour was taken from the question has a health care practitioner , header , or pharmacist being visited in the last 4 weeks ? medical care - seeking behaviour therefore was coded as a binary measure where 1 = yes and 0 = otherwise . table 1 examines social and health variables of samples for 2002 and 2007 . for 2002 , a sample of 8,078 respondents was extracted and this was 2,224 respondents for 2007 . over 2002 , the number of respondents who reported an illness in 2007 increased by 1.4% . in 2007 over 2002 , increases were recorded in number of respondents being diagnosed with cold ( + 8.4% ) , asthma ( + 5.7 ) , diabetes mellitus ( + 7.5% ) , and unspecified health conditions ( + 7.4% ) . conversely , reductions were seen in hypertensive ( -29.4% ) and arthritic cases ( -12.0% ) . demographic characteristics of sample , 2002 and 2007 there was a strong significant statistical correlation between self - reported health status and self - reported health conditions ( df=4 ) = 531.7 , p < 0.001 , correlation coefficient = 0.446 . the cross - tabulation revealed that respondents who had indicated that they had an illness ( dysfunction or health condition ) were more likely to report moderate to poor health status . nine percent of those with health conditions recorded very good health status compared to 41% of those who did not report illness who indicated good health status . a significant statistical association was found between self - reported illness and marital status of respondents for both years the findings showed that the widowed recorded the greatest percentage of illness of all the marital statuses . conversely , those who were never married recorded the lowest illnesses . in 2002 , married respondents recorded 2.2 times more illnesses than men who were never married , and this increased to 2.7 times more illnesses in 2007 . however , this was the reverse in 2007 , with the latter group registering 4.3% more illnesses . a significant statistical correlation existed between medical care - seeking behaviour and marital status of respondents in 2002 . conversely , there was no statistical association between the two aforementioned variables for 2007 self - reported diagnosed health conditions by marital status self - reported health conditions by marital status table 4 represents information on social and biological determinants of good health status of the sample . the model is a good fit for the data : hosmer and lemeshow = 3.88 , p = 0.87 : 76.8% of the data were correctly classified , with 56.1% of those classified in good self - reported health and 88.5% in poor health status . stepwise logistic regression : correlates of self - reported good health status ( n=1952 ) table 5 highlights information on social determinants of self - reported health conditions . seven social determinants accounted for 7.6% of the variability in self - reported health conditions . the model is a good fit for the data : hosmer and lemeshow = 5.6 , p = 0.69 : 83.4% of the data were correctly classified , with 99.7% of those classified in health and 76.5% in poor health status . table 1 examines social and health variables of samples for 2002 and 2007 . for 2002 , a sample of 8,078 respondents was extracted and this was 2,224 respondents for 2007 . over 2002 , the number of respondents who reported an illness in 2007 increased by 1.4% . in 2007 over 2002 , increases were recorded in number of respondents being diagnosed with cold ( + 8.4% ) , asthma ( + 5.7 ) , diabetes mellitus ( + 7.5% ) , and unspecified health conditions ( + 7.4% ) . conversely , reductions were seen in hypertensive ( -29.4% ) and arthritic cases ( -12.0% ) . demographic characteristics of sample , 2002 and 2007 there was a strong significant statistical correlation between self - reported health status and self - reported health conditions ( df=4 ) = 531.7 , p < 0.001 , correlation coefficient = 0.446 . the cross - tabulation revealed that respondents who had indicated that they had an illness ( dysfunction or health condition ) were more likely to report moderate to poor health status . nine percent of those with health conditions recorded very good health status compared to 41% of those who did not report illness who indicated good health status . a significant statistical association was found between self - reported illness and marital status of respondents for both years the findings showed that the widowed recorded the greatest percentage of illness of all the marital statuses . conversely , those who were never married recorded the lowest illnesses . in 2002 , married respondents recorded 2.2 times more illnesses than men who were never married , and this increased to 2.7 times more illnesses in 2007 . however , this was the reverse in 2007 , with the latter group registering 4.3% more illnesses . a significant statistical correlation existed between medical care - seeking behaviour and marital status of respondents in 2002 . conversely , there was no statistical association between the two aforementioned variables for 2007 self - reported diagnosed health conditions by marital status self - reported health conditions by marital status table 4 represents information on social and biological determinants of good health status of the sample . the model is a good fit for the data : hosmer and lemeshow = 3.88 , p = 0.87 : 76.8% of the data were correctly classified , with 56.1% of those classified in good self - reported health and 88.5% in poor health status . stepwise logistic regression : correlates of self - reported good health status ( n=1952 ) table 5 highlights information on social determinants of self - reported health conditions . seven social determinants accounted for 7.6% of the variability in self - reported health conditions . the model is a good fit for the data : hosmer and lemeshow = 5.6 , p = 0.69 : 83.4% of the data were correctly classified , with 99.7% of those classified in health and 76.5% in poor health status . table 1 examines social and health variables of samples for 2002 and 2007 . for 2002 , a sample of 8,078 respondents was extracted and this was 2,224 respondents for 2007 . over 2002 , the number of respondents who reported an illness in 2007 increased by 1.4% . in 2007 over 2002 , increases were recorded in number of respondents being diagnosed with cold ( + 8.4% ) , asthma ( + 5.7 ) , diabetes mellitus ( + 7.5% ) , and unspecified health conditions ( + 7.4% ) . conversely , reductions were seen in hypertensive ( -29.4% ) and arthritic cases ( -12.0% ) . demographic characteristics of sample , 2002 and 2007 there was a strong significant statistical correlation between self - reported health status and self - reported health conditions ( df=4 ) = 531.7 , p < 0.001 , correlation coefficient = 0.446 . the cross - tabulation revealed that respondents who had indicated that they had an illness ( dysfunction or health condition ) were more likely to report moderate to poor health status . nine percent of those with health conditions recorded very good health status compared to 41% of those who did not report illness who indicated good health status . a significant statistical association was found between self - reported illness and marital status of respondents for both years the findings showed that the widowed recorded the greatest percentage of illness of all the marital statuses . conversely , those who were never married recorded the lowest illnesses . in 2002 , married respondents recorded 2.2 times more illnesses than men who were never married , and this increased to 2.7 times more illnesses in 2007 . however , this was the reverse in 2007 , with the latter group registering 4.3% more illnesses . a significant statistical correlation existed between medical care - seeking behaviour and marital status of respondents in 2002 . conversely , there was no statistical association between the two aforementioned variables for 2007 self - reported diagnosed health conditions by marital status self - reported health conditions by marital status table 4 represents information on social and biological determinants of good health status of the sample . the model is a good fit for the data : hosmer and lemeshow = 3.88 , p = 0.87 : 76.8% of the data were correctly classified , with 56.1% of those classified in good self - reported health and 88.5% in poor health status . stepwise logistic regression : correlates of self - reported good health status ( n=1952 ) table 5 highlights information on social determinants of self - reported health conditions . seven social determinants accounted for 7.6% of the variability in self - reported health conditions . the model is a good fit for the data : hosmer and lemeshow = 5.6 , p = 0.69 : 83.4% of the data were correctly classified , with 99.7% of those classified in health and 76.5% in poor health status . there are many empirical studies which have established that married people have a better self - reported health status ( or self - reported health , subjective wellbeing ) and/or a lower mortality than non - married people[4755 ] . the current study disagrees with the literature as it revealed that there was no significant statistical difference between the self - reported good health status of married and unmarried men . however , married men have a greater probability of reporting an illness than those who were never married , with those who were separated , divorced and widowed having a greater probability of reporting illness compared to married and never married men . concurrently , this study went further than the other research that examined health status of married and unmarried men by examining the shift in self - reported illnesses . it was revealed that hypertension continues to be the leading cause of illness among men except for men who were never married and divorced men . another critical finding which emerged from this study is the dissipation of statistical difference among the different marital statuses and medical care - seeking behaviour of men . even when there were significant differences among the marital statuses , separated men utilised medical care facilities more than married men , and unmarried men visited the least . half a decade later ( 2007 ) , there is equality among men of different marital statuses and medical care - seeking behaviour . education is one of the socio - demographic correlates of health , and this is well established in health literature . there is a paradox which emerged from this study as educational attainment is not significantly associated with good health status ; however , it is correlated with health conditions . the current research found that as men become more educated , they are less likely to report health conditions . embedded in this finding good health status , as typologies of reported dysfunctions are lifestyle causing , suggesting that the while education is aiding in reducing poor health status , it is not translating into better health for men as they still not adhering to healthy lifestyle practices . grossman , smith & kington , marmot , and other scholars have found that income is strongly correlated with good health . the current study found that income is not correlated with good health status , but rather with health conditions . concurrently , medical care - seeking behaviour is based on 1 ) one 's ability to afford it , 2 ) perception of the illness and its effect on life , 3 ) severity of illness , and 4 ) other factors . although income does not directly influence good health status for men , it indirectly affects it through medical care - seeking behaviour . men who seek medical care are 0.60 times less likely to report health conditions , suggesting that medical care - seeking behaviour is a preventative measure against poor health . income affords people the ability to visit medical care facilities , and it should be noted here that jamaican men are more likely to visit private health care centres . income , therefore , can not buy good health as smith & kington opined , as the group with the wealthy men in jamaica were 1.32 times more likely to report more health conditions in reference to those in the two poorest quintiles . embedded in this finding is erosion of good health by having income beyond a certain amount , as wealthy men in jamaica are involved in unhealthy lifestyle practices which accounted for their health status being lower than that of poor men . while income is able to afford particular things , including better medical care , the wealthy involvement in unhealthy lifestyle behaviour is reducing the gains of income on health for men . the literature has shown that area of residence is correlated to health status , and this concurs with that finding . however , area of residence was not significantly associated with good health but rather with health status . unlike other studies that found urban dwellers had the greatest health status , the current research found no significant statistical difference between self - reported health conditions experienced by urban and rural men . on the other hand , men who resided in other town areas were less likely to report illness than rural men . the number of people living in a household is well established as being associated with health and this study concurs with that finding . the current work however found that crowding is associated with health conditions but not related to good health status . the more people dwelling in a household denotes that men will be 0.15 times less likely to report ailments , suggesting that family and relatives positively contribute to men 's health . the contribution comes in the form of assistance in seeking medical care , financial assistance to seeking care , advice on seeking care , and provision of care . separated , divorced and widowed men are the most likely to record illness than men of other marital status . the current findings also revealed that separated men were more likely to record having had hypertension , diarrhoea and arthritis compared to other marital statuses . this suggests that separation increases health conditions in men , and higher rates in the aforementioned chronic illnesses indicates the poor lifestyle choices made by this group of individuals . a study by ben - shlomo et al . was greatest among divorced / separated men and that alcoholic beverages accounted for between 21 to 30% of mortality resulting from respiratory and non - cardiovascular / neo - plastic diseases . concurrently , current smokers were highest among separated and divorced men , and mortality caused by lung cancer was 1.83 times more for the former than for married men which was the highest among the different marital statuses . 's work provides some explanation for the highest levels of self - reported illness , in particular chronic dysfunctions , affecting separated men in jamaica , as separation results in a change towards unhealthy lifestyle practices among these men . it can be extrapolated from the current findings that separation of men from their spouses results in unhealthy practices . the unhealthy choices that are made by these men increase their risk of coronary heart disease , stroke and diabetes mellitus and this reinforces the disease risk for separated , divorced , and widowed men in jamaica as they become indulgent ( or increase indulgence ) in unhealthy lifestyle practices . unhealthy life choices of divorced , widowed or separated men including tobacco use , physical inactivity , alcohol consumption , and unhealthy dietary intake further erode life expectancy of these individuals . these lifestyle practices may begin in early adulthood and with the new marital status begin to be practiced increasingly by some men . hence , separated and divorced men who remain in these categorisations for a long time are more likely to experience premature death caused by suicide or other issues such as lung cancer , resulting from their increased unhealthy lifestyle choices . they are also more likely to relapse into depression and commit suicide divorced and separated men are 2.4 times more likely to commit suicide than married men . a recently conducted research by abel et al . found that the suicide rates among men was 9 times more than among women and while they do not disaggregate this by marital status , a part of this is owing to the choices they make and their inability to live with these choices . an important question that needs to be addressed is whether there are differences in suicide rates among marital statuses in jamaica , as this will provide some answers to the cost of separation and divorce on men . those unhealthy choices are problematic not only for men but their families , including their children and particularly the young children[6669 ] . this disruption in the family is correlated with increased suicide , greater risk of illnesses such as asthma , headaches , delinquency and poverty for the children who are now a part of the separation process . separation and divorce not only affects young children . a study on children 18 to 22 year s old whose parents separated or divorced were more likely to have poor relationships with their parents as well as reported psychosocial behaviour problems . separation and divorce extends to the wider society as the general society will be required to pay the medical costs for care of those who visit public health care facilities as well as the uninsured , including elderly separated and divorced men . in sum , the current study revealed that married men do not have greater health status than unmarried men in jamaica , and that there are epidemiological shifts in illness from hypertension to unspecified conditions for widowed men and those who were never married . while income correlates with health conditions , it does not directly influence good health status of men . income has an indirect correlation with good health status through its relationship with illness and medical care - seeking behaviour . marriage is beneficial for men , and once they become separated , divorced or widowed , the separation from their spouses positively correlates with increased health conditions . this study therefore provides a platform upon which future studies can commence as we begin to examine men 's health in jamaica , and this will be critical to public health practitioners in effectively carrying out their mandate as well as for future research .

background : since 1988 , when jamaica began collecting data on the living conditions of its people , men have reported seeking less health care than women . despite this fact , the group has never been studied by researchers . the same is true about the health status of married and non - married men.objectives:the current study will 1 ) evaluate the changing epidemiological patterns of diseases affecting men in jamaica ; 2 ) determine factors that correlate with good health status of men ; 3 ) compare and contrast the differences in health status of men , in particular marital status ; and 4 ) determine which marital status has the greater health status.materials and methods : the data for this research were taken from two secondary cross - sectional surveys . a sample of 8,078 respondents 15 years and older was extracted from the 2002 survey ( n=25,018 respondents ) and 2,224 respondents from the 2007 sample ( n=6,783 respondents ) . spss for windows 16.0 was used to store , retrieve and analyse the data . chi - square , analysis of variance , t - test and logistic regression were used in this paper.results:married men are more likely to report an illness than never married ( or = 1.68 , 95% ci = 1.45 - 1.95 ) , separated , divorced or widowed men ( or = 2.62 , 95% ci = 2.06 - 3.33 ) . no significant statistical difference existed between the self - rated health status of married and unmarried men.conclusion:this study provides a platform upon which future studies can commence as we begin to examine men 's health in jamaica .

Introduction Materials and Methods Results None Demographic characteristics Multivariate analyses Discussion Conclusion

in the english - speaking caribbean , even among the new and emerging themes , studies have avoided men 's health status , men 's health conditions , and issues affecting men 's health except statistics on mortality and morbidity . in a recently published text titled health issues in the caribbean , the coverage of topics included child health , reproductive health , the elderly , chronic non - communicable diseases , disability , health care - delivery and health issues in the caribbean , which reinforces the claim of the lack of research on men 's health . another text entitled gender in the 21 century : caribbean perspectives , visions and possibilities had articles on new and emerging themes , redefining masculinities and femininities , but none that examined gender and health in the region , particularly men 's health in the 21 century and the challenges of ill - health of the sexes . men 's health is therefore in need of research as it remains relatively unexamined in the region . a comprehensive search of health literature in the english - speaking caribbean revealed a lack of study regarding men 's health , men 's health care - seeking behaviour , correlates of good health status of men , and the epidemiological shifts in health conditions of men . this denotes neglect of men 's health although they have had a greater mortality than their female counterparts . concurrently , since 1988 when the planning institute of jamaica and the statistical institute of jamaica began collecting data on jamaicans living conditions , women have been outnumbering men in health care utilisation . life expectancy for the sexes in jamaica revealed that women continue to outlive men , and this is as much as 6 years in 2004 . it can be extrapolated from the aforementioned statistics that the lack of health care - seeking behaviour is accounting for 1 ) premature mortality of men , 2 ) men spending more time getting care when becoming ill , and 3 ) men having poorer health status than women . there is a paradox in the health statistics for the sexes as males in jamaica report fewer illnesses than females , yet they tend to live 6 fewer years than women . is it that 1 ) males are underreporting their illnesses , and 2 ) that there are issues surrounding subjective health data ? self - reported health conditions have been widely used to measure the health of people or a population[2022 ] . using self - reported illness to measure health status in jamaica , is there a validity issue with self - reported health to assess health ? there are some fundamental challenges associated with the utilisation of subjective measures in the evaluation of health and these include 1 ) subjectivity , 2 ) response bias , 3 ) recall bias , and 4 ) validity . studies have revealed that current illness is strongly correlated with health status , self - reported illness and life expectancy , self - reported health and mortality , and self - reported health and functional ability , indicating some validity in subjective measures in assessing objective health status . one study found that self - rated health status is a highly reliable measure to proxy health and that this successfully crosses cultural lines . concurrently , odonnell & tait found that self - reported health status can be used to assess health status as they found that all respondents who had chronic diseases reported very poor health . it can not be denied that the discourse on subjective wellbeing , using survey data , is based on person 's judgement , and therefore must be prone to systematic and non - systematic biases . this highlights the biasness , therefore , that remains in cross - sectional surveys asking people to remember over a long time . the literature has provided some empiricism to the use of self - reported health conditions or self - reported health in assessing health status of people . within the context that subjective indexes are good in measuring health status , this approach will be used to examine men 's health in jamaica . the objectives of the current study are to 1 ) evaluate the changing epidemiological patterns of diseases affecting men in jamaica ; 2 ) ascertain factors that correlate with good health status of men ; 3 ) compare and contrast the differences in health status of men , in particular marital status ; and 4 ) determine which marital status group has the greatest health status . population this study utilised secondary cross - sectional datasets for 2002 and 2007 taken from the jamaica survey of living conditions ( jslc ) . the jslc is a joint publication from the planning institute of jamaica ( pioj ) and the statistical institute of jamaica ( statin ) for analysis[3234 ] . the jslc began in 1988 to collect data on the living conditions of jamaicans in order to measure government policies . these cross - sectional surveys were conducted between may and october of each year across the 14 parishes of jamaica . the current study extracted 8,078 respondents who were 15 years and older from the 2002 sample ( n=25,018 respondents ) and 2,224 from the 2007 sample of 6,783 respondents . statistical methods descriptive statistics were used to analyse the socio - demographic characteristics of the samples . chi - square analyses were used to examine the association between non - metric variables for area of residence , and gender of respondents . t - test statistic and analysis of variance were used to evaluate metric and either a dichotomous or non - dichotomous variable respectively . logistic regression analyses examined the 1 ) association between good health status and some socio - demographic , economic and biological variables ; as well as 2 ) a correlation between self - reported health conditions ( illnesses , dysfunctions or ailments ) and some socio - demographic , economic and biological variables . spss for windows 16.0 ( spss inc ; chicago , il , usa ) was used to store , retrieve and analyse the data . the only selection criterion for this study was based on the respondents being 15 years and older and male . for the model , the selection criteria were based on the 1 ) literature review , 2 ) low correlations , and 3 ) non - response rate . forward stepwise logistic regression technique was used to determine the magnitude ( or contribution ) of each statistically significant variable in comparison with the others , and the odds ratios ( or ) aided the interpretation of each significant variable . models the current study will employ multivariate analyses in the study of self - reported health status and self - reported health conditions . , self - rated health conditions in time t ) is a function of age of respondents ai ; sex of individual i , gi ; household head of individual i , hhi ; area of residence , ari ; logged consumption per person per household member , lnc ; education level of individual i , ed i ; marital status of person i , mr i ; social class of person i , s i ; summation of medical expenditure of individual i in time period t , mc t ; self - reported illness , sri i , and an error term ( i.e. medical care - seeking behaviour was taken from the question has a health care practitioner , header , or pharmacist being visited in the last 4 weeks ? for 2002 , a sample of 8,078 respondents was extracted and this was 2,224 respondents for 2007 . over 2002 , the number of respondents who reported an illness in 2007 increased by 1.4% . demographic characteristics of sample , 2002 and 2007 there was a strong significant statistical correlation between self - reported health status and self - reported health conditions ( df=4 ) = 531.7 , p < 0.001 , correlation coefficient = 0.446 . the cross - tabulation revealed that respondents who had indicated that they had an illness ( dysfunction or health condition ) were more likely to report moderate to poor health status . nine percent of those with health conditions recorded very good health status compared to 41% of those who did not report illness who indicated good health status . a significant statistical association was found between self - reported illness and marital status of respondents for both years the findings showed that the widowed recorded the greatest percentage of illness of all the marital statuses . a significant statistical correlation existed between medical care - seeking behaviour and marital status of respondents in 2002 . conversely , there was no statistical association between the two aforementioned variables for 2007 self - reported diagnosed health conditions by marital status self - reported health conditions by marital status table 4 represents information on social and biological determinants of good health status of the sample . the model is a good fit for the data : hosmer and lemeshow = 3.88 , p = 0.87 : 76.8% of the data were correctly classified , with 56.1% of those classified in good self - reported health and 88.5% in poor health status . stepwise logistic regression : correlates of self - reported good health status ( n=1952 ) table 5 highlights information on social determinants of self - reported health conditions . the model is a good fit for the data : hosmer and lemeshow = 5.6 , p = 0.69 : 83.4% of the data were correctly classified , with 99.7% of those classified in health and 76.5% in poor health status . for 2002 , a sample of 8,078 respondents was extracted and this was 2,224 respondents for 2007 . over 2002 , the number of respondents who reported an illness in 2007 increased by 1.4% . conversely , reductions were seen in hypertensive ( -29.4% ) and arthritic cases ( -12.0% ) . demographic characteristics of sample , 2002 and 2007 there was a strong significant statistical correlation between self - reported health status and self - reported health conditions ( df=4 ) = 531.7 , p < 0.001 , correlation coefficient = 0.446 . the cross - tabulation revealed that respondents who had indicated that they had an illness ( dysfunction or health condition ) were more likely to report moderate to poor health status . nine percent of those with health conditions recorded very good health status compared to 41% of those who did not report illness who indicated good health status . a significant statistical association was found between self - reported illness and marital status of respondents for both years the findings showed that the widowed recorded the greatest percentage of illness of all the marital statuses . a significant statistical correlation existed between medical care - seeking behaviour and marital status of respondents in 2002 . conversely , there was no statistical association between the two aforementioned variables for 2007 self - reported diagnosed health conditions by marital status self - reported health conditions by marital status table 4 represents information on social and biological determinants of good health status of the sample . the model is a good fit for the data : hosmer and lemeshow = 3.88 , p = 0.87 : 76.8% of the data were correctly classified , with 56.1% of those classified in good self - reported health and 88.5% in poor health status . stepwise logistic regression : correlates of self - reported good health status ( n=1952 ) table 5 highlights information on social determinants of self - reported health conditions . the model is a good fit for the data : hosmer and lemeshow = 5.6 , p = 0.69 : 83.4% of the data were correctly classified , with 99.7% of those classified in health and 76.5% in poor health status . for 2002 , a sample of 8,078 respondents was extracted and this was 2,224 respondents for 2007 . over 2002 , the number of respondents who reported an illness in 2007 increased by 1.4% . demographic characteristics of sample , 2002 and 2007 there was a strong significant statistical correlation between self - reported health status and self - reported health conditions ( df=4 ) = 531.7 , p < 0.001 , correlation coefficient = 0.446 . the cross - tabulation revealed that respondents who had indicated that they had an illness ( dysfunction or health condition ) were more likely to report moderate to poor health status . nine percent of those with health conditions recorded very good health status compared to 41% of those who did not report illness who indicated good health status . a significant statistical association was found between self - reported illness and marital status of respondents for both years the findings showed that the widowed recorded the greatest percentage of illness of all the marital statuses . a significant statistical correlation existed between medical care - seeking behaviour and marital status of respondents in 2002 . conversely , there was no statistical association between the two aforementioned variables for 2007 self - reported diagnosed health conditions by marital status self - reported health conditions by marital status table 4 represents information on social and biological determinants of good health status of the sample . the model is a good fit for the data : hosmer and lemeshow = 3.88 , p = 0.87 : 76.8% of the data were correctly classified , with 56.1% of those classified in good self - reported health and 88.5% in poor health status . stepwise logistic regression : correlates of self - reported good health status ( n=1952 ) table 5 highlights information on social determinants of self - reported health conditions . the model is a good fit for the data : hosmer and lemeshow = 5.6 , p = 0.69 : 83.4% of the data were correctly classified , with 99.7% of those classified in health and 76.5% in poor health status . there are many empirical studies which have established that married people have a better self - reported health status ( or self - reported health , subjective wellbeing ) and/or a lower mortality than non - married people[4755 ] . the current study disagrees with the literature as it revealed that there was no significant statistical difference between the self - reported good health status of married and unmarried men . however , married men have a greater probability of reporting an illness than those who were never married , with those who were separated , divorced and widowed having a greater probability of reporting illness compared to married and never married men . concurrently , this study went further than the other research that examined health status of married and unmarried men by examining the shift in self - reported illnesses . it was revealed that hypertension continues to be the leading cause of illness among men except for men who were never married and divorced men . another critical finding which emerged from this study is the dissipation of statistical difference among the different marital statuses and medical care - seeking behaviour of men . even when there were significant differences among the marital statuses , separated men utilised medical care facilities more than married men , and unmarried men visited the least . there is a paradox which emerged from this study as educational attainment is not significantly associated with good health status ; however , it is correlated with health conditions . the current research found that as men become more educated , they are less likely to report health conditions . embedded in this finding good health status , as typologies of reported dysfunctions are lifestyle causing , suggesting that the while education is aiding in reducing poor health status , it is not translating into better health for men as they still not adhering to healthy lifestyle practices . grossman , smith & kington , marmot , and other scholars have found that income is strongly correlated with good health . the current study found that income is not correlated with good health status , but rather with health conditions . concurrently , medical care - seeking behaviour is based on 1 ) one 's ability to afford it , 2 ) perception of the illness and its effect on life , 3 ) severity of illness , and 4 ) other factors . although income does not directly influence good health status for men , it indirectly affects it through medical care - seeking behaviour . men who seek medical care are 0.60 times less likely to report health conditions , suggesting that medical care - seeking behaviour is a preventative measure against poor health . income affords people the ability to visit medical care facilities , and it should be noted here that jamaican men are more likely to visit private health care centres . income , therefore , can not buy good health as smith & kington opined , as the group with the wealthy men in jamaica were 1.32 times more likely to report more health conditions in reference to those in the two poorest quintiles . embedded in this finding is erosion of good health by having income beyond a certain amount , as wealthy men in jamaica are involved in unhealthy lifestyle practices which accounted for their health status being lower than that of poor men . however , area of residence was not significantly associated with good health but rather with health status . unlike other studies that found urban dwellers had the greatest health status , the current research found no significant statistical difference between self - reported health conditions experienced by urban and rural men . on the other hand , men who resided in other town areas were less likely to report illness than rural men . the current work however found that crowding is associated with health conditions but not related to good health status . the more people dwelling in a household denotes that men will be 0.15 times less likely to report ailments , suggesting that family and relatives positively contribute to men 's health . separated , divorced and widowed men are the most likely to record illness than men of other marital status . the current findings also revealed that separated men were more likely to record having had hypertension , diarrhoea and arthritis compared to other marital statuses . was greatest among divorced / separated men and that alcoholic beverages accounted for between 21 to 30% of mortality resulting from respiratory and non - cardiovascular / neo - plastic diseases . concurrently , current smokers were highest among separated and divorced men , and mortality caused by lung cancer was 1.83 times more for the former than for married men which was the highest among the different marital statuses . 's work provides some explanation for the highest levels of self - reported illness , in particular chronic dysfunctions , affecting separated men in jamaica , as separation results in a change towards unhealthy lifestyle practices among these men . it can be extrapolated from the current findings that separation of men from their spouses results in unhealthy practices . the unhealthy choices that are made by these men increase their risk of coronary heart disease , stroke and diabetes mellitus and this reinforces the disease risk for separated , divorced , and widowed men in jamaica as they become indulgent ( or increase indulgence ) in unhealthy lifestyle practices . these lifestyle practices may begin in early adulthood and with the new marital status begin to be practiced increasingly by some men . hence , separated and divorced men who remain in these categorisations for a long time are more likely to experience premature death caused by suicide or other issues such as lung cancer , resulting from their increased unhealthy lifestyle choices . they are also more likely to relapse into depression and commit suicide divorced and separated men are 2.4 times more likely to commit suicide than married men . an important question that needs to be addressed is whether there are differences in suicide rates among marital statuses in jamaica , as this will provide some answers to the cost of separation and divorce on men . a study on children 18 to 22 year s old whose parents separated or divorced were more likely to have poor relationships with their parents as well as reported psychosocial behaviour problems . in sum , the current study revealed that married men do not have greater health status than unmarried men in jamaica , and that there are epidemiological shifts in illness from hypertension to unspecified conditions for widowed men and those who were never married . while income correlates with health conditions , it does not directly influence good health status of men . income has an indirect correlation with good health status through its relationship with illness and medical care - seeking behaviour . marriage is beneficial for men , and once they become separated , divorced or widowed , the separation from their spouses positively correlates with increased health conditions . this study therefore provides a platform upon which future studies can commence as we begin to examine men 's health in jamaica , and this will be critical to public health practitioners in effectively carrying out their mandate as well as for future research .

tuberculosis ( tb ) is an infectious disease caused by mycobacterium tuberculosis or the other members of mycobacterium complex such as mycobacterium africanum and mycobacterium bovis , known to affect the humans . since the times immemorial , it has affected many civilizations around the world and has been a major reason for many death tolls during the earlier days . it remains as the number one killer infectious disease among the adult population in developing countries even today . the who s 1990 global disease burden report ranked tb in the seventh position and expected to continue the same until 2020 in terms of morbidity . , tb continues to be a devastating health crisis with more than 3 , 00,000 deaths , 2.2 million new cases with an economic loss of $ 23bn ( 14.9bn ; 20.3bn ) each year . in spite of noticeable progress achieved tb always offers newer challenges such multidrug - resistant tb ( mdr - tb ) , extensively drug - resistant tb ( xdr - tb ) along with debilitating side effects of anti - tb drugs . in india , tb in the community is managed by a centrally sponsored tb control program known as revised national tb control programme ( rntcp ) . the main target of this program is 85% cure rate and 70% case detection rate . since 2007 although india has achieved the global targets but the problem does not end there owing to a multiplicity of problems . despite being a centrally sponsored program and free treatment and diagnostic services people still visit private practitioners at their first point of contact when a symptom arises . studies report that around 50 - 80% of the patients visit private practitioners for tb treatment . currently , ayurvedic drugs are not a part of rntcp . however , private practitioners use various types of ayurvedic medicines , both general practitioners and chest physicians , to support tb management . this ancient medical science can offer some solutions to these problems hence a systematic review was carried out to assess the role of ayurveda for the management of tb . in addition , there is inevitable metabolic dysfunction ( dhatwagninasana ) , in which rasa ( tissue fluid ) , rakta ( blood ) , mamsa ( muscle ) , meda ( adipose tissue ) , and sukra ( generative tissue ) are lost . an unusual metabolic change occur leading to loss of various dhatus ( tissue ) such as ojokshaya , sukra , meda dhatus to rasa dhatu preceding each other , which is known as pratilomakshaya . in addition , there is inevitable metabolic dysfunction ( dhatwagninasana ) , in which rasa ( tissue fluid ) , rakta ( blood ) , mamsa ( muscle ) , meda ( adipose tissue ) , and sukra ( generative tissue ) are lost . an unusual metabolic change occur leading to loss of various dhatus ( tissue ) such as ojokshaya , sukra , meda dhatus to rasa dhatu preceding each other , which is known as pratilomakshaya . pubmed ( us national library of medicine , usa ) for the main search process . the key words used for the purpose of pubmed search include ayurveda , role and tb . no limits were adopted such as ; journals , years of publication , language , types of articles , or authors , but the articles published in english language only were included for the purpose of review . this was done purposefully to obtain a comprehensive list of articles published until april 2015 without the above - mentioned limits . in the second stage , the total articles obtained from searching the database using the above search criteria was pooled together , and articles were initially screened by reading the title and thereafter the abstracts . studies not satisfying the inclusion criteria were excluded at these stages . the remaining articles were screened in the final stage by reading the full - text and those not meeting inclusion criteria were excluded . the figure 1 gives an idea about the flow of information through different phases of this systematic review as per preferred reporting items for systematic review and mata analysis ( prisma ) guidelines ( http://www.prisma-statement.org/ ) . flow of information through different phases of this systematic review ( as per prisma guidelines ) in this section , the results are mentioned in two phases . in the first phase studies related to the ayurvedic management of tb as an adjunct therapy are delineated and in the next part the studies related to ayurvedic drugs showing in - vitro anti - tb properties are described . four different studies conducted in different clinical settings in india evaluated the properties of some of the ayurvedic therapeutics as adjunct to anti - tb treatment . table 1 summarizes the properties of some of the ayurvedic therapeutics as adjunct to anti - tb drugs . conducted a single blind controlled trial to evaluate the adjunct properties of rasayana compound among 133 tb patients receiving the rasayana used in this study is composed of amalaki ( emblica officinalis gaertn.)-pericarp , 1 part , guduchi ( tinospora cordifolia willd.)-stem , 1 part , ashwagandha ( withania somnifera l.)-root , 1 part , yashtimadhu ( glycyrrhiza glabra linn.)-root , 1 part , pippali ( piper longum linn.)-fruit , parts , sariva ( hemidesmus indicus r.br.)-root , parts , kustha ( saussurea lappa falc.)-root , parts , haridra ( curcuma longa linn.)-rhizome , parts and kulinjan ( alpinia galangal linn.)-rhizome , parts and administered in capsule form . they conducted the study at opd level at three different hospitals ; ( 1 ) institute of post graduate training and research in ayurveda ( ipgt and ra ) , hospital , gujarat ayurveda university , jamnagar , ( 2 ) district tb centre and hospital , jamnagar , and ( 3 ) guru govind singh hospital , p. n. marg , jamnagar . the study was carried out for 60 days , and the capsule was administered at a dose of 450 mg . only sputum smear - positive patients from category i of ptb or extra ptb and age group of > 13 years were selected for the study , rest excluded from the study . the study found that the compound is helpful in alleviating the associated symptoms of ptb in the treatment group ( tg ) compared to the control group ( cg ) . the rasayana compound was found to decrease cough ( 83% ) , fever ( 93% ) , dyspnea ( 71.3% ) , hemoptysis ( 87% ) , and increase body weight ( 7.7% ) ayurvedic management of tb as an adjunct therapy dornala and dornala conducted a study to evaluate the clinical efficacy of bhringarajasava as naimittika rasayana in ptb . the study was conducted at the out patient department ( opd ) of the state tb training and demonstration centre , s.r . nagar , hyderabad , andhra pradesh , india among 60 ptb patients who were already under directly observed treatment short course ( dots ) . each patient was administered 30 ml of bhringarajasava with an equal quantity of water , an hour after food , thrice a day during the intensive phase of treatment , and followed up to 6 - 8 months based on treatment category . bhringarajasava is a liquid formulation composed of bhringaraja ( eclipta prostrate linn . ) , as active ingredient along with haritaki ( terminalia chebula retz . ) , pippali ( piper longum linn . ) , jatiphala ( myristica fragrance houtt . ) , lavanga ( sygizium aromaticum linn . ) , twak ( cinnamomum zeylanicum ) , ela ( elatteria cardamomum ) , tamalapatra ( cinnamomum tamala ) , nagakesara ( messua ferrea ) , and gudam ( old cane jaggery ) . the investigators evaluated the role of bhringarajasava in 15 subjective parameters and 3 objective parameters . the role of this formulation on these subjective parameters is as follows ; the improvement is mentioned in both tg and in cg ; amsaparsabhitapah ( pain in costal and scapular region ) - 35% in cg and 100% in tg , samtapakarapadayoh ( burning sensation in palms and soles ) - 50% in cg and 75% in tg and complete relief in cat - iii , patients , jwara ( pyrexia ) - < 75% in cg and 100% in tg , bhaktadwesha ( anorexia)-mild in cg and complete in tg , swasa ( dyspnea ) - < 65% in cg and > 90% in tg , kasa ( cough ) - moderate relief in cg and progressive relief , later occasional - nonproductive and easy expectoration in tg after 15 days of treatment , shonita darshanam ( hemoptysis ) - the relief is not statistically significant among cg and were given another drug but encouraged results were observed in tg , swarabheda ( hoarseness of voice ) - the relief is not statistically significant among cg but encouraged results were observed in tg , anilath shula ( pain in visceral organs ) - complete relief in tg , sankochamsaparshyoh ( shoulder and scapular emaciation ) - very encouraging in tg patients over the cg , daha ( burning sensation ) - complete relief in tg , atisara ( diarrhea ) - no manifestation among tg , pittaraktasyachagama ( hematemesis ) - manifested in 2 patients and controlled immediately , sirasaha paripoonata ( heaviness in the head ) - relieved in 10 days among tg and in cg after 3 - 4 months , kantadwamsa ( tracheal shift ) - observed only in one case with fibrosed and consolidated lung . the role in objective parameters is as follows ; wight loss - in cg there was further weight loss but in tg there was no weight loss and is statistically significant , sputum conversion - sputum conversion was found among tg after intensive phase but in cg 10 patients out of 30 again put on intensive phase , skiagram ( chest x - ray [ cxr ] ) - density of the opacity in the cxr was less than on previous studies , with lessening of the cavities and resolution of fibrotic changes noted in the tg patients . conducted a study to assess the use of adjunct therapy of ayurvedic medicine with anti - tubercular drugs in the therapeutic management of ptb . it primarily aimed to evaluate the toxicity reduction and early restoration by adjunct therapy of ayurvedic drugs by increasing the bioavailability of atds . the study was conducted among 99 newly diagnosed ptb patients from both the sexes aged between 10 and 65 years . the subjects were administered 500 mg of ashwagandha -2 caps , twice daily and chyawanprash ( as per indian pharmacopeia)-10 g , thrice daily for a period of 28 days . the study reported amelioration of symptoms , improvement of body weight , normalization of erythrocyte sedimentation rate , appreciable change in iga and igm patterns , and significant increase in bioavailability of isoniazid and pyrazinamide . bioavailability of isoniazid and pyrazinamide showed 7 - 10% increased value after 28 days of treatment . sharma et al . conducted a study to assess the hepato - protective properties of ayurvedic herbs among patients receiving anti - tb treatment . the study was conducted among three groups of subjects , each with 10 subjects aged 15 - 70 years , for a period of 90 days with normal liver anatomy and physiology confirmed by ultrasonography and biochemical analysis at the time of the investigation . group-1 received capsule liv-600 , thrice a day composed of 200 mg of hydro alcoholic extract of berberis aristata , solanum nigrum , and aloe vera , group-2 received freshly prepared decoction of bhumyamalaki ( phyllanthus fraternus ) prepared from 10 g of aerial plant , and the group-3 received 600 mg of starch powder as placebo . to evaluate , the hepato - protective efficacies of the above drugs the investigators used subjective and objective parameters such as liver function test including serum bilirubin , alanine transaminase ( alt ) , aspartate aminotransferase ( ast ) , and alkaline phosphatase . no significant elevation in ast and alt were observed in tg compared to cg , whereas no significant elevation was observed for serum total bilirubin and alkaline phosphatase in both tg and cg . table 2 summarizes the in vitro anti - tb properties of some of the ayurvedic herbs . conducted a study to evaluate the in vitro anti - tb properties of the leaves of five legumes . non - polar and polar solvent extracts of leaves of these medicinal legumes were tested against m. tb h37rv , and minimum inhibitory concentrations ( mics ) were determined by the agar - based proportion assay . isoniazid was used as positive control to evaluate the anti - tb activity of the crude extracts of medicinal legumes . except i. cassioides methanol extracts of the leaves of all the five legumes completely inhibited the growth of m. tb at the concentration of 50 g / ml . ethyl acetate extract of two legume leaves , k. pinnatum and d. scandens and chloroform extract of three legume leaves of h. brunonis , c. mimosoides , and d. scandens showed anti - tb activity . however , petroleum ether extract of only i. cassioides showed the activity , similarly , the methanol extract of only h. brunonis leaves showed the activity . gupta et al . conducted a study to evaluate in vitro anti - tb activity of five medicinal plants viz . , acalypha indica , adhatoda vasica , allium cepa , allium sativum , and aloe vera . aqueous extracts of leaves of a. indica , a. vasica , bulbs of a. cepa , cloves of a. sativum , and pure gel of a. vera leaves , were tested in vitro . percentage inhibition was used to evaluate the activity in l - j ( lowenstein - jensen ) medium which was calculated by mean reduction in a number of colonies on extract containing as compared to extract free controls . extracts of all the five plants a. vasica , a. indica , a. cepa , a. vera , and a. sativum exhibited anti - tb activity in l - j medium , the proportion of inhibition of these plants extract in respect to that mentioned above is 95 , 32 , 37 , 72 , 32% , respectively , for mdr isolate dku-156 and 68 , 86 , 79 , 72 , 85% , respectively , for another mdr isolate jal-1236 , while for sensitive m. tb h37rv , inhibition was found to be 68 , 70 , 35 , 63 , and 41% , at 4% v / v concentration in l - j medium . in bact / alert also extracts of these plants showed significant inhibition against m. tb . four different studies conducted in different clinical settings in india evaluated the properties of some of the ayurvedic therapeutics as adjunct to anti - tb treatment . table 1 summarizes the properties of some of the ayurvedic therapeutics as adjunct to anti - tb drugs . conducted a single blind controlled trial to evaluate the adjunct properties of rasayana compound among 133 tb patients receiving the rasayana used in this study is composed of amalaki ( emblica officinalis gaertn.)-pericarp , 1 part , guduchi ( tinospora cordifolia willd.)-stem , 1 part , ashwagandha ( withania somnifera l.)-root , 1 part , yashtimadhu ( glycyrrhiza glabra linn.)-root , 1 part , pippali ( piper longum linn.)-fruit , parts , sariva ( hemidesmus indicus r.br.)-root , parts , kustha ( saussurea lappa falc.)-root , parts , haridra ( curcuma longa linn.)-rhizome , parts and kulinjan ( alpinia galangal linn.)-rhizome , parts and administered in capsule form . they conducted the study at opd level at three different hospitals ; ( 1 ) institute of post graduate training and research in ayurveda ( ipgt and ra ) , hospital , gujarat ayurveda university , jamnagar , ( 2 ) district tb centre and hospital , jamnagar , and ( 3 ) guru govind singh hospital , p. n. marg , jamnagar . the study was carried out for 60 days , and the capsule was administered at a dose of 450 mg . only sputum smear - positive patients from category i of ptb or extra ptb and age group of > 13 years were selected for the study , rest excluded from the study . the study found that the compound is helpful in alleviating the associated symptoms of ptb in the treatment group ( tg ) compared to the control group ( cg ) . the rasayana compound was found to decrease cough ( 83% ) , fever ( 93% ) , dyspnea ( 71.3% ) , hemoptysis ( 87% ) , and increase body weight ( 7.7% ) ayurvedic management of tb as an adjunct therapy dornala and dornala conducted a study to evaluate the clinical efficacy of bhringarajasava as naimittika rasayana in ptb . the study was conducted at the out patient department ( opd ) of the state tb training and demonstration centre , s.r . nagar , hyderabad , andhra pradesh , india among 60 ptb patients who were already under directly observed treatment short course ( dots ) . each patient was administered 30 ml of bhringarajasava with an equal quantity of water , an hour after food , thrice a day during the intensive phase of treatment , and followed up to 6 - 8 months based on treatment category . bhringarajasava is a liquid formulation composed of bhringaraja ( eclipta prostrate linn . ) , as active ingredient along with haritaki ( terminalia chebula retz . ) , pippali ( piper longum linn . ) , jatiphala ( myristica fragrance houtt . ) , lavanga ( sygizium aromaticum linn . ) , twak ( cinnamomum zeylanicum ) , ela ( elatteria cardamomum ) , tamalapatra ( cinnamomum tamala ) , nagakesara ( messua ferrea ) , and gudam ( old cane jaggery ) . the investigators evaluated the role of bhringarajasava in 15 subjective parameters and 3 objective parameters . the role of this formulation on these subjective parameters is as follows ; the improvement is mentioned in both tg and in cg ; amsaparsabhitapah ( pain in costal and scapular region ) - 35% in cg and 100% in tg , samtapakarapadayoh ( burning sensation in palms and soles ) - 50% in cg and 75% in tg and complete relief in cat - iii , patients , jwara ( pyrexia ) - < 75% in cg and 100% in tg , bhaktadwesha ( anorexia)-mild in cg and complete in tg , swasa ( dyspnea ) - < 65% in cg and > 90% in tg , kasa ( cough ) - moderate relief in cg and progressive relief , later occasional - nonproductive and easy expectoration in tg after 15 days of treatment , shonita darshanam ( hemoptysis ) - the relief is not statistically significant among cg and were given another drug but encouraged results were observed in tg , swarabheda ( hoarseness of voice ) - the relief is not statistically significant among cg but encouraged results were observed in tg , anilath shula ( pain in visceral organs ) - complete relief in tg , sankochamsaparshyoh ( shoulder and scapular emaciation ) - very encouraging in tg patients over the cg , daha ( burning sensation ) - complete relief in tg , atisara ( diarrhea ) - no manifestation among tg , pittaraktasyachagama ( hematemesis ) - manifested in 2 patients and controlled immediately , sirasaha paripoonata ( heaviness in the head ) - relieved in 10 days among tg and in cg after 3 - 4 months , kantadwamsa ( tracheal shift ) - observed only in one case with fibrosed and consolidated lung . the role in objective parameters is as follows ; wight loss - in cg there was further weight loss but in tg there was no weight loss and is statistically significant , sputum conversion - sputum conversion was found among tg after intensive phase but in cg 10 patients out of 30 again put on intensive phase , skiagram ( chest x - ray [ cxr ] ) - density of the opacity in the cxr was less than on previous studies , with lessening of the cavities and resolution of fibrotic changes noted in the tg patients . conducted a study to assess the use of adjunct therapy of ayurvedic medicine with anti - tubercular drugs in the therapeutic management of ptb . it primarily aimed to evaluate the toxicity reduction and early restoration by adjunct therapy of ayurvedic drugs by increasing the bioavailability of atds . the study was conducted among 99 newly diagnosed ptb patients from both the sexes aged between 10 and 65 years . the subjects were administered 500 mg of ashwagandha -2 caps , twice daily and chyawanprash ( as per indian pharmacopeia)-10 g , thrice daily for a period of 28 days . the study reported amelioration of symptoms , improvement of body weight , normalization of erythrocyte sedimentation rate , appreciable change in iga and igm patterns , and significant increase in bioavailability of isoniazid and pyrazinamide . bioavailability of isoniazid and pyrazinamide showed 7 - 10% increased value after 28 days of treatment . sharma et al . conducted a study to assess the hepato - protective properties of ayurvedic herbs among patients receiving anti - tb treatment . the study was conducted among three groups of subjects , each with 10 subjects aged 15 - 70 years , for a period of 90 days with normal liver anatomy and physiology confirmed by ultrasonography and biochemical analysis at the time of the investigation . group-1 received capsule liv-600 , thrice a day composed of 200 mg of hydro alcoholic extract of berberis aristata , solanum nigrum , and aloe vera , group-2 received freshly prepared decoction of bhumyamalaki ( phyllanthus fraternus ) prepared from 10 g of aerial plant , and the group-3 received 600 mg of starch powder as placebo . to evaluate , the hepato - protective efficacies of the above drugs the investigators used subjective and objective parameters such as liver function test including serum bilirubin , alanine transaminase ( alt ) , aspartate aminotransferase ( ast ) , and alkaline phosphatase . no significant elevation in ast and alt were observed in tg compared to cg , whereas no significant elevation was observed for serum total bilirubin and alkaline phosphatase in both tg and cg . table 2 summarizes the in vitro anti - tb properties of some of the ayurvedic herbs . conducted a study to evaluate the in vitro anti - tb properties of the leaves of five legumes . non - polar and polar solvent extracts of leaves of these medicinal legumes were tested against m. tb h37rv , and minimum inhibitory concentrations ( mics ) were determined by the agar - based proportion assay . isoniazid was used as positive control to evaluate the anti - tb activity of the crude extracts of medicinal legumes . except i. cassioides methanol extracts of the leaves of all the five legumes completely inhibited the growth of m. tb at the concentration of 50 g / ml . ethyl acetate extract of two legume leaves , k. pinnatum and d. scandens and chloroform extract of three legume leaves of h. brunonis , c. mimosoides , and d. scandens showed anti - tb activity . however , petroleum ether extract of only i. cassioides showed the activity , similarly , the methanol extract of only h. brunonis leaves showed the activity . gupta et al . conducted a study to evaluate in vitro anti - tb activity of five medicinal plants viz . , acalypha indica , adhatoda vasica , allium cepa , allium sativum , and aloe vera . aqueous extracts of leaves of a. indica , a. vasica , bulbs of a. cepa , cloves of a. sativum , and pure gel of a. vera leaves , were tested in vitro . percentage inhibition was used to evaluate the activity in l - j ( lowenstein - jensen ) medium which was calculated by mean reduction in a number of colonies on extract containing as compared to extract free controls . extracts of all the five plants a. vasica , a. indica , a. cepa , a. vera , and a. sativum exhibited anti - tb activity in l - j medium , the proportion of inhibition of these plants extract in respect to that mentioned above is 95 , 32 , 37 , 72 , 32% , respectively , for mdr isolate dku-156 and 68 , 86 , 79 , 72 , 85% , respectively , for another mdr isolate jal-1236 , while for sensitive m. tb h37rv , inhibition was found to be 68 , 70 , 35 , 63 , and 41% , at 4% v / v concentration in l - j medium . there was no inhibition against rapid grower mycobacterium fortuitum ( tmc-1529 ) . in bact / alert it was observed that a couple of single drugs and compound drugs are useful for the management of tb . however , none of the studies could reflect the true anti - tb activities of any drug , both single and compound . two studies revealed in vitro anti - tb properties of some herbs which can potentially be brought into the realm of a clinical trial to test their efficacy in a human subject . most of these therapeutic preparations studied at different clinical set ups reflected their adjunct properties for the management of tb . these drugs were able to possibly reduce associated symptoms and the adverse drug effects of atds . some of the preparations showed potential hepato - protective properties that can be used as adjunct to atds . the first study primarily focused on the use of a rasayana drug as an adjunct with the simultaneous administration of atds . it provided better physical and mental well - being to the patients by potentiating therapeutic efficacy of atds and also counteracting the unwanted effects caused by atds . rasayana therapy has an anti - oxidant effect along with nutritive value , immune - modulator , immune - protective properties , and free radical scavenging property . in the second study , the authors focused on the role of a naimittika rasayana , a unique concept used in chronic diseases described in ayurveda . it promotes vigor and vitality and instills the ability to hold out disastrous outcomes of diseases as in the case of ptb which has similar untoward effects due to the disease itself and the effects of atds as well . most importantly , the second study delineated an objective parameter , sputum conversion , which creates some degree of skepticism . as the authors have mentioned that the tg got sputum conversion after the intensive phase which was not seen among some patients in cg poses a question , whether it is mere coincidence or the rasayana has really some effect on m. tb . similarly , the study conducted by debnath et al . also reflected on the reduction of the bacterial load with the patients who were on adjunct therapy along with atds . study reported a very significant finding of increased bioavailability of isoniazid and pyrazinamide , which is potentially a great hope in today s situation of drug resistance . furthermore , tb is associated with social determinants such as poor living condition , overcrowding , poor nutrition , and above all poverty . again poverty leads to under nutrition , which itself is affected by both scarcity of food and intra - household distribution . a poor nutritional status also affects drug absorption , resulting in sub - therapeutic serum drug levels , which may lead to non - response to drug therapy . hence , supplementation of these drugs ( ashwagandha and chyawanprash ) along with first line atds could improve bioavailability and help in combating the problem of drug resistance and improve curability . currently at the national level such initiatives are not a part of tb control program which can seriously be thought of looking at the current scenario of widespread drug resistance . most importantly , in all the studies , the commonest improvement as a result of ayurvedic intervention is gain in body weight , which is undoubtedly a cardinal improvement in the part of a ptb patient . rifampicin is an enzyme inducer and promotes the formation of reactive metabolites and thus hepatotoxic in the form of impairment of uptake of bilirubin and acute cellular necrosis . hence , the adjunct intake of liv-600 capsule and decoction of bhumyamalaki could be extremely beneficial in combating hepatotoxicity as a consequence of administration of atds . in addition , in the post - trial analysis , it was observed that the effect of freshly prepared decoction of bhumyamalaki is more effective than liv-600 composed of three drugs . the activity is attributable to their anti - cholestatic action , reduction in free radical damage and cell protein necrosis as well . hence , freshly prepared decoction of bhumyamalaki can be promoted , as a home remedy , at the community level as a hepato - protective agent with the co - administration of atds , which appears to be a cost - effective measure to combat hepatotoxicity of atds . in addition to clinical trials , few herbal drugs showed potential anti - tb activities in vitro . the study conducted by kumar et al . showed anti - tb properties of five different legumes . the leaf extracts of the legumes completely inhibited the growth of m. tb at mic level of 50 g / ml . this is acceptable as activity is considered significant if mic values are below 100 g / ml for crude extract and moderate when mic is between 100 to 625 g / ml . the frontline anti - tb drugs have mics in the range of 0.025 - 2.0 g / ml and if the activity of any new entity is to be compared ( compound / extract ) , its activity should also be in the same range . however , since extracts are crude preparations they may be considered active at higher concentrations also . gupta et al . conducted a study to evaluate the anti - tb properties of some herbs on two mdr m. tb isolates . in addition to some other first line and second line drugs , these mdr isolates were earlier found to be resistant to at least rifampicin and isoniazid . hence , further studies should be carried out using various fractions of crude extracts of these plants as well as their semi - purified / purified principles responsible for anti - tb activity ( specially for mdr and xdr isolates of m. tb ) to find out the mic in suitable broth based media as mics defined in broth are more accurate . most importantly in vitro studies could be brought into the arena of the clinical trial to test their efficacy in human subjects in today s situation of mdr and xdr tb . studies reveal that chest physicians are treating tb patients using few ayurvedic preparations along with atds . the chest physicians are of the opinion that these medications act as immune modulators if given along with second line anti - tb drugs . furthermore , there is a growing interest in identifying the compounds responsible for the anti - mycobacterial activity of traditional medicine and developing them as potential tb drugs . many drugs of the indian system of medicine have come to the realm of national health programs for the management of common ailments , as in national health mission . similarly , few drugs could also be brought into the same platform and be implemented in the national tb control program . tb has been a major public health crisis for the developing world including india . due to increase in mdr and xdr strains of m. tb distressingly research on the role of ayurveda in the management of tb is very scanty and mostly limited to adjunct or supportive therapy . however , the adjunct role of ayurveda drugs can not be simply neglected for not qualifying as agents to combat the m. tb rather their role as agents of increasing bioavailability of atds and counteracting the adverse drug interactions should be properly utilized . being a global public health crisis and having the state of current drug resistance , it is highly recommended to carry out clinical trials on tb patients using ayurvedic drugs and therapeutic regimens . the drugs those proved potent in combating the adverse drug reactions of both the first line and second line anti - tb drugs could be successfully added to the drug regimens of tb for better curability and to reduce drug resistance . similarly , the drugs which showed potential anti - tb properties in vitro could also be useful in today s world of drug resistance .

background : tuberculosis ( tb ) is a global public health crisis . 25% of world s tb cases are found in india . ayurveda , an ancient medical science may offer some solution to this problem . hence , a systematic review was carried out to assess the role of ayurveda for the management of tb.methodology:a systematic review was carried out using published literature obtained through pubmed until april 2015 . the key words used for literature search include ayurveda , role and tb.results and discussion : it was observed that a couple of single and compound drugs have been used for the management of tb . however , none of the studies could reflect the true anti - tb activities of any drug , both single and compound . two of the studies revealed in vitro anti - tb properties of some herbs which can potentially be brought into the realm of a clinical trial to test their efficacy in a human subject . most of these ayurvedic therapeutic preparations studied in different clinical settings primarily reflected their adjunct properties for the management of tb . these studies revealed that ayurvedic therapeutics was able to reduce associated symptoms and the adverse drug effects of atds ( anti - tb drugs ) . furthermore , some of the preparations showed potential hepato - protective properties that can be simultaneously administered with atds.conclusion:distressingly research on the role of ayurveda in the management of tb is very scanty and mostly limited to adjunct or supportive therapy . being a global public health crisis , it is highly recommended to carry out clinical trials on tb patients using ayurvedic drugs and therapeutic regimens .

INTRODUCTION Ayurvedic Concept of Pulmonary TB (PTB) METHODOLOGY RESULTS Ayurvedic Management of TB (Adjunct and Supportive) None DISCUSSION CONCLUSION

tuberculosis ( tb ) is an infectious disease caused by mycobacterium tuberculosis or the other members of mycobacterium complex such as mycobacterium africanum and mycobacterium bovis , known to affect the humans . in spite of noticeable progress achieved tb always offers newer challenges such multidrug - resistant tb ( mdr - tb ) , extensively drug - resistant tb ( xdr - tb ) along with debilitating side effects of anti - tb drugs . in india , tb in the community is managed by a centrally sponsored tb control program known as revised national tb control programme ( rntcp ) . however , private practitioners use various types of ayurvedic medicines , both general practitioners and chest physicians , to support tb management . this ancient medical science can offer some solutions to these problems hence a systematic review was carried out to assess the role of ayurveda for the management of tb . the key words used for the purpose of pubmed search include ayurveda , role and tb . this was done purposefully to obtain a comprehensive list of articles published until april 2015 without the above - mentioned limits . in the first phase studies related to the ayurvedic management of tb as an adjunct therapy are delineated and in the next part the studies related to ayurvedic drugs showing in - vitro anti - tb properties are described . four different studies conducted in different clinical settings in india evaluated the properties of some of the ayurvedic therapeutics as adjunct to anti - tb treatment . table 1 summarizes the properties of some of the ayurvedic therapeutics as adjunct to anti - tb drugs . conducted a single blind controlled trial to evaluate the adjunct properties of rasayana compound among 133 tb patients receiving the rasayana used in this study is composed of amalaki ( emblica officinalis gaertn. the study was carried out for 60 days , and the capsule was administered at a dose of 450 mg . the study found that the compound is helpful in alleviating the associated symptoms of ptb in the treatment group ( tg ) compared to the control group ( cg ) . the rasayana compound was found to decrease cough ( 83% ) , fever ( 93% ) , dyspnea ( 71.3% ) , hemoptysis ( 87% ) , and increase body weight ( 7.7% ) ayurvedic management of tb as an adjunct therapy dornala and dornala conducted a study to evaluate the clinical efficacy of bhringarajasava as naimittika rasayana in ptb . the investigators evaluated the role of bhringarajasava in 15 subjective parameters and 3 objective parameters . the role of this formulation on these subjective parameters is as follows ; the improvement is mentioned in both tg and in cg ; amsaparsabhitapah ( pain in costal and scapular region ) - 35% in cg and 100% in tg , samtapakarapadayoh ( burning sensation in palms and soles ) - 50% in cg and 75% in tg and complete relief in cat - iii , patients , jwara ( pyrexia ) - < 75% in cg and 100% in tg , bhaktadwesha ( anorexia)-mild in cg and complete in tg , swasa ( dyspnea ) - < 65% in cg and > 90% in tg , kasa ( cough ) - moderate relief in cg and progressive relief , later occasional - nonproductive and easy expectoration in tg after 15 days of treatment , shonita darshanam ( hemoptysis ) - the relief is not statistically significant among cg and were given another drug but encouraged results were observed in tg , swarabheda ( hoarseness of voice ) - the relief is not statistically significant among cg but encouraged results were observed in tg , anilath shula ( pain in visceral organs ) - complete relief in tg , sankochamsaparshyoh ( shoulder and scapular emaciation ) - very encouraging in tg patients over the cg , daha ( burning sensation ) - complete relief in tg , atisara ( diarrhea ) - no manifestation among tg , pittaraktasyachagama ( hematemesis ) - manifested in 2 patients and controlled immediately , sirasaha paripoonata ( heaviness in the head ) - relieved in 10 days among tg and in cg after 3 - 4 months , kantadwamsa ( tracheal shift ) - observed only in one case with fibrosed and consolidated lung . the role in objective parameters is as follows ; wight loss - in cg there was further weight loss but in tg there was no weight loss and is statistically significant , sputum conversion - sputum conversion was found among tg after intensive phase but in cg 10 patients out of 30 again put on intensive phase , skiagram ( chest x - ray [ cxr ] ) - density of the opacity in the cxr was less than on previous studies , with lessening of the cavities and resolution of fibrotic changes noted in the tg patients . conducted a study to assess the use of adjunct therapy of ayurvedic medicine with anti - tubercular drugs in the therapeutic management of ptb . it primarily aimed to evaluate the toxicity reduction and early restoration by adjunct therapy of ayurvedic drugs by increasing the bioavailability of atds . conducted a study to assess the hepato - protective properties of ayurvedic herbs among patients receiving anti - tb treatment . to evaluate , the hepato - protective efficacies of the above drugs the investigators used subjective and objective parameters such as liver function test including serum bilirubin , alanine transaminase ( alt ) , aspartate aminotransferase ( ast ) , and alkaline phosphatase . table 2 summarizes the in vitro anti - tb properties of some of the ayurvedic herbs . conducted a study to evaluate the in vitro anti - tb properties of the leaves of five legumes . isoniazid was used as positive control to evaluate the anti - tb activity of the crude extracts of medicinal legumes . ethyl acetate extract of two legume leaves , k. pinnatum and d. scandens and chloroform extract of three legume leaves of h. brunonis , c. mimosoides , and d. scandens showed anti - tb activity . conducted a study to evaluate in vitro anti - tb activity of five medicinal plants viz . aqueous extracts of leaves of a. indica , a. vasica , bulbs of a. cepa , cloves of a. sativum , and pure gel of a. vera leaves , were tested in vitro . extracts of all the five plants a. vasica , a. indica , a. cepa , a. vera , and a. sativum exhibited anti - tb activity in l - j medium , the proportion of inhibition of these plants extract in respect to that mentioned above is 95 , 32 , 37 , 72 , 32% , respectively , for mdr isolate dku-156 and 68 , 86 , 79 , 72 , 85% , respectively , for another mdr isolate jal-1236 , while for sensitive m. tb h37rv , inhibition was found to be 68 , 70 , 35 , 63 , and 41% , at 4% v / v concentration in l - j medium . four different studies conducted in different clinical settings in india evaluated the properties of some of the ayurvedic therapeutics as adjunct to anti - tb treatment . table 1 summarizes the properties of some of the ayurvedic therapeutics as adjunct to anti - tb drugs . conducted a single blind controlled trial to evaluate the adjunct properties of rasayana compound among 133 tb patients receiving the rasayana used in this study is composed of amalaki ( emblica officinalis gaertn. the study was carried out for 60 days , and the capsule was administered at a dose of 450 mg . the study found that the compound is helpful in alleviating the associated symptoms of ptb in the treatment group ( tg ) compared to the control group ( cg ) . the rasayana compound was found to decrease cough ( 83% ) , fever ( 93% ) , dyspnea ( 71.3% ) , hemoptysis ( 87% ) , and increase body weight ( 7.7% ) ayurvedic management of tb as an adjunct therapy dornala and dornala conducted a study to evaluate the clinical efficacy of bhringarajasava as naimittika rasayana in ptb . the investigators evaluated the role of bhringarajasava in 15 subjective parameters and 3 objective parameters . the role of this formulation on these subjective parameters is as follows ; the improvement is mentioned in both tg and in cg ; amsaparsabhitapah ( pain in costal and scapular region ) - 35% in cg and 100% in tg , samtapakarapadayoh ( burning sensation in palms and soles ) - 50% in cg and 75% in tg and complete relief in cat - iii , patients , jwara ( pyrexia ) - < 75% in cg and 100% in tg , bhaktadwesha ( anorexia)-mild in cg and complete in tg , swasa ( dyspnea ) - < 65% in cg and > 90% in tg , kasa ( cough ) - moderate relief in cg and progressive relief , later occasional - nonproductive and easy expectoration in tg after 15 days of treatment , shonita darshanam ( hemoptysis ) - the relief is not statistically significant among cg and were given another drug but encouraged results were observed in tg , swarabheda ( hoarseness of voice ) - the relief is not statistically significant among cg but encouraged results were observed in tg , anilath shula ( pain in visceral organs ) - complete relief in tg , sankochamsaparshyoh ( shoulder and scapular emaciation ) - very encouraging in tg patients over the cg , daha ( burning sensation ) - complete relief in tg , atisara ( diarrhea ) - no manifestation among tg , pittaraktasyachagama ( hematemesis ) - manifested in 2 patients and controlled immediately , sirasaha paripoonata ( heaviness in the head ) - relieved in 10 days among tg and in cg after 3 - 4 months , kantadwamsa ( tracheal shift ) - observed only in one case with fibrosed and consolidated lung . the role in objective parameters is as follows ; wight loss - in cg there was further weight loss but in tg there was no weight loss and is statistically significant , sputum conversion - sputum conversion was found among tg after intensive phase but in cg 10 patients out of 30 again put on intensive phase , skiagram ( chest x - ray [ cxr ] ) - density of the opacity in the cxr was less than on previous studies , with lessening of the cavities and resolution of fibrotic changes noted in the tg patients . conducted a study to assess the use of adjunct therapy of ayurvedic medicine with anti - tubercular drugs in the therapeutic management of ptb . it primarily aimed to evaluate the toxicity reduction and early restoration by adjunct therapy of ayurvedic drugs by increasing the bioavailability of atds . conducted a study to assess the hepato - protective properties of ayurvedic herbs among patients receiving anti - tb treatment . to evaluate , the hepato - protective efficacies of the above drugs the investigators used subjective and objective parameters such as liver function test including serum bilirubin , alanine transaminase ( alt ) , aspartate aminotransferase ( ast ) , and alkaline phosphatase . table 2 summarizes the in vitro anti - tb properties of some of the ayurvedic herbs . conducted a study to evaluate the in vitro anti - tb properties of the leaves of five legumes . isoniazid was used as positive control to evaluate the anti - tb activity of the crude extracts of medicinal legumes . ethyl acetate extract of two legume leaves , k. pinnatum and d. scandens and chloroform extract of three legume leaves of h. brunonis , c. mimosoides , and d. scandens showed anti - tb activity . conducted a study to evaluate in vitro anti - tb activity of five medicinal plants viz . aqueous extracts of leaves of a. indica , a. vasica , bulbs of a. cepa , cloves of a. sativum , and pure gel of a. vera leaves , were tested in vitro . extracts of all the five plants a. vasica , a. indica , a. cepa , a. vera , and a. sativum exhibited anti - tb activity in l - j medium , the proportion of inhibition of these plants extract in respect to that mentioned above is 95 , 32 , 37 , 72 , 32% , respectively , for mdr isolate dku-156 and 68 , 86 , 79 , 72 , 85% , respectively , for another mdr isolate jal-1236 , while for sensitive m. tb h37rv , inhibition was found to be 68 , 70 , 35 , 63 , and 41% , at 4% v / v concentration in l - j medium . in bact / alert it was observed that a couple of single drugs and compound drugs are useful for the management of tb . however , none of the studies could reflect the true anti - tb activities of any drug , both single and compound . two studies revealed in vitro anti - tb properties of some herbs which can potentially be brought into the realm of a clinical trial to test their efficacy in a human subject . most of these therapeutic preparations studied at different clinical set ups reflected their adjunct properties for the management of tb . these drugs were able to possibly reduce associated symptoms and the adverse drug effects of atds . some of the preparations showed potential hepato - protective properties that can be used as adjunct to atds . the first study primarily focused on the use of a rasayana drug as an adjunct with the simultaneous administration of atds . rasayana therapy has an anti - oxidant effect along with nutritive value , immune - modulator , immune - protective properties , and free radical scavenging property . in the second study , the authors focused on the role of a naimittika rasayana , a unique concept used in chronic diseases described in ayurveda . it promotes vigor and vitality and instills the ability to hold out disastrous outcomes of diseases as in the case of ptb which has similar untoward effects due to the disease itself and the effects of atds as well . also reflected on the reduction of the bacterial load with the patients who were on adjunct therapy along with atds . furthermore , tb is associated with social determinants such as poor living condition , overcrowding , poor nutrition , and above all poverty . hence , supplementation of these drugs ( ashwagandha and chyawanprash ) along with first line atds could improve bioavailability and help in combating the problem of drug resistance and improve curability . currently at the national level such initiatives are not a part of tb control program which can seriously be thought of looking at the current scenario of widespread drug resistance . most importantly , in all the studies , the commonest improvement as a result of ayurvedic intervention is gain in body weight , which is undoubtedly a cardinal improvement in the part of a ptb patient . hence , the adjunct intake of liv-600 capsule and decoction of bhumyamalaki could be extremely beneficial in combating hepatotoxicity as a consequence of administration of atds . in addition , in the post - trial analysis , it was observed that the effect of freshly prepared decoction of bhumyamalaki is more effective than liv-600 composed of three drugs . hence , freshly prepared decoction of bhumyamalaki can be promoted , as a home remedy , at the community level as a hepato - protective agent with the co - administration of atds , which appears to be a cost - effective measure to combat hepatotoxicity of atds . in addition to clinical trials , few herbal drugs showed potential anti - tb activities in vitro . showed anti - tb properties of five different legumes . the frontline anti - tb drugs have mics in the range of 0.025 - 2.0 g / ml and if the activity of any new entity is to be compared ( compound / extract ) , its activity should also be in the same range . conducted a study to evaluate the anti - tb properties of some herbs on two mdr m. tb isolates . hence , further studies should be carried out using various fractions of crude extracts of these plants as well as their semi - purified / purified principles responsible for anti - tb activity ( specially for mdr and xdr isolates of m. tb ) to find out the mic in suitable broth based media as mics defined in broth are more accurate . most importantly in vitro studies could be brought into the arena of the clinical trial to test their efficacy in human subjects in today s situation of mdr and xdr tb . studies reveal that chest physicians are treating tb patients using few ayurvedic preparations along with atds . the chest physicians are of the opinion that these medications act as immune modulators if given along with second line anti - tb drugs . furthermore , there is a growing interest in identifying the compounds responsible for the anti - mycobacterial activity of traditional medicine and developing them as potential tb drugs . many drugs of the indian system of medicine have come to the realm of national health programs for the management of common ailments , as in national health mission . similarly , few drugs could also be brought into the same platform and be implemented in the national tb control program . tb has been a major public health crisis for the developing world including india . due to increase in mdr and xdr strains of m. tb distressingly research on the role of ayurveda in the management of tb is very scanty and mostly limited to adjunct or supportive therapy . however , the adjunct role of ayurveda drugs can not be simply neglected for not qualifying as agents to combat the m. tb rather their role as agents of increasing bioavailability of atds and counteracting the adverse drug interactions should be properly utilized . being a global public health crisis and having the state of current drug resistance , it is highly recommended to carry out clinical trials on tb patients using ayurvedic drugs and therapeutic regimens . the drugs those proved potent in combating the adverse drug reactions of both the first line and second line anti - tb drugs could be successfully added to the drug regimens of tb for better curability and to reduce drug resistance . similarly , the drugs which showed potential anti - tb properties in vitro could also be useful in today s world of drug resistance .

the use of generic drugs is steadily increasing internationally as a result of economic pressure on drug budgets . generic drugs provide the opportunity for major savings in healthcare expenditure since they are usually substantially lower in price than the innovator brands.however , physicians are apprehensive regarding the quality of generic drugsand have concerns about their reliability as well as interchange of certain drug categories.although the generic medicines are bio - equivalents of their innovator counterparts and are produced in similar facilities according to good manufacturing practices , these are widely believed as inferior in their therapeutic efficacy and quality to branded products.marketing practices adopted by manufacturers of imported branded medicines also propagate the belief that generics are of inferior quality as reported from countries in central and eastern europe and independent countries emerged from former soviet union.the present study was conducted to compare the quality and price of generic ( branded - generic ) drug products to their expensive popular brand ( branded ) products manufactured by the same pharmaceutical company in india . currently , almost all medicines in india are sold under a brand ( trade ) name and medicines are called as branded medicines or branded - generic . in real sense , indian market does not have branded medicines ( a name commonly given to an innovator product ) because till january 2005 product patent was not applicable in india . in india , many pharmaceutical companies manufacture two types of products for the same molecule , i.e. the branded product which they advertise and push through doctors and branded - generic which they expect retailers to push in the market . the so - called branded medicines in india are manufactured and promoted by multinationals or by reputed indian manufacturers . branded - generics , on the other hand , are not promoted or advertized by the manufacturer . patients and doctors perception for all branded - generics irrespective of company is the same . in india , generic substitution is legally not allowed so patients awareness about generics is limited and doctors and patients do not want pharmacist to change the trade name written by doctor . hence , consumer awareness for the generics , variety of trade names available in the market , and price variation is very limited . hence , there is need to conduct a study that can document the price structure and quality of the branded product and their branded - generic versions manufactured in india . pair of product from the same company was chosen to appreciate the price structure and mark - ups for the two versions . quality and price of medicines were studied to evaluate the two versions of the same therapeutic molecule . five commonly used medicines from different classes were selected whose branded ( popular brand ) and branded - generic versions were manufactured by the same pharmaceutical company . the five medicines chosen were alprazolam , ( 0.25 mg ) , cetirizine ( 10 mg ) , ciprofloxacin ( 500 mg ) , fluoxetine ( 20 mg ) , and lansoprazole ( 30 mg ) . price - to - patient and price - to - retailer ( ptr ) were analyzed . maximum retail price ( mrp ) is the price - to - patient and is always printed on the package in india . details of the five " paired " drug products sold under different trade names with their mrp were checked physically with the private retail pharmacies ( chemist shop ) . ptr is the price at which wholesaler ( distributor ) sells the product to the retailer and the bill ( voucher ) given to retailer by wholesaler mentions the ptr . this price was checked and confirmed from form v ( under dpco , 1995 ) available at the distributors of the company . it is mandatory for all the companies to give form v that gives details of the product with mrp , ptr , taxes paid , etc to their distributors . the test samples were procured from the licensed authorized chemist dealers through valid purchase invoice . the sample size comprised 1010 tablets / capsules of both branded and branded - generic versions of each drug product . efforts were made to procure these test samples ( pairs ) with identical date of manufacture to rule out the possibility of difference in assay because of different dates of manufacturing . the qualitative as well as quantitative analysis was carried out in a government - approved laboratory following the methods prescribed in the indian pharmacopoeia , ( 2007 ) as per the standards laid down in the drugs and cosmetics act 1940 and rules 1945 . identification test : identity of the drug molecule was established by performing the identification test through instrumental analysis using hplc ( high pressure liquid chromatography ) or ir ( infra - red spectroscopy ) as per method prescribed for each medicine.chemical composition test\ : the samples were subjected to quantitative analysis using hplc instrumental analytical methods as provided in indian pharmacopoeia 2007.uniformity of content test : to confirm the uniformity of contents in the batch , the sampled dosage units were subjected to uniformity of contents test wherein assay on 10 units of dosage form were performed individually using instrumental analytical methods . the test for uniformity of content is not applicable to tablets / capsules containing more than 10 mg ; it was conducted only for alprazolam ( 0.25 mg ) and cetirizine ( 10 mg).uniformity of weight : all 10 units of sample were tested for uniformity of weight as prescribed.tests for dissolution : the samples were subjected to dissolution studies to evaluate their drug release pattern . these studies were performed in the dissolution media specified in the individual monograph of the indian pharmacopoeia 2007 on six dosage units and were indicative of the in vivo availability of active drug moiety from the dosage form , i.e. tablet or capsule . identification test : identity of the drug molecule was established by performing the identification test through instrumental analysis using hplc ( high pressure liquid chromatography ) or ir ( infra - red spectroscopy ) as per method prescribed for each medicine . chemical composition test\ : the samples were subjected to quantitative analysis using hplc instrumental analytical methods as provided in indian pharmacopoeia 2007 . uniformity of content test : to confirm the uniformity of contents in the batch , the sampled dosage units were subjected to uniformity of contents test wherein assay on 10 units of dosage form were performed individually using instrumental analytical methods . the test for uniformity of content is not applicable to tablets / capsules containing more than 10 mg ; it was conducted only for alprazolam ( 0.25 mg ) and cetirizine ( 10 mg ) . uniformity of weight : all 10 units of sample were tested for uniformity of weight as prescribed . tests for dissolution : the samples were subjected to dissolution studies to evaluate their drug release pattern . these studies were performed in the dissolution media specified in the individual monograph of the indian pharmacopoeia 2007 on six dosage units and were indicative of the in vivo availability of active drug moiety from the dosage form , i.e. tablet or capsule . five commonly used medicines from different classes were selected whose branded ( popular brand ) and branded - generic versions were manufactured by the same pharmaceutical company . the five medicines chosen were alprazolam , ( 0.25 mg ) , cetirizine ( 10 mg ) , ciprofloxacin ( 500 mg ) , fluoxetine ( 20 mg ) , and lansoprazole ( 30 mg ) . price - to - patient and price - to - retailer ( ptr ) were analyzed . maximum retail price ( mrp ) is the price - to - patient and is always printed on the package in india . details of the five " paired " drug products sold under different trade names with their mrp were checked physically with the private retail pharmacies ( chemist shop ) . ptr is the price at which wholesaler ( distributor ) sells the product to the retailer and the bill ( voucher ) given to retailer by wholesaler mentions the ptr . this price was checked and confirmed from form v ( under dpco , 1995 ) available at the distributors of the company . it is mandatory for all the companies to give form v that gives details of the product with mrp , ptr , taxes paid , etc to their distributors . the test samples were procured from the licensed authorized chemist dealers through valid purchase invoice . the sample size comprised 1010 tablets / capsules of both branded and branded - generic versions of each drug product . efforts were made to procure these test samples ( pairs ) with identical date of manufacture to rule out the possibility of difference in assay because of different dates of manufacturing . the qualitative as well as quantitative analysis was carried out in a government - approved laboratory following the methods prescribed in the indian pharmacopoeia , ( 2007 ) as per the standards laid down in the drugs and cosmetics act 1940 and rules 1945 . identification test : identity of the drug molecule was established by performing the identification test through instrumental analysis using hplc ( high pressure liquid chromatography ) or ir ( infra - red spectroscopy ) as per method prescribed for each medicine.chemical composition test\ : the samples were subjected to quantitative analysis using hplc instrumental analytical methods as provided in indian pharmacopoeia 2007.uniformity of content test : to confirm the uniformity of contents in the batch , the sampled dosage units were subjected to uniformity of contents test wherein assay on 10 units of dosage form were performed individually using instrumental analytical methods . the test for uniformity of content is not applicable to tablets / capsules containing more than 10 mg ; it was conducted only for alprazolam ( 0.25 mg ) and cetirizine ( 10 mg).uniformity of weight : all 10 units of sample were tested for uniformity of weight as prescribed.tests for dissolution : the samples were subjected to dissolution studies to evaluate their drug release pattern . these studies were performed in the dissolution media specified in the individual monograph of the indian pharmacopoeia 2007 on six dosage units and were indicative of the in vivo availability of active drug moiety from the dosage form , i.e. tablet or capsule . identification test : identity of the drug molecule was established by performing the identification test through instrumental analysis using hplc ( high pressure liquid chromatography ) or ir ( infra - red spectroscopy ) as per method prescribed for each medicine . chemical composition test\ : the samples were subjected to quantitative analysis using hplc instrumental analytical methods as provided in indian pharmacopoeia 2007 . uniformity of content test : to confirm the uniformity of contents in the batch , the sampled dosage units were subjected to uniformity of contents test wherein assay on 10 units of dosage form were performed individually using instrumental analytical methods . the test for uniformity of content is not applicable to tablets / capsules containing more than 10 mg ; it was conducted only for alprazolam ( 0.25 mg ) and cetirizine ( 10 mg ) . uniformity of weight : all 10 units of sample were tested for uniformity of weight as prescribed . tests for dissolution : the samples were subjected to dissolution studies to evaluate their drug release pattern . these studies were performed in the dissolution media specified in the individual monograph of the indian pharmacopoeia 2007 on six dosage units and were indicative of the in vivo availability of active drug moiety from the dosage form , i.e. tablet or capsule . five commonly used medicines from different classes were selected whose branded ( popular brand ) and branded - generic versions were manufactured by the same pharmaceutical company . the five medicines chosen were alprazolam , ( 0.25 mg ) , cetirizine ( 10 mg ) , ciprofloxacin ( 500 mg ) , fluoxetine ( 20 mg ) , and lansoprazole ( 30 mg ) . price - to - patient and price - to - retailer ( ptr ) were analyzed . maximum retail price ( mrp ) is the price - to - patient and is always printed on the package in india . details of the five " paired " drug products sold under different trade names with their mrp were checked physically with the private retail pharmacies ( chemist shop ) . ptr is the price at which wholesaler ( distributor ) sells the product to the retailer and the bill ( voucher ) given to retailer by wholesaler mentions the ptr . this price was checked and confirmed from form v ( under dpco , 1995 ) available at the distributors of the company . it is mandatory for all the companies to give form v that gives details of the product with mrp , ptr , taxes paid , etc to their distributors . the test samples were procured from the licensed authorized chemist dealers through valid purchase invoice . the sample size comprised 1010 tablets / capsules of both branded and branded - generic versions of each drug product . efforts were made to procure these test samples ( pairs ) with identical date of manufacture to rule out the possibility of difference in assay because of different dates of manufacturing . the qualitative as well as quantitative analysis was carried out in a government - approved laboratory following the methods prescribed in the indian pharmacopoeia , ( 2007 ) as per the standards laid down in the drugs and cosmetics act 1940 and rules 1945 . identification test : identity of the drug molecule was established by performing the identification test through instrumental analysis using hplc ( high pressure liquid chromatography ) or ir ( infra - red spectroscopy ) as per method prescribed for each medicine.chemical composition test\ : the samples were subjected to quantitative analysis using hplc instrumental analytical methods as provided in indian pharmacopoeia 2007.uniformity of content test : to confirm the uniformity of contents in the batch , the sampled dosage units were subjected to uniformity of contents test wherein assay on 10 units of dosage form were performed individually using instrumental analytical methods . the test for uniformity of content is not applicable to tablets / capsules containing more than 10 mg ; it was conducted only for alprazolam ( 0.25 mg ) and cetirizine ( 10 mg).uniformity of weight : all 10 units of sample were tested for uniformity of weight as prescribed.tests for dissolution : the samples were subjected to dissolution studies to evaluate their drug release pattern . these studies were performed in the dissolution media specified in the individual monograph of the indian pharmacopoeia 2007 on six dosage units and were indicative of the in vivo availability of active drug moiety from the dosage form , i.e. tablet or capsule . identification test : identity of the drug molecule was established by performing the identification test through instrumental analysis using hplc ( high pressure liquid chromatography ) or ir ( infra - red spectroscopy ) as per method prescribed for each medicine . chemical composition test\ : the samples were subjected to quantitative analysis using hplc instrumental analytical methods as provided in indian pharmacopoeia 2007 . uniformity of content test : to confirm the uniformity of contents in the batch , the sampled dosage units were subjected to uniformity of contents test wherein assay on 10 units of dosage form were performed individually using instrumental analytical methods . the test for uniformity of content is not applicable to tablets / capsules containing more than 10 mg ; it was conducted only for alprazolam ( 0.25 mg ) and cetirizine ( 10 mg ) . uniformity of weight : all 10 units of sample were tested for uniformity of weight as prescribed . tests for dissolution : the samples were subjected to dissolution studies to evaluate their drug release pattern . these studies were performed in the dissolution media specified in the individual monograph of the indian pharmacopoeia 2007 on six dosage units and were indicative of the in vivo availability of active drug moiety from the dosage form , i.e. tablet or capsule . details of five pairs of medicines including their trade name as sold in the indian market , strength , dosage form , and the pharmaceutical company that manufactures these products are given in table 1 . price - to - patient ( mrp ) and price - to - retailer ( ptr ) found for all the five ptr for the branded product of cetirizine was 11 times the price for branded - generic by the same company . retailer is earning inr 22.76 for 10 tablets of branded - generic cetirizine versus rs 8.16 for the branded version from the same company . for ciprofloxacin , the mrp of both the branded and branded - generic product was same but the branded - generic was available to retailer at 3.6 times less price than branded medicine from the same company . comparative price structure of branded and branded - generic medicines the price - to - patient ( mrp ) of the branded product for the five medicines evaluated was 41% , 33% , 0% , 14% , and 31% higher than the mrp of the branded - generic version of the same company . on the other hand , ptr for branded - generic was 1112% , 397% , 266% , 170% , and 439% less than the branded version of cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam , respectively . retailer mark - ups for five pair of medicines for branded versus branded - generic : cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam were 30% versus 1016% , 25% versus 367% , 25% versus 357% , 27% versus 201% , and 25% versus 415% , respectively [ table 1 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26].chemical composition test : the quantitative analysis conducted using the hplc method showed each unit of the tested samples to be well within the prescribed range [ tables 26].uniformity of content : this test was done for alprazolam and cetirizine and results for both the versions of medicines were within the prescribed range [ tables 2 and 3].uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26]dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26 ] . chemical composition test : the quantitative analysis conducted using the hplc method showed each unit of the tested samples to be well within the prescribed range [ tables 26 ] . uniformity of content : this test was done for alprazolam and cetirizine and results for both the versions of medicines were within the prescribed range [ tables 2 and 3 ] . uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26 ] dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . comparative analytical evaluation of branded and branded - generic drug product alprazolam tablets ( 0.25 mg ) comparative analytical evaluation of branded and branded generic drug product cetirizine tablets ( 10 mg ) comparative analytical evaluation of branded and brandedgeneric drug product ciprofloxacin tablets ( 500 mg ) comparative analytical evaluation of branded and brandedgeneric drug product fluoxetine capsules ( 20 mg ) comparative analytical evaluation of branded / branded - generic drug product lansoprazole capsules ( 30 mg ) details of five pairs of medicines including their trade name as sold in the indian market , strength , dosage form , and the pharmaceutical company that manufactures these products are given in table 1 . price - to - patient ( mrp ) and price - to - retailer ( ptr ) found for all the five ptr for the branded product of cetirizine was 11 times the price for branded - generic by the same company . retailer is earning inr 22.76 for 10 tablets of branded - generic cetirizine versus rs 8.16 for the branded version from the same company . for ciprofloxacin , the mrp of both the branded and branded - generic product was same but the branded - generic was available to retailer at 3.6 times less price than branded medicine from the same company . comparative price structure of branded and branded - generic medicines the price - to - patient ( mrp ) of the branded product for the five medicines evaluated was 41% , 33% , 0% , 14% , and 31% higher than the mrp of the branded - generic version of the same company . on the other hand , ptr for branded - generic was 1112% , 397% , 266% , 170% , and 439% less than the branded version of cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam , respectively . retailer mark - ups for five pair of medicines for branded versus branded - generic : cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam were 30% versus 1016% , 25% versus 367% , 25% versus 357% , 27% versus 201% , and 25% versus 415% , respectively [ table 1 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26].chemical composition test : the quantitative analysis conducted using the hplc method showed each unit of the tested samples to be well within the prescribed range [ tables 26].uniformity of content : this test was done for alprazolam and cetirizine and results for both the versions of medicines were within the prescribed range [ tables 2 and 3].uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26]dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26 ] . chemical composition test : the quantitative analysis conducted using the hplc method showed each unit of the tested samples to be well within the prescribed range [ tables 26 ] . uniformity of content : this test was done for alprazolam and cetirizine and results for both the versions of medicines were within the prescribed range [ tables 2 and 3 ] . uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26 ] dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . comparative analytical evaluation of branded and branded - generic drug product alprazolam tablets ( 0.25 mg ) comparative analytical evaluation of branded and branded generic drug product cetirizine tablets ( 10 mg ) comparative analytical evaluation of branded and brandedgeneric drug product ciprofloxacin tablets ( 500 mg ) comparative analytical evaluation of branded and brandedgeneric drug product fluoxetine capsules ( 20 mg ) comparative analytical evaluation of branded / branded - generic drug product lansoprazole capsules ( 30 mg ) details of five pairs of medicines including their trade name as sold in the indian market , strength , dosage form , and the pharmaceutical company that manufactures these products are given in table 1 . price - to - patient ( mrp ) and price - to - retailer ( ptr ) found for all the five ptr for the branded product of cetirizine was 11 times the price for branded - generic by the same company . retailer is earning inr 22.76 for 10 tablets of branded - generic cetirizine versus rs 8.16 for the branded version from the same company . for ciprofloxacin , the mrp of both the branded and branded - generic product was same but the branded - generic was available to retailer at 3.6 times less price than branded medicine from the same company . comparative price structure of branded and branded - generic medicines the price - to - patient ( mrp ) of the branded product for the five medicines evaluated was 41% , 33% , 0% , 14% , and 31% higher than the mrp of the branded - generic version of the same company . on the other hand , ptr for branded - generic was 1112% , 397% , 266% , 170% , and 439% less than the branded version of cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam , respectively . retailer mark - ups for five pair of medicines for branded versus branded - generic : cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam were 30% versus 1016% , 25% versus 367% , 25% versus 357% , 27% versus 201% , and 25% versus 415% , respectively [ table 1 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26].chemical composition test : the quantitative analysis conducted using the hplc method showed each unit of the tested samples to be well within the prescribed range [ tables 26].uniformity of content : this test was done for alprazolam and cetirizine and results for both the versions of medicines were within the prescribed range [ tables 2 and 3].uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26]dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26 ] . chemical composition test : the quantitative analysis conducted using the hplc method showed each unit of the tested samples to be well within the prescribed range [ tables 26 ] . uniformity of content : this test was done for alprazolam and cetirizine and results for both the versions of medicines were within the prescribed range [ tables 2 and 3 ] . uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26 ] dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . comparative analytical evaluation of branded and branded - generic drug product alprazolam tablets ( 0.25 mg ) comparative analytical evaluation of branded and branded generic drug product cetirizine tablets ( 10 mg ) comparative analytical evaluation of branded and brandedgeneric drug product ciprofloxacin tablets ( 500 mg ) comparative analytical evaluation of branded and brandedgeneric drug product fluoxetine capsules ( 20 mg ) comparative analytical evaluation of branded / branded - generic drug product lansoprazole capsules ( 30 mg ) this is one of the first studies in india conducted to systematic evaluate the price - to - patient and retailer mark - up for the branded and branded - generic versions of the same therapeutic molecule manufactured by the same company . findings of the study revealed that there are huge mark - ups for retailer on branded - generic medicines . the retailer margin for five branded medicines studied was in the range of 25 - 30% , but for their branded - generics version manufactured by the same company it was in the range of 201 - 1016% . both versions of all five medicines cleared all the quantitative and qualitative parameters as prescribed in indian pharmacopoeia , 2007 . there exists a widespread belief among people and dispensing chemists that a branded product is better in terms of quality and safety than the generic.[1012]a systematic review has shown that generic and brand name cardio - vascular drugs were similar for nearly all clinical outcomes . this study concluded that there is no evidence of superiority of brand preparations to generic drugs.such studies may be helpful in promoting generic drug use that reduces unnecessary spending without improving clinical outcome . in most developed countries , generic medicines are promoted by competition - enhancing policies operating through health - care reimbursements to contain expenditure and encourage efficient use of resources . results of our study revealed that price - to - patient for the branded - generic version was not much less than to its branded counterpart ; branded - generic was available at 70 - 100% cost of the branded product . medicine prices are under the purview of department of pharmaceuticals which itself is under ministry of chemicals and fertilizers . the drug price control order ( dpco ) identifies active pharmaceutical ingredients ( apis ) for which a pricing formula is used to set the mrp . there are only 74 bulk drugs which are under price controland are called scheduled medicines . for all other medicines --- called non - scheduled medicines---the manufacturer sets the price and registers that price with the national pharmaceutical pricing authority ( nppa ) under department of pharmaceuticals.the medicines are sold at the printed mrp on their label and dispensing pharmacist can not charge a price exceeding mrp printed on the pack , as per the provisions under paragraph 16 of the dpco , 1995 . for scheduled medicines , the nppa pricing formula sets the 8% mark - up for wholesalers and 16% for retailers . for non - scheduled medicines , these markups are not set , but it is agreed by the partners of the trade that for branded medicines average mark - up would be around 10% and 20% for wholesalers and retailers , respectively . in our sample of medicines , only ciprofloxacin is under price control and other four belong to non - schedule medicine . the study revealed that even for the branded version of both schedule and non - scheduled medicines , the retailer margin was more than the established margin , it was in the range of 25 - 30% . for the branded - generic version , the retailer margins were very large , 201 - 1016% . pharmaceutical companies decide not only the final price ( mrp ) to the patient but also the mark - up for the retailer . if the marketing is done by the company as for the " branded " version then the major mark - ups are for the company ; if the marketing and promotion are done by the retailer as in the case of branded - generic then the ptr is less , but mrp is not much different . therefore , the branded - generics are promoted by the retailers for monetary considerations in total disregard to the patient 's interests . the ultimate consumer , i.e. patient is not benefited much by preferring branded - generic versions to its branded version . a newspaper reports the huge profit margins for retailers ranging from 500% to1000% on generic medicines in india.the high mark - ups on generics are totally negating the very concept of affordable generic medicines for patients . unlike developed countries , people in developing countries more than 80% health financing is borne by patients.india is known to export medicines to various countries at low cost , but faces the challenge of access to affordable and quality medicines for its own population.hence , the government should have a policy whereby the prices of branded - generic drugs can be made realistic and affordable to common man . the profit margins presently being shared by traders must be passed to consumer . in india , a dispensing pharmacist is not authorized to substitute a branded medicine with a branded - generic ( or generic ) as per the provisions under rule 65 of the drugs and cosmetics act , 1940 and rules , 1945 , which also add to the patient 's burden . in the usa substitution is allowed and patients accept generic substitution if physician approves of the same . generic substitution rates have increased remarkably there , probably due to greater acceptance by physicians and pharmacists as well as encouragement from the third party payers.cheaper generics are one of the important factors to reduce health - care cost . the practice of generic substitution is strongly supported by health authorities in many developed countries.use of generic drugs , which are bio - equivalent to brand name drug , can help contain prescription drug spending.government of india has opened few generic drug stores in some states that sell generic medicines manufactured by public sector companies.the quality of generic medicines available on these stores at cheaper rates should be tested and compared with popular brands and results should be widely published . studies involving comparative evaluation on quality of branded and their generic counterpart may be made mandatory for the generic ( or branded - generic ) manufacturer and their reports should be made public to promote generic use and prescriptions . one inherent limitation of this study is that we have tested pair of branded and branded - generic medicines that were manufactured by the same reputed company . though it is expected that both the versions should have the same quality but perception for any branded - generic is same among doctors and patients . so to start with , we have taken both branded and branded - generic products of the same company and studied not only the quality but also the price structure . findings of the present study indicate that both the branded and branded - generic versions of the five paired medicines had identical quality and they fulfilled all the criteria prescribed by the statutory standards . hence , the general notion and doubt regarding the quality of the branded - generic version of medicines needs to be erased conducting more such studies and publishing them widely.suitable changes in the drug price policy may be made to have lower prices for branded - generic versions . transparency in fixing the mrp by the manufacturer and clear guidelines for mark - ups at least for branded - generics is required in pharmaceutical trade . the government must take up generic promotional schemes , general awareness programs on quality of generics to build confidence among prescribers , pharmacists , and consumers . availability of generics or branded - generics in the market with lower price tag and assured quality is essential to make the medicines affordable .

objective : to compare and evaluate the price and quality of branded and branded - generic equivalents of some commonly used medicines manufactured by the same pharmaceutical company in india.materials and methods : five commonly used medicines : alprazolam , cetirizine , ciprofloxacin , fluoxetine , and lansoprazole manufactured in branded and branded - generic versions by the same company were selected . price - to - patient and price - to - retailers were found for five pair of medicines . both quantitative and qualitative analysis were performed following the methods prescribed in the indian pharmacopoeia 2007 on five pair of medicines . the tests performed were identification test , chemical composition estimation test , uniformity of contents test , uniformity of weight , and dissolution studies.main outcome measures : price - to - patient , retailer mark - up and qualitative analysis of branded and branded - generic medicines.results:retailer margin for five branded medicines were in the range of 25 - 30% but for their branded - generics version manufactured by the same company it was in the range of 201 - 1016% . price - to - patient for the branded version of cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprozolam was higher by 41% , 33% , 0% , 14% , and 31% than branded - generic . both versions of five medicines were within their permissible range for all the quantitative and qualitative parameters as prescribed in indian pharmacopoeia.conclusion:difference in price - to - patient was not as huge as it is expected for generics but margins for retailer were very high for branded - generics . quality of branded - generics is same as for their branded version . the study highlights the need to modify the drug price policy , regulate the mark - ups in generic supply chain , conduct and widely publicize the quality testing of generics for awareness of all stakeholders .

Introduction Methods None Selection of medicines Medicine price Quality testing Results None Comparative Price and Mark-Up for Quality of Discussion Conclusions

generic drugs provide the opportunity for major savings in healthcare expenditure since they are usually substantially lower in price than the innovator brands.however , physicians are apprehensive regarding the quality of generic drugsand have concerns about their reliability as well as interchange of certain drug categories.although the generic medicines are bio - equivalents of their innovator counterparts and are produced in similar facilities according to good manufacturing practices , these are widely believed as inferior in their therapeutic efficacy and quality to branded products.marketing practices adopted by manufacturers of imported branded medicines also propagate the belief that generics are of inferior quality as reported from countries in central and eastern europe and independent countries emerged from former soviet union.the present study was conducted to compare the quality and price of generic ( branded - generic ) drug products to their expensive popular brand ( branded ) products manufactured by the same pharmaceutical company in india . hence , there is need to conduct a study that can document the price structure and quality of the branded product and their branded - generic versions manufactured in india . pair of product from the same company was chosen to appreciate the price structure and mark - ups for the two versions . quality and price of medicines were studied to evaluate the two versions of the same therapeutic molecule . five commonly used medicines from different classes were selected whose branded ( popular brand ) and branded - generic versions were manufactured by the same pharmaceutical company . the five medicines chosen were alprazolam , ( 0.25 mg ) , cetirizine ( 10 mg ) , ciprofloxacin ( 500 mg ) , fluoxetine ( 20 mg ) , and lansoprazole ( 30 mg ) . maximum retail price ( mrp ) is the price - to - patient and is always printed on the package in india . the sample size comprised 1010 tablets / capsules of both branded and branded - generic versions of each drug product . the qualitative as well as quantitative analysis was carried out in a government - approved laboratory following the methods prescribed in the indian pharmacopoeia , ( 2007 ) as per the standards laid down in the drugs and cosmetics act 1940 and rules 1945 . identification test : identity of the drug molecule was established by performing the identification test through instrumental analysis using hplc ( high pressure liquid chromatography ) or ir ( infra - red spectroscopy ) as per method prescribed for each medicine.chemical composition test\ : the samples were subjected to quantitative analysis using hplc instrumental analytical methods as provided in indian pharmacopoeia 2007.uniformity of content test : to confirm the uniformity of contents in the batch , the sampled dosage units were subjected to uniformity of contents test wherein assay on 10 units of dosage form were performed individually using instrumental analytical methods . these studies were performed in the dissolution media specified in the individual monograph of the indian pharmacopoeia 2007 on six dosage units and were indicative of the in vivo availability of active drug moiety from the dosage form , i.e. five commonly used medicines from different classes were selected whose branded ( popular brand ) and branded - generic versions were manufactured by the same pharmaceutical company . the five medicines chosen were alprazolam , ( 0.25 mg ) , cetirizine ( 10 mg ) , ciprofloxacin ( 500 mg ) , fluoxetine ( 20 mg ) , and lansoprazole ( 30 mg ) . maximum retail price ( mrp ) is the price - to - patient and is always printed on the package in india . the sample size comprised 1010 tablets / capsules of both branded and branded - generic versions of each drug product . the qualitative as well as quantitative analysis was carried out in a government - approved laboratory following the methods prescribed in the indian pharmacopoeia , ( 2007 ) as per the standards laid down in the drugs and cosmetics act 1940 and rules 1945 . identification test : identity of the drug molecule was established by performing the identification test through instrumental analysis using hplc ( high pressure liquid chromatography ) or ir ( infra - red spectroscopy ) as per method prescribed for each medicine.chemical composition test\ : the samples were subjected to quantitative analysis using hplc instrumental analytical methods as provided in indian pharmacopoeia 2007.uniformity of content test : to confirm the uniformity of contents in the batch , the sampled dosage units were subjected to uniformity of contents test wherein assay on 10 units of dosage form were performed individually using instrumental analytical methods . these studies were performed in the dissolution media specified in the individual monograph of the indian pharmacopoeia 2007 on six dosage units and were indicative of the in vivo availability of active drug moiety from the dosage form , i.e. five commonly used medicines from different classes were selected whose branded ( popular brand ) and branded - generic versions were manufactured by the same pharmaceutical company . the five medicines chosen were alprazolam , ( 0.25 mg ) , cetirizine ( 10 mg ) , ciprofloxacin ( 500 mg ) , fluoxetine ( 20 mg ) , and lansoprazole ( 30 mg ) . maximum retail price ( mrp ) is the price - to - patient and is always printed on the package in india . the qualitative as well as quantitative analysis was carried out in a government - approved laboratory following the methods prescribed in the indian pharmacopoeia , ( 2007 ) as per the standards laid down in the drugs and cosmetics act 1940 and rules 1945 . identification test : identity of the drug molecule was established by performing the identification test through instrumental analysis using hplc ( high pressure liquid chromatography ) or ir ( infra - red spectroscopy ) as per method prescribed for each medicine.chemical composition test\ : the samples were subjected to quantitative analysis using hplc instrumental analytical methods as provided in indian pharmacopoeia 2007.uniformity of content test : to confirm the uniformity of contents in the batch , the sampled dosage units were subjected to uniformity of contents test wherein assay on 10 units of dosage form were performed individually using instrumental analytical methods . these studies were performed in the dissolution media specified in the individual monograph of the indian pharmacopoeia 2007 on six dosage units and were indicative of the in vivo availability of active drug moiety from the dosage form , i.e. price - to - patient ( mrp ) and price - to - retailer ( ptr ) found for all the five ptr for the branded product of cetirizine was 11 times the price for branded - generic by the same company . retailer is earning inr 22.76 for 10 tablets of branded - generic cetirizine versus rs 8.16 for the branded version from the same company . for ciprofloxacin , the mrp of both the branded and branded - generic product was same but the branded - generic was available to retailer at 3.6 times less price than branded medicine from the same company . comparative price structure of branded and branded - generic medicines the price - to - patient ( mrp ) of the branded product for the five medicines evaluated was 41% , 33% , 0% , 14% , and 31% higher than the mrp of the branded - generic version of the same company . on the other hand , ptr for branded - generic was 1112% , 397% , 266% , 170% , and 439% less than the branded version of cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam , respectively . retailer mark - ups for five pair of medicines for branded versus branded - generic : cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam were 30% versus 1016% , 25% versus 367% , 25% versus 357% , 27% versus 201% , and 25% versus 415% , respectively [ table 1 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26].chemical composition test : the quantitative analysis conducted using the hplc method showed each unit of the tested samples to be well within the prescribed range [ tables 26].uniformity of content : this test was done for alprazolam and cetirizine and results for both the versions of medicines were within the prescribed range [ tables 2 and 3].uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26]dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26 ] . uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26 ] dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . comparative analytical evaluation of branded and branded - generic drug product alprazolam tablets ( 0.25 mg ) comparative analytical evaluation of branded and branded generic drug product cetirizine tablets ( 10 mg ) comparative analytical evaluation of branded and brandedgeneric drug product ciprofloxacin tablets ( 500 mg ) comparative analytical evaluation of branded and brandedgeneric drug product fluoxetine capsules ( 20 mg ) comparative analytical evaluation of branded / branded - generic drug product lansoprazole capsules ( 30 mg ) details of five pairs of medicines including their trade name as sold in the indian market , strength , dosage form , and the pharmaceutical company that manufactures these products are given in table 1 . price - to - patient ( mrp ) and price - to - retailer ( ptr ) found for all the five ptr for the branded product of cetirizine was 11 times the price for branded - generic by the same company . retailer is earning inr 22.76 for 10 tablets of branded - generic cetirizine versus rs 8.16 for the branded version from the same company . for ciprofloxacin , the mrp of both the branded and branded - generic product was same but the branded - generic was available to retailer at 3.6 times less price than branded medicine from the same company . comparative price structure of branded and branded - generic medicines the price - to - patient ( mrp ) of the branded product for the five medicines evaluated was 41% , 33% , 0% , 14% , and 31% higher than the mrp of the branded - generic version of the same company . on the other hand , ptr for branded - generic was 1112% , 397% , 266% , 170% , and 439% less than the branded version of cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam , respectively . retailer mark - ups for five pair of medicines for branded versus branded - generic : cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam were 30% versus 1016% , 25% versus 367% , 25% versus 357% , 27% versus 201% , and 25% versus 415% , respectively [ table 1 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26].chemical composition test : the quantitative analysis conducted using the hplc method showed each unit of the tested samples to be well within the prescribed range [ tables 26].uniformity of content : this test was done for alprazolam and cetirizine and results for both the versions of medicines were within the prescribed range [ tables 2 and 3].uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26]dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26 ] . uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26 ] dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . comparative analytical evaluation of branded and branded - generic drug product alprazolam tablets ( 0.25 mg ) comparative analytical evaluation of branded and branded generic drug product cetirizine tablets ( 10 mg ) comparative analytical evaluation of branded and brandedgeneric drug product ciprofloxacin tablets ( 500 mg ) comparative analytical evaluation of branded and brandedgeneric drug product fluoxetine capsules ( 20 mg ) comparative analytical evaluation of branded / branded - generic drug product lansoprazole capsules ( 30 mg ) details of five pairs of medicines including their trade name as sold in the indian market , strength , dosage form , and the pharmaceutical company that manufactures these products are given in table 1 . price - to - patient ( mrp ) and price - to - retailer ( ptr ) found for all the five ptr for the branded product of cetirizine was 11 times the price for branded - generic by the same company . retailer is earning inr 22.76 for 10 tablets of branded - generic cetirizine versus rs 8.16 for the branded version from the same company . for ciprofloxacin , the mrp of both the branded and branded - generic product was same but the branded - generic was available to retailer at 3.6 times less price than branded medicine from the same company . comparative price structure of branded and branded - generic medicines the price - to - patient ( mrp ) of the branded product for the five medicines evaluated was 41% , 33% , 0% , 14% , and 31% higher than the mrp of the branded - generic version of the same company . on the other hand , ptr for branded - generic was 1112% , 397% , 266% , 170% , and 439% less than the branded version of cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam , respectively . retailer mark - ups for five pair of medicines for branded versus branded - generic : cetirizine , fluoxetine , ciprofloxacin , lansoprazole , and alprazolam were 30% versus 1016% , 25% versus 367% , 25% versus 357% , 27% versus 201% , and 25% versus 415% , respectively [ table 1 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26].chemical composition test : the quantitative analysis conducted using the hplc method showed each unit of the tested samples to be well within the prescribed range [ tables 26].uniformity of content : this test was done for alprazolam and cetirizine and results for both the versions of medicines were within the prescribed range [ tables 2 and 3].uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26]dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . identification test : all the five paired medicines of branded and branded - generics gave positive identification tests when tested on hplc or ir establishing their chemical identity [ tables 26 ] . uniformity of weight : each unit of the sample was within the prescribed range for all the five pair of medicines [ tables 26 ] dissolution test : the dissolution test for all the five paired medicines were within the permissible limits of the statutory standards [ tables 26 ] . comparative analytical evaluation of branded and branded - generic drug product alprazolam tablets ( 0.25 mg ) comparative analytical evaluation of branded and branded generic drug product cetirizine tablets ( 10 mg ) comparative analytical evaluation of branded and brandedgeneric drug product ciprofloxacin tablets ( 500 mg ) comparative analytical evaluation of branded and brandedgeneric drug product fluoxetine capsules ( 20 mg ) comparative analytical evaluation of branded / branded - generic drug product lansoprazole capsules ( 30 mg ) this is one of the first studies in india conducted to systematic evaluate the price - to - patient and retailer mark - up for the branded and branded - generic versions of the same therapeutic molecule manufactured by the same company . findings of the study revealed that there are huge mark - ups for retailer on branded - generic medicines . the retailer margin for five branded medicines studied was in the range of 25 - 30% , but for their branded - generics version manufactured by the same company it was in the range of 201 - 1016% . both versions of all five medicines cleared all the quantitative and qualitative parameters as prescribed in indian pharmacopoeia , 2007 . results of our study revealed that price - to - patient for the branded - generic version was not much less than to its branded counterpart ; branded - generic was available at 70 - 100% cost of the branded product . the study revealed that even for the branded version of both schedule and non - scheduled medicines , the retailer margin was more than the established margin , it was in the range of 25 - 30% . for the branded - generic version , the retailer margins were very large , 201 - 1016% . if the marketing is done by the company as for the " branded " version then the major mark - ups are for the company ; if the marketing and promotion are done by the retailer as in the case of branded - generic then the ptr is less , but mrp is not much different . one inherent limitation of this study is that we have tested pair of branded and branded - generic medicines that were manufactured by the same reputed company . though it is expected that both the versions should have the same quality but perception for any branded - generic is same among doctors and patients . so to start with , we have taken both branded and branded - generic products of the same company and studied not only the quality but also the price structure . findings of the present study indicate that both the branded and branded - generic versions of the five paired medicines had identical quality and they fulfilled all the criteria prescribed by the statutory standards . hence , the general notion and doubt regarding the quality of the branded - generic version of medicines needs to be erased conducting more such studies and publishing them widely.suitable changes in the drug price policy may be made to have lower prices for branded - generic versions . transparency in fixing the mrp by the manufacturer and clear guidelines for mark - ups at least for branded - generics is required in pharmaceutical trade .

infections with feline immunodeficiency virus ( fiv ) and feline leukemia virus ( felv ) have been reported from a number of countries and are important conditions in cats . felv infection is also commonly acquired via the oronasal route through mutual grooming , nursing , or sharing of dishes . the known risk factors for acquiring these infections are male sex , adulthood , and exposure to outdoors , whereas being neutered and indoor lifestyle are known protective factors . recent studies estimate a seroprevalence of 2.3% ( felv ) and 2.5% ( fiv ) in the united states ( us ) and 3.4% ( felv ) and 4.3% ( fiv ) in canada . despite decades of discovery , clinical management of cats infected with therefore , better ways to control the infections and prophylactic management is the mainstay of disease prevention strategy for these infections . a number of previous studies have suggested that the prevalence of retroviral infections in domestic cat populations varies by regions and maybe attributed to variable population density , reproductive status , age , gender , and housing conditions [ 511 ] . for the us and canada , spatial variation in fiv and felv seroprevalence has been reported in previous studies that generated data for this research [ 1 , 4 ] . here we attempt to extend the findings by applying spatial statistical methods to illustrate geographic variation in the distribution of fiv and felv infections and assess the relationship with group - level risk factors . spatial epidemiological methods are commonly used to identify , describe , and quantify spatial patterns in the distribution of health events . spatial patterns commonly of interest include trends , clustering , and detection of clusters in the occurrence of health events in a population . furthermore , geographic correlation studies can be important tools to evaluate the association of spatial or environmental risk factors with the occurrence of health events after adjusting for confounders . the identification of such spatial patterns may provide clues for further testable hypotheses about an unknown disease etiology . ecological studies , such as geographic correlation studies , are particularly valuable when an individual level association between infection and risk factors is evident and a group - level association is assessed to determine the population health impact . to this effect , spatial analysis of fiv and felv infections can be a valuable tool in epidemiological understanding of these infections . due to lack of individual level data and client confidentiality and to create meaningful units for data analysis , aggregated or area level data may be used to carry out such spatial epidemiological studies . however , the way areal units are defined can influence the results and inferences based on aggregated data . specifically , the number or size of areas used and how the area boundaries are drawn can influence spatial data analysis . this has been termed the modifiable areal unit problem ( maup ) and is a long known phenomenon [ 14 , 15 ] in the geographical literature . the maup stems from the fact that areal units are usually arbitrarily determined and can be modified to form units of different sizes or spatial arrangements . the scale effect is the variation in results obtained when the areal data comprising smaller areal units is grouped to form increasingly larger units . the zoning effect , on the other hand , is the variation in results obtained due to alternative formations of areal units where the number of areal units is constant , that is , analysis comprising the same number of areal units but different area shapes [ 14 , 17 , 18 ] . the goal of this study was to evaluate the association of seroprevalence of fiv and felv with ecological risk factors in a spatial regression model . specific objectives of the study were to examine the maup effects on ( a ) the spatial clustering of fiv and felv infections ; ( b ) the occurrence of high - risk clusters of fiv and felv infections ; and ( c ) the relationship between area level seroprevalence and risk factors in context of aggregated covariates . a dataset consisting of diagnostic test results from 29,182 cats tested for fiv and felv between august and november of the years 2004 and 2007 from the us and canada was obtained from previous cross - sectional studies [ 1 , 4 ] . the cats included in this study were conveniently sampled from veterinary clinics and animal shelters across 40 contiguous states of the us and 9 canadian provinces encompassing 641 us zip codes and canadian forward sortation areas in 346 us counties and canadian census divisions . the testing for fiv and felv was carried out in house or in laboratory employing a commercially available elisa ( snap combo felv antigen / fiv antibody , petcheck fiv antibody , and petcheck felv antigen ; idexx laboratories ) using blood , serum , or plasma . information on postal code of testing facility , type of testing facility ( clinic or shelter ) , age of the cat ( juvenile < 6 months or adult ) , sex , and neuter status ( sexually intact female , spayed female , sexually intact male , or castrated male ) , access to outdoors ( indoors or outdoors ) , and general health at time of testing ( healthy or sick ) was also retrieved from the dataset ( table 1(a ) ) . the three spatial aggregation scales of interest in this study were postal codes , counties and states . the us five - digit zip code and canadian forward sortation areas ( fsa ) were designated as postal codes , statcan ( statistics canada ) census divisions ( cds ) were defined as corresponding to us counties , and canadian provinces were defined as states . the counts of positive test results and number of tests for each area were aggregated to these three spatial aggregation scales of interest ( 641 postal codes , 346 counties , and 49 states ) . in addition , group - level risk factors , constructed from individual risk factors , included the proportion of juvenile cats ( < 6 months ) , intact males , intact females , cats that were exclusively indoors , cats tested at clinics , cats that were healthy at the time of testing , and the seroprevalence of fiv and felv . these covariates were constructed for respective scales using categories of individual data presented in table 1(a ) . in order to spatially reference the postal codes , counties , and states , the geographic coordinates ( as centroids ) of the us zip codes , counties , states , and the canadian fsas each canadian fsa was assigned to the respective county and state based on the postal code conversion file ( pccf ) available from statcan . to investigate disease clustering ( i.e. , the presence of spatial autocorrelation in the data ) , moran 's i test was applied . given the infectious nature of fiv and felv , clustering was assumed to be present . the interest in this study was to evaluate whether aggregation of data from postal code level ( where the data was collected ) to county and states had any effect on strength and presence of clustering . in this regard , the presence and strength of spatial clustering of fiv and felv infections for each level of aggregation were assessed by moran 's i test on the smoothed seroprevalence estimates using empirical bayesian smoothing . since the number of tested cats varied among the areas , smoothed seroprevalence estimates were estimated from crude seroprevalence ( number of cats testing positive / number of cats tested ) for each area using the empirical bayesian ( eb ) estimation such that the area specific seroprevalence estimates were adjusted towards the overall mean . the null hypothesis of moran 's i test states that there is no spatial autocorrelation of fiv or felv seroprevalence between areas , and the respective moran 's i coefficient summarizes the degree to which similar observations ( i.e. , seroprevalence of fiv or felv ) tend to occur near each other . the moran 's i coefficient was estimated as follows : ( 1)i = ni=1ni = jnwij(yiy)(yjy)(i=1n(yiy)2)(ijwij ) , where n : number of areas , wij : measure of spatial proximity between areas i and j , yi : poisson model based eb smoothed seroprevalence of fiv or felv in area i , yj : poisson model based eb smoothed seroprevalence of fiv or felv in area j , and y : overall eb smoothed seroprevalence . w ij is the spatial weights matrix which considers three nearest neighbours ( wij is 1 if area i and j are within a distance of three nearest neighbours and zero if otherwise ) . the moran 's i test was applied using the spdep package of statistical software r . while moran 's i summarizes the overall clustering pattern in the study area , disease cluster detection methods are used to identify the locations of clusters and thus are location specific . of various methods proposed for cluster detection , here , the maup effect on the spatial scan test was investigated with respect to fiv and felv infections . furthermore , the spatial scan test can be extended to detect clusters after adjustment for known risk factors or confounders for fiv and felv infections . therefore , the presence of statistically significant high - risk clusters of fiv ( or felv ) infection was investigated using a spatial scan test adjusted for risk factors under the poisson assumption , as implemented in satscan version 9.0 . the spatial scan test identifies potential clusters using circular windows of varying radius ( size ) and location ( area centroids ) across the study area . to apply the poisson model , it was assumed under the null hypothesis that the number of fiv or felv cases in each area followed poisson distribution with the expected number of cases in each area proportional to the covariate ( risk factor ) adjusted tested cat population . high - risk cluster detection was performed by comparing the observed number of cases within the scanning window with the expected number , that is , if cases were to be distributed randomly in space . in other words , detection of high - risk clusters would indicate the prevalence of fiv ( or felv ) inside the circular window as significantly higher than outside the window . the statistical significance of the clusters was established by monte carlo hypothesis testing using 999 monte carlo replications with a significance level set to = 5% . the significance of multiple clusters was tested sequentially conditional on the presence of the previously detected clusters such that secondary clusters were tested and reported only if the more likely clusters were significant . the size of the scanning window in the spatial scan statistic was allowed to increase from individual areas and expanded to include neighbouring areas until a maximum of 50% of the total tested population . detected clusters were visualized by plotting respective circles on a map of the study area . the characteristics of detected clusters were compared across aggregation levels to assess the maup effect . apart from describing the spatial patterns of disease in terms of clustering and cluster , geographic correlation analysis ( or spatial regression modeling ) for spatial data was carried out to quantify the effect of spatially referenced group - level risk factors on the spatial distribution of disease events , that is , fiv and felv infections [ 17 , 29 ] . while these studies are similar to ecological regression methods , it is critical to adjust for the spatial autocorrelation in the data in order to prevent type i errors , that is , providing statistically significant results when none exists . among many proposed methods for spatial regression modeling for areal data [ 17 , 29 , 31 , 32 ] , poisson distributed counts for rare disease or infections such as fiv and felv can be effectively modeled to assess its relationship with group - level risk factors using generalized linear mixed models ( glmm ) with spatially correlated random effects , also known as spatial glmm . in this study , interest was to evaluate group - level risk factors for fiv and felv infections as well as to quantify the effect of maup as change in magnitude and significance of regression parameters with spatial aggregation scale . for each aggregation level , the count of fiv and felv infections in each area was modelled as a function of the group - level covariates in a poisson regression model framework with the log of number of tested cats as the offset . prior to inclusion of covariates in the regression models , the relationship between the outcome and covariates was assessed for linearity by plotting the log of the seroprevalence of infection for both fiv and felv against the covariate using a locally weighted regression . the covariates were modelled as dichotomized variables if the relationship was deemed to be nonlinear . covariates were modeled as dichotomous variables with cut - offs for low and high categories set at median value ( 50% ) of the respective covariates . when modeled as predictor variable and not the outcome , the cut - off for categories of covariate fiv and felv seroprevalence was set at 3% , 8% and over 8% . the cut - off of 3% is the general prevalence of fiv and felv in cats in north america . since all the covariates are deemed clinically important risk factors , they were included as fixed effects in a multivariable model , with no interactions . further , the same model was fit to data at all 3 levels of aggregation ( state , county , and postal code ) to avoid any influence of the selection method or covariate(s ) exclusion in the comparison of models . for state level aggregation , covariates with sample size less than five were omitted . in order to account for spatial autocorrelation and overdispersion in the models , an exponential spatial covariance structure was introduced and the models were rerun using penalized quasilikelihood ( pql ) estimation [ 32 , 34 ] . an exponential covariance structure was based on a semivariogram fitted to the deviance residuals of the poisson regression models and was deemed biologically appropriate because , for infectious agents such as fiv and felv , areas in proximity are expected to be similar with respect to disease prevalence . the presence of overdispersion in ( nonspatial ) poisson regression models was evaluated by testing the model deviance against degrees of freedom using a distribution and a 5% significance level . multicollinearity was tested among the covariates in the multivariable model by estimating the variance inflation factor ( vif ) , and all variables with a vif value of 10 or above were considered collinear . a total of 28,914 test results were included in this study from 688 veterinary clinics and 158 animal shelters from 40 states of the us and 9 canadian provinces encompassing 346 counties and 641 postal codes . a total of 634 recorded postal codes ( out of 648 ) were accurately matched during geocoding . geographic coordinates were retrieved for 641 postal codes ( out of 648 ) for 28,914 cats ( out of 29,182 cats ) . the individual characteristics of fiv and felv infected cats and descriptive statistics of area wise counts are presented in tables i(a ) and i(b ) . overall the observed seroprevalence of fiv was higher than that of felv , 3.16% and 2.71% , respectively . the mean and variability in number of cats with positive test results for both infections and the number of cats tested increased with higher level of aggregation but decreased for seroprevalence ( table 1(b ) ) . the seroprevalence of fiv infection for postal codes , counties and states ranged from 0100% , 050% , and 013% respectively , while the seroprevalence of felv ranged from 0100% , 033% , and 020% for postal code , county , and state levels , respectively . the results of moran 's i clustering test on eb smoothed seroprevalence is presented in table 2 . moran 's i statistic indicated significant spatial clustering in seroprevalence of infection for fiv at postal code and county level aggregations ( i = 0.09 and i = 0.15 resp . , p < 0.01 ) , likewise , spatial clustering was identified for felv at postal code and county level aggregations ( i = 0.12 and 0.15 , resp . , p < 0.01 ) . at state level of aggregation no spatial clustering was detected . tables 3(a)-3(b ) and figures 1 and 2 display detailed information for all clusters identified by the spatial scan statistic . for both fiv and felv infections , however , the numbers of clusters detected for fiv and felv infections varied with the level of aggregation . for fiv infections , one cluster was detected for state , five for county , and six for postal code level aggregations . some clusters identified at postal code level were not detected at county level and state level aggregations ( table 3(a ) and figures 1(a)1(c ) ) . for felv , three clusters each for state , county , and postal code levels were identified , with location and size of the clusters slightly varying by spatial scale ( table 3(b ) and figures 2(a)2(c ) ) . figures 2(a)2(c ) indicate that felv clusters were about the same size and in the same location for postal code and county levels of aggregation , whereas clusters at the state level differed with respect to size and , more importantly , location . spatial poisson regression indicated that the seroprevalence of felv infections was observed to be lower among areas with greater proportion of cats that were young and indoors ( table 4(a ) ) . conversely , seroprevalence of felv infections was higher among areas with a greater proportion of intact males and cats tested at clinics and with a higher seroprevalence of fiv ( table 4(a ) ) . similarly , seroprevalence of fiv infection was higher among areas with a greater proportion of cats tested at clinics and with a higher seroprevalence of felv ( table 4(b ) ) . the seroprevalence of fiv , however , was lower in areas with greater proportion of intact females . associations seen at postal code and county levels may not be evident at the state level ( e.g. , percentage of juvenile cats in an area and felv ) . or conversely , associations observed at state level were not detected at lower levels ( e.g. , percentage of male intact in an area and felv ) . this study showed that commonly used spatial epidemiological methods ( moran 's i , spatial scan test , and spatial regression modeling ) are sensitive to choice of the spatial aggregation scale for analysis , that is , affected by the maup . recognizing the importance of bias due to the maup is important for the validity of spatial epidemiological inferences . however , the strength and significance of clustering varied across spatial aggregation levels . given the infectious nature of both retroviruses , areas near each other are expected to have similar seroprevalence levels . therefore , positive autocorrelation in fiv and felv seroprevalence was expected . as the data are aggregated , variations at lower levels of aggregation dissolve to form more homogenous areas in terms of population characteristics . with the postal codes aggregated to counties and states , the variability in seroprevalence estimates evident at the scale of postal code and counties likely diminished as the seroprevalence estimates were averaged ( table 1(b ) ) . generally , spatial aggregation is expected to increase spatial correlations . however , in this study the opposite effect was observed , and the spatial autocorrelation present at postal and county levels disappeared at state level . this may imply that the biological processes which are associated with the clustering of infected cats at local levels ( i.e. , postal codes and counties ) become irrelevant or unobservable at higher aggregation levels ( i.e. , states ) . it is important to note that there is a random aspect to the effects of the maup and it may be difficult to generalize about how different datasets with different spatial units are affected by the maup . furthermore , the aggregation process itself may induce positive spatial autocorrelation , particularly if the aggregation process allows overlapping units such as postal code to form counties . unfortunately , not all postal code areas or counties in the us and canada were sampled for this study and moran 's i test was based on a neighbourhood specification of three nearest neighbours . areas too distant from a biological perspective on infection , which would tend to aggravate variability and reduce autocorrelation at lower levels of aggregation . evidence of high - risk areas for fiv and felv infections as detected by the spatial scan test adjusted for known confounders suggests that yet unknown spatial factors may exist . this study also indicates that the results from the spatial scan test can be influenced by spatial aggregation as evident from the difference in size , number , and location of clusters for both fiv and felv infections . despite differences in cluster characteristics with respect to size and location across aggregation levels for felv , no clusters were detected in the western parts of united states and canada indicating that these areas had lower prevalence of infection compared to the rest of the study area . the results at county and postal code area levels were similar with respect to cluster size and locations . the sampled counties and postal codes were not very different with respect to population characteristics and thus may be insensitive to aggregation effects . however , it is most likely an artificial effect as most counties had only a few postal codes sampled within them . while multiple clusters were detected for fiv at the postal code level , these were not detected at higher aggregation levels ( figures 1(a)1(c ) ) . spatial aggregation reduced the sample size from 641 postal codes to 346 counties and 49 states ( or provinces ) in this study . aggregation of data may smooth out local effects but may also lead to reduced power to detect small clusters while stabilizing rates that may be unstable in smaller areas due to smaller at - risk - populations in the denominator [ 39 , 40 ] . the results from spatial poisson regression modeling indicate that the seroprevalence of both infections is higher in areas with the greater proportion of cats tested at clinics than at shelters . although the seroprevalence of fiv and felv in shelter cat populations mirrors that of cat populations served by veterinary clinics , the reasons for testing may be different . testing in shelters is driven by housing considerations and potential for adoption , whereas at clinics mostly sick cats are tested . thus , the seroprevalence estimates in populations tested at clinics may be inflated . an increase in seroprevalence of felv this is expected since both infections share similar risk factors and as a result would have similar infection rates . furthermore , the seroprevalence of felv in an area was negatively associated with a higher proportion of young cats , indoor cats , and neutered males . consequently , the seroprevalence would be higher in areas with greater proportions of adults , outdoor cats , and intact cats due to social interactions related to roaming , breeding , and fighting . therefore , the areas populated with cats of these characteristics can be expected to harbour cats with higher risk of acquiring retroviral infections . areas with greater proportions of intact female cats had a lower seroprevalence for fiv than areas with greater proportions of spayed female cats . this finding seems to be counterintuitive from a biological perspective , as similar to areas with greater proportions of intact males ; populations with greater proportions of intact females might be expected to be more susceptible to acquire an infection as a result of higher probabilities of animals fighting . however , the predictors that are derived variables ( variables constructed as summaries of individual characteristics ) in the group level analysis can not distinguish the individual - level effect of the variable from its contextual or group level effect . derived variables are constructed mathematically by summarizing the individual characteristics in a group , for example , proportion of males in an area . the significance and magnitude of associations between health status and risk factors ( or predictor variables ) are governed by the scale of spatial aggregation . the associations observed at one scale should be used with caution when inferences are made at another scale . except for fiv and felv seroprevalence , this study did not identify any covariate consistently associated with the outcome across all three aggregation levels . the geographic scales on which these two variables are meaningful factors probably include scales larger than postal code . this is likely true , since veterinary clinics generally service areas that overlap several postal code areas or occasionally across county barriers . for other variables , the choice of the aggregation scale seems to affect the significance and magnitude of observed associations . generally , most of the predictor variables were only significant at lower levels of aggregation . suggesting that seroprevalence of fiv and felv at higher levels of aggregation depend on further group - level factors not considered in this study . it is important that this spatial scale dependence is not overinterpreted as a sole maup effect as multivariable analysis is a complex subject and nevertheless can be prone to missing but confounding variables . this study utilized an ecological regression framework based on covariates as derived variables from individual level data . it is necessary to be cautious in extrapolating these findings to the individual level due to the potential for ecological bias . currently , there are no solutions to fully overcome the effects of maup and related methodological issues have not yet been adequately addressed . recommendations have been made to minimize maup effects in statistical inference by analyzing the aggregated covariates in hierarchical levels of areal units from the finest spatial resolution possible to a coarser resolution , verifying consistent model results across different scales , avoiding ecological fallacy , collecting data at the scale at which inferences is to be made , and using scale invariant statistics to make inferences [ 17 , 4446 ] . this study demonstrated the importance of study design in the context of spatial epidemiological studies . inference from spatial epidemiological studies dealing with aggregated data could potentially be affected by the modifiable areal unit problem ( maup ) . the maup can result in overlooking or conversely overstating the effect of risk factors and influence statistics designed to test for clustering and clusters . in the present study of fiv and felv seroprevalence among cats across the us and canada it was found that disease clusters may become unidentifiable when data are aggregated . therefore , it is of utmost importance that investigators define the appropriate scale for data collection and analysis with respect to their research questions .

the knowledge of the spatial distribution feline immunodeficiency virus and feline leukemia virus infections , which are untreatable , can inform on their risk factors and high - risk areas to enhance control . however , when spatial analysis involves aggregated spatial data , results may be influenced by the spatial scale of aggregation , an effect known as the modifiable areal unit problem ( maup ) . in this study , area level risk factors for both infections in 28,914 cats tested with elisa were investigated by multivariable spatial poisson regression models along with maup effect on spatial clustering and cluster detection ( for postal codes , counties , and states ) by moran 's i test and spatial scan test , respectively . the study results indicate that the significance and magnitude of the association of risk factors with both infections varied with aggregation scale . further more , moran 's i test only identified spatial clustering at postal code and county levels of aggregation . similarly , the spatial scan test indicated that the number , size , and location of clusters varied over aggregation scales . in conclusion , the association between infection and area was influenced by the choice of spatial scale and indicates the importance of study design and data analysis with respect to specific research questions .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion

infections with feline immunodeficiency virus ( fiv ) and feline leukemia virus ( felv ) have been reported from a number of countries and are important conditions in cats . the known risk factors for acquiring these infections are male sex , adulthood , and exposure to outdoors , whereas being neutered and indoor lifestyle are known protective factors . a number of previous studies have suggested that the prevalence of retroviral infections in domestic cat populations varies by regions and maybe attributed to variable population density , reproductive status , age , gender , and housing conditions [ 511 ] . here we attempt to extend the findings by applying spatial statistical methods to illustrate geographic variation in the distribution of fiv and felv infections and assess the relationship with group - level risk factors . spatial patterns commonly of interest include trends , clustering , and detection of clusters in the occurrence of health events in a population . furthermore , geographic correlation studies can be important tools to evaluate the association of spatial or environmental risk factors with the occurrence of health events after adjusting for confounders . ecological studies , such as geographic correlation studies , are particularly valuable when an individual level association between infection and risk factors is evident and a group - level association is assessed to determine the population health impact . due to lack of individual level data and client confidentiality and to create meaningful units for data analysis , aggregated or area level data may be used to carry out such spatial epidemiological studies . however , the way areal units are defined can influence the results and inferences based on aggregated data . specifically , the number or size of areas used and how the area boundaries are drawn can influence spatial data analysis . this has been termed the modifiable areal unit problem ( maup ) and is a long known phenomenon [ 14 , 15 ] in the geographical literature . the goal of this study was to evaluate the association of seroprevalence of fiv and felv with ecological risk factors in a spatial regression model . specific objectives of the study were to examine the maup effects on ( a ) the spatial clustering of fiv and felv infections ; ( b ) the occurrence of high - risk clusters of fiv and felv infections ; and ( c ) the relationship between area level seroprevalence and risk factors in context of aggregated covariates . a dataset consisting of diagnostic test results from 29,182 cats tested for fiv and felv between august and november of the years 2004 and 2007 from the us and canada was obtained from previous cross - sectional studies [ 1 , 4 ] . the cats included in this study were conveniently sampled from veterinary clinics and animal shelters across 40 contiguous states of the us and 9 canadian provinces encompassing 641 us zip codes and canadian forward sortation areas in 346 us counties and canadian census divisions . information on postal code of testing facility , type of testing facility ( clinic or shelter ) , age of the cat ( juvenile < 6 months or adult ) , sex , and neuter status ( sexually intact female , spayed female , sexually intact male , or castrated male ) , access to outdoors ( indoors or outdoors ) , and general health at time of testing ( healthy or sick ) was also retrieved from the dataset ( table 1(a ) ) . the three spatial aggregation scales of interest in this study were postal codes , counties and states . the us five - digit zip code and canadian forward sortation areas ( fsa ) were designated as postal codes , statcan ( statistics canada ) census divisions ( cds ) were defined as corresponding to us counties , and canadian provinces were defined as states . the counts of positive test results and number of tests for each area were aggregated to these three spatial aggregation scales of interest ( 641 postal codes , 346 counties , and 49 states ) . in addition , group - level risk factors , constructed from individual risk factors , included the proportion of juvenile cats ( < 6 months ) , intact males , intact females , cats that were exclusively indoors , cats tested at clinics , cats that were healthy at the time of testing , and the seroprevalence of fiv and felv . in order to spatially reference the postal codes , counties , and states , the geographic coordinates ( as centroids ) of the us zip codes , counties , states , and the canadian fsas each canadian fsa was assigned to the respective county and state based on the postal code conversion file ( pccf ) available from statcan . , the presence of spatial autocorrelation in the data ) , moran 's i test was applied . the interest in this study was to evaluate whether aggregation of data from postal code level ( where the data was collected ) to county and states had any effect on strength and presence of clustering . in this regard , the presence and strength of spatial clustering of fiv and felv infections for each level of aggregation were assessed by moran 's i test on the smoothed seroprevalence estimates using empirical bayesian smoothing . since the number of tested cats varied among the areas , smoothed seroprevalence estimates were estimated from crude seroprevalence ( number of cats testing positive / number of cats tested ) for each area using the empirical bayesian ( eb ) estimation such that the area specific seroprevalence estimates were adjusted towards the overall mean . the null hypothesis of moran 's i test states that there is no spatial autocorrelation of fiv or felv seroprevalence between areas , and the respective moran 's i coefficient summarizes the degree to which similar observations ( i.e. the moran 's i coefficient was estimated as follows : ( 1)i = ni=1ni = jnwij(yiy)(yjy)(i=1n(yiy)2)(ijwij ) , where n : number of areas , wij : measure of spatial proximity between areas i and j , yi : poisson model based eb smoothed seroprevalence of fiv or felv in area i , yj : poisson model based eb smoothed seroprevalence of fiv or felv in area j , and y : overall eb smoothed seroprevalence . the moran 's i test was applied using the spdep package of statistical software r . while moran 's i summarizes the overall clustering pattern in the study area , disease cluster detection methods are used to identify the locations of clusters and thus are location specific . of various methods proposed for cluster detection , here , the maup effect on the spatial scan test was investigated with respect to fiv and felv infections . furthermore , the spatial scan test can be extended to detect clusters after adjustment for known risk factors or confounders for fiv and felv infections . therefore , the presence of statistically significant high - risk clusters of fiv ( or felv ) infection was investigated using a spatial scan test adjusted for risk factors under the poisson assumption , as implemented in satscan version 9.0 . the spatial scan test identifies potential clusters using circular windows of varying radius ( size ) and location ( area centroids ) across the study area . to apply the poisson model , it was assumed under the null hypothesis that the number of fiv or felv cases in each area followed poisson distribution with the expected number of cases in each area proportional to the covariate ( risk factor ) adjusted tested cat population . high - risk cluster detection was performed by comparing the observed number of cases within the scanning window with the expected number , that is , if cases were to be distributed randomly in space . in other words , detection of high - risk clusters would indicate the prevalence of fiv ( or felv ) inside the circular window as significantly higher than outside the window . the significance of multiple clusters was tested sequentially conditional on the presence of the previously detected clusters such that secondary clusters were tested and reported only if the more likely clusters were significant . the size of the scanning window in the spatial scan statistic was allowed to increase from individual areas and expanded to include neighbouring areas until a maximum of 50% of the total tested population . detected clusters were visualized by plotting respective circles on a map of the study area . apart from describing the spatial patterns of disease in terms of clustering and cluster , geographic correlation analysis ( or spatial regression modeling ) for spatial data was carried out to quantify the effect of spatially referenced group - level risk factors on the spatial distribution of disease events , that is , fiv and felv infections [ 17 , 29 ] . while these studies are similar to ecological regression methods , it is critical to adjust for the spatial autocorrelation in the data in order to prevent type i errors , that is , providing statistically significant results when none exists . among many proposed methods for spatial regression modeling for areal data [ 17 , 29 , 31 , 32 ] , poisson distributed counts for rare disease or infections such as fiv and felv can be effectively modeled to assess its relationship with group - level risk factors using generalized linear mixed models ( glmm ) with spatially correlated random effects , also known as spatial glmm . in this study , interest was to evaluate group - level risk factors for fiv and felv infections as well as to quantify the effect of maup as change in magnitude and significance of regression parameters with spatial aggregation scale . for each aggregation level , the count of fiv and felv infections in each area was modelled as a function of the group - level covariates in a poisson regression model framework with the log of number of tested cats as the offset . prior to inclusion of covariates in the regression models , the relationship between the outcome and covariates was assessed for linearity by plotting the log of the seroprevalence of infection for both fiv and felv against the covariate using a locally weighted regression . covariates were modeled as dichotomous variables with cut - offs for low and high categories set at median value ( 50% ) of the respective covariates . when modeled as predictor variable and not the outcome , the cut - off for categories of covariate fiv and felv seroprevalence was set at 3% , 8% and over 8% . further , the same model was fit to data at all 3 levels of aggregation ( state , county , and postal code ) to avoid any influence of the selection method or covariate(s ) exclusion in the comparison of models . an exponential covariance structure was based on a semivariogram fitted to the deviance residuals of the poisson regression models and was deemed biologically appropriate because , for infectious agents such as fiv and felv , areas in proximity are expected to be similar with respect to disease prevalence . the presence of overdispersion in ( nonspatial ) poisson regression models was evaluated by testing the model deviance against degrees of freedom using a distribution and a 5% significance level . a total of 28,914 test results were included in this study from 688 veterinary clinics and 158 animal shelters from 40 states of the us and 9 canadian provinces encompassing 346 counties and 641 postal codes . geographic coordinates were retrieved for 641 postal codes ( out of 648 ) for 28,914 cats ( out of 29,182 cats ) . the mean and variability in number of cats with positive test results for both infections and the number of cats tested increased with higher level of aggregation but decreased for seroprevalence ( table 1(b ) ) . the seroprevalence of fiv infection for postal codes , counties and states ranged from 0100% , 050% , and 013% respectively , while the seroprevalence of felv ranged from 0100% , 033% , and 020% for postal code , county , and state levels , respectively . the results of moran 's i clustering test on eb smoothed seroprevalence is presented in table 2 . moran 's i statistic indicated significant spatial clustering in seroprevalence of infection for fiv at postal code and county level aggregations ( i = 0.09 and i = 0.15 resp . , p < 0.01 ) , likewise , spatial clustering was identified for felv at postal code and county level aggregations ( i = 0.12 and 0.15 , resp . at state level of aggregation no spatial clustering was detected . tables 3(a)-3(b ) and figures 1 and 2 display detailed information for all clusters identified by the spatial scan statistic . for both fiv and felv infections , however , the numbers of clusters detected for fiv and felv infections varied with the level of aggregation . for fiv infections , one cluster was detected for state , five for county , and six for postal code level aggregations . some clusters identified at postal code level were not detected at county level and state level aggregations ( table 3(a ) and figures 1(a)1(c ) ) . for felv , three clusters each for state , county , and postal code levels were identified , with location and size of the clusters slightly varying by spatial scale ( table 3(b ) and figures 2(a)2(c ) ) . figures 2(a)2(c ) indicate that felv clusters were about the same size and in the same location for postal code and county levels of aggregation , whereas clusters at the state level differed with respect to size and , more importantly , location . spatial poisson regression indicated that the seroprevalence of felv infections was observed to be lower among areas with greater proportion of cats that were young and indoors ( table 4(a ) ) . similarly , seroprevalence of fiv infection was higher among areas with a greater proportion of cats tested at clinics and with a higher seroprevalence of felv ( table 4(b ) ) . associations seen at postal code and county levels may not be evident at the state level ( e.g. this study showed that commonly used spatial epidemiological methods ( moran 's i , spatial scan test , and spatial regression modeling ) are sensitive to choice of the spatial aggregation scale for analysis , that is , affected by the maup . recognizing the importance of bias due to the maup is important for the validity of spatial epidemiological inferences . however , the strength and significance of clustering varied across spatial aggregation levels . as the data are aggregated , variations at lower levels of aggregation dissolve to form more homogenous areas in terms of population characteristics . with the postal codes aggregated to counties and states , the variability in seroprevalence estimates evident at the scale of postal code and counties likely diminished as the seroprevalence estimates were averaged ( table 1(b ) ) . however , in this study the opposite effect was observed , and the spatial autocorrelation present at postal and county levels disappeared at state level . this may imply that the biological processes which are associated with the clustering of infected cats at local levels ( i.e. it is important to note that there is a random aspect to the effects of the maup and it may be difficult to generalize about how different datasets with different spatial units are affected by the maup . furthermore , the aggregation process itself may induce positive spatial autocorrelation , particularly if the aggregation process allows overlapping units such as postal code to form counties . unfortunately , not all postal code areas or counties in the us and canada were sampled for this study and moran 's i test was based on a neighbourhood specification of three nearest neighbours . areas too distant from a biological perspective on infection , which would tend to aggravate variability and reduce autocorrelation at lower levels of aggregation . evidence of high - risk areas for fiv and felv infections as detected by the spatial scan test adjusted for known confounders suggests that yet unknown spatial factors may exist . this study also indicates that the results from the spatial scan test can be influenced by spatial aggregation as evident from the difference in size , number , and location of clusters for both fiv and felv infections . despite differences in cluster characteristics with respect to size and location across aggregation levels for felv , no clusters were detected in the western parts of united states and canada indicating that these areas had lower prevalence of infection compared to the rest of the study area . the results at county and postal code area levels were similar with respect to cluster size and locations . the sampled counties and postal codes were not very different with respect to population characteristics and thus may be insensitive to aggregation effects . however , it is most likely an artificial effect as most counties had only a few postal codes sampled within them . spatial aggregation reduced the sample size from 641 postal codes to 346 counties and 49 states ( or provinces ) in this study . aggregation of data may smooth out local effects but may also lead to reduced power to detect small clusters while stabilizing rates that may be unstable in smaller areas due to smaller at - risk - populations in the denominator [ 39 , 40 ] . the results from spatial poisson regression modeling indicate that the seroprevalence of both infections is higher in areas with the greater proportion of cats tested at clinics than at shelters . although the seroprevalence of fiv and felv in shelter cat populations mirrors that of cat populations served by veterinary clinics , the reasons for testing may be different . thus , the seroprevalence estimates in populations tested at clinics may be inflated . an increase in seroprevalence of felv this is expected since both infections share similar risk factors and as a result would have similar infection rates . furthermore , the seroprevalence of felv in an area was negatively associated with a higher proportion of young cats , indoor cats , and neutered males . consequently , the seroprevalence would be higher in areas with greater proportions of adults , outdoor cats , and intact cats due to social interactions related to roaming , breeding , and fighting . however , the predictors that are derived variables ( variables constructed as summaries of individual characteristics ) in the group level analysis can not distinguish the individual - level effect of the variable from its contextual or group level effect . the significance and magnitude of associations between health status and risk factors ( or predictor variables ) are governed by the scale of spatial aggregation . for other variables , the choice of the aggregation scale seems to affect the significance and magnitude of observed associations . generally , most of the predictor variables were only significant at lower levels of aggregation . suggesting that seroprevalence of fiv and felv at higher levels of aggregation depend on further group - level factors not considered in this study . it is important that this spatial scale dependence is not overinterpreted as a sole maup effect as multivariable analysis is a complex subject and nevertheless can be prone to missing but confounding variables . this study utilized an ecological regression framework based on covariates as derived variables from individual level data . recommendations have been made to minimize maup effects in statistical inference by analyzing the aggregated covariates in hierarchical levels of areal units from the finest spatial resolution possible to a coarser resolution , verifying consistent model results across different scales , avoiding ecological fallacy , collecting data at the scale at which inferences is to be made , and using scale invariant statistics to make inferences [ 17 , 4446 ] . this study demonstrated the importance of study design in the context of spatial epidemiological studies . inference from spatial epidemiological studies dealing with aggregated data could potentially be affected by the modifiable areal unit problem ( maup ) . the maup can result in overlooking or conversely overstating the effect of risk factors and influence statistics designed to test for clustering and clusters . therefore , it is of utmost importance that investigators define the appropriate scale for data collection and analysis with respect to their research questions .

the chemistry of heavier carbene analogues has attracted the attention of both experimentally and theoretically oriented chemists over the last decades . the fundamental differences in electronic ground states , reactivities , and structures between carbenes and their heavier counterparts are certainly a major reason for this attraction . heavy tetrylenes usually possess a singlet ground state with an increasing singlet triplet gap with higher atomic number . responsible for this ground - state preference is a progressing reluctance to form hybrid orbitals . the s - electrons of the thus preferred ( ns)(np ) configuration remain paired . as a consequence of this , dimerization of divalent species most heavier tetrylene dimers do not feature -bonds , as common for olefins , but rather exist as dimeric donor acceptor adducts . attachment of electropositive substituents to the divalent group 14 atoms forces some mixing of their s- and p - orbitals . this way the singlet triplet gap can significantly be diminished . sekiguchi s distannene ( bu2mesi)2sn = sn(simebu2)2 , which , despite rather bulky groups on the tin atoms , does not dissociate into monomers in solution , is a good example for this behavior . descending group 14 further to lead , the reluctance to form dimeric compounds becomes even more pronounced . this is nicely illustrated by the difference between bis[tris(trimethylsilyl)silyl]tin and the analogous lead compound . while both compounds exist as monomers in solution , the stannylene crystallizes as a distannene , while the plumbylene retains its monomeric structure in the solid state . recently , we could show that the bidentate tetrakis(trimethylsilyl)tetramethyltetrasilanylene ligand can be employed to stabilize divalent tin and lead compounds . these can be isolated either as the respective base adducts ( 1 and 2 ) or as dimers ( 3 and 4 ) . the dimeric stannylene and plumbylene compounds 3 and 4 exhibit considerable structural differences . while the tin compound 3 is the result of a dimerization rearrangement process of a disilylated stannylene and exists as an endocyclic bicyclic distannene , the plumbylene appears as a monomer in solution but crystallizes as a weak donor we report on an extension of these studies to explore the coordination chemistry of disilylated stannylenes and plumbylenes with early transition metals and in particular with group 4 metallocenes . only few examples of related compounds are known so far . in a seminal study piers and co more recently reactions of c , n - chelated tin(ii ) and lead(ii ) compounds to zirconocene were reported by ruzicka and co - workers . in the latter case the employed plumbylene decomposed to elemental lead and free ligand during the reaction , thus no compound with a zr pb bond was observed . in this context also the formation of a hafnocene silylene complex , reported recently by sekiguchi and co - workers , should be noted . that the heavier tetrylenes can display quite different bonding motifs to transition metals for the synthesis of group 4 metallocene plumbylene and stannylene complexes a new general approach was sought . in the mentioned examples , the zirconocene stannylene complexes were prepared by warming negishi zirconocene cp2zrcl22buli in the presence of 2 equiv of stannylene from 80 c to room temperature , thus limiting the scope of the reaction to zirconium . alternatively , we found that reductions of group 4 metallocene dichlorides with magnesium in the presence of stannylene or plumbylene phosphine adducts ( 1 and 2 ) provided smooth conversion to the desired complexes ( 510 ) ( scheme 1 ) . isolated yields after crystallization from pentane of these highly colored compounds were above 80% . the plumbylene complexes of titanocene ( 8) , zirconocene ( 9 ) , and hafnocene ( 10 ) represent the first examples of compounds with group 4 metal in general it should be noted that the number of known plumbylene transition - metal complexes is quite small . by reaction of 1,2-dipotassiodisilanes and -digermanes with metallocene dichlorides , we recently succeeded in the synthesis of group 4 disilene and digermene complexes . when this reaction , however , was carried out with the respective 1,2-dipotassiodistannane , instead of the expected distannene complexes , metallacyclotetrastannanes were isolated as the only tin containing products . this behavior of tin was attributed to its greater preference for the divalent state compared to its lighter congeneres . as the distannene unit of 3 ( scheme 2 ) is held together by two bridging tetrasilanylene ligands , we reasoned that the dissociation to stannylenes might be blocked , and thus an equimolar amount of 3 could serve as a precursor for a distannene complex . however , when cp2hfcl2 was reduced with magnesium in the presence of an equimolar amount of 3 , the only pentane soluble product was the hafnocene bis(stannylene ) complex 11 ( scheme 2 ) . its formation is apparently facilitated by the reversible rearrangement of 3 to the exocyclic distannene isomer , which is the direct dimerization product of the monomeric stannylene 12 ( table 2 ) . coordination of the exocyclic distannene to the hafnocene would then favor the distannylene over the distannene complex as observed previously . complex 11 was isolated in about 60% yield as thin almost black needles . briefly mentioned the possibility of replacing just one stannylene ligand in their zirconocene bis(stannylene ) complex with pme3 , but no detailed information on this reaction was provided . reaction of 11 with 2 equiv of pet3 in fact led to selective formation of 7 and 1 ( scheme 2 ) . reaction of 3 with cp2ticl2 and magnesium yielded the titanocene bis(stannylene ) complex 13 ( scheme 2 ) , but in this case , starting material 3 was contaminated with kn(sime3)2 leading in addition to the formation of the trivalent titanium compound cp2tin(sime3)2 ( 14 ) . an interesting aspect of these reactions is that attempts to react 3 with excess metallocene dichloride did not lead to monostannylene complexes but selectively to the distannylene compounds . under very similar conditions , employing magnesium reduction of hafnocene dichloride in the presence of 2 equiv of 4 even the hafnocene bis(plumbylene ) complex 15 could be obtained in moderate yield ( scheme 3 ) . again , addition of pet3 to 15 led to the selective formation of plumbylene phosphine adduct 2 and hafnocene complex 10 . due to the very high solubility in pentane of both 11 and 15 , they were difficult to isolate , and unfortunately only rather low - quality crystals could be obtained and used for x - ray diffraction experiments . a reaction of cp2zrcl2 and magnesium with a substoichiometric amount of 4 ( scheme 4 ) was carried out to check whether the preference for the formation of ditetrylene complexes extends to lead . the formation of the zirconocene plumbylene complex 16 , where the vacant coordination site at zirconium is occupied by a thf molecule , showed this not to be the case . in order to assess the general applicability of this simple access to group 4 metal tetrylenes , we reacted tacl5 as a group 5 compound with magnesium in the presence of stannylene adduct 1 ( scheme 5 ) . the successful formation of a stannylene complex of tacl3 , 17 , was confirmed by nmr spectroscopy , and a low - quality crystal structure , which revealed a distorted octahedral coordination geometry around tantalum with two trans - oriented pet3 ligands . distannene 3 exhibits a typical distannene sn nmr shift of 545 ppm , strongly indicating the persistence of the sn = sn double bond in solution . sn bond in solution is the presence of sn satellites in the sn nmr spectrum of 3 . the coupling constant of j(sn / sn ) = 1240 hz is relatively small compared to a value of 2930 hz found for masamune s tetraaryldistannene , suggesting only weak bonding of donor acceptor type . by coordination of a phosphine ligand to the stannylene ( 1 ) , electron octet configuration is achieved . therefore , chemical shifts far upfield from those expected for the free stannylene 12 are observed . in 1 the sn resonance was found at 224 ppm , consistent with the very recent report by escudi and co - workers for the nhc adduct of bis[tris(trimethylsilyl)silyl]tin ( = 197 ppm ) . the sn nmr signal of 1 is split into a doublet by the adjacent p nucleus with a coupling constant of 2220 hz . the group 4 metallocene stannylene complexes the chemical shift of titanocene complex 5 was found to be 1635 ppm with the signal being a doublet with jsnp = 276 hz coupling to phosphorus . the respective zirconocene and hafnocene complexes 6 and 7 exhibit their sn signals at considerably higher field at 1263 ppm ( 6 , jpsn = 132 hz ) and 1080 ppm ( 7 , jpsn = 92 hz ) with smaller coupling constants . the same trend can also be observed in the p spectra of these complexes , as the chemical shift ranges from 50.2 ppm in the titanocene complex ( 5 ) down to 38.5 ppm for zirconium ( 6 ) to finally 34.8 ppm in the hafnium case ( 7 ) ( table 1 ) . nmr spectroscopy thus clearly suggests an increasing degree of -back - donation when descending group 4 . differences in the published values of tabulated covalent radii of elements lead to different sums . obtained by searching for m for the hafnium bis(stannylene ) complex 11 the sn nmr resonance was found at a much lower field at 1785 ppm . this is in good agreement with the value of 1677 ppm observed by piers and co - workers for their zirconocene bis(stannylene ) complex . the zirconocene bis(stannylene ) complex published by ruzicka and co - workers on the other hand resonates considerably upfield at 923 ppm . however , the different behavior of the latter can be explained by intramolecular donation of electron density into the empty tin p - orbital from the attached amino groups of the parent stannylene . the difference between the sn nmr chemical shifts of 7 and 11 can be rationalized on the basis of -back - donation . in 7 there is one phosphine and one stannylene ligand present , of which the first can be considered to be mainly a -donor . for this reason -back - donation to tin is enhanced in this system , whereas in 11 the situation can be described as competition of two -donor -acceptor ligands for the same electron pair . therefore , the electronic environment approaches that of the free stannylene 12 and exhibits the pronounced downfield shift . while it is not completely clear how valid a comparison of the sn nmr shift of the tantalum compound 17 with the group 4 metallocene complexes of the same stannylene is , it seems fair to state that the chemical shift of = 1985 ppm is in the same region . the associated downfield shift compared to = 1080 ppm found for 7 , which contains also a third row transition metal and a phosphine ligand , is likely caused by a combination of several factors . together with the fact that compound 17 is a 14 electron complex , the electronegative chloride substituents further diminish electron density at the metal . this certainly affects the ability of the tantalum atom to engage in back - donation . four signals in the si nmr spectrum of complex 11 indicate the equivalence of both five - membered rings in the complex . however , the two faces of each ring are not equivalent as two signals for the four sime3 groups were observed . the same molecular symmetry was also derived from the respective h and c nmr spectra . this face differentiation suggests hindered rotation around the zr sn bond in compound 11 . the pb nmr spectroscopic results for the plumbylene complexes 810 show a similar trend as observed for their stannylene counterparts ( 57 ) . a steady decrease in the chemical shift value descending group 4 was found : titanocene plumbylene complex 8 resonates at = 5299 ppm , zirconocene complex 9 at = 4165 ppm , and finally hafnocene plumbylene 10 at = 3462 ppm . the expected splitting into doublets caused by coupling to the p nucleus of the phosphine ligand was not observed . the weaker bonds between pb and the respective metal , compared to the analogous stannylene complexes , in addition to the fact that the pb signals are comparably broad [ values for full width at half - maximum ( fwhm ) range from 130 to 220 hz ] seem to impede the observation of the jpbp coupling . p nmr spectroscopic results for compounds 810 parallel the behavior observed for their lighter tin congeners . the p nmr shifts are = 57.6 , 46.3 , and 45.5 ppm for 810 , respectively . the observed trend points again to a higher degree of back - bonding between the group 4 metal and pb in zr and hf compounds 9 and 10 compared to titanocene complex 8 . the dependence of the m e back - bonding from the additional phosphine ligands can be estimated from a comparison of the pb nmr chemical shifts of the plumbylene zirconocene 9( = 4165 ppm ) with the respective thf adduct 16 ( = 5770 ) . a downfield shift of ca . pb = 1600 ppm illustrates the superior electron - donating ability of the phosphine compared to thf . compared to a pb nmr chemical shift of = 3587 ppm reported for [ fe(co)4]4pb , the strongly downfield shifted resonances between = 34625770 ppm for compounds 810 and 16 seem to indicate a marked plumbylene character . however , these chemical shifts have to be seen in the context of the free plumbylene , 18 , for which an extremely downfield shifted pb resonance at = 19516 ppm was found . compared to this , the plumbylene character of 810 and 16 seems to be not so pronounced . the increasing electronic saturation of the pb atom also can be observed in the si nmr spectra . in 8 the central silicon atoms resonate at = 16 ppm . with stronger -back - bonding from the d transition metal to the plumbylene , this resonance shifts upfield to = 42 and 54 ppm for 9 and 10 , respectively . consistent with this argumentation , the comparison of 9 and 16 revealed a downfield shift of the si resonance of the silicon attached to pb from = 42 ppm for 9 to = 37 ppm for 16 . the si signals for the more remote silicon atoms in 810 are found at almost identical positions , suggesting a very similar chemical environment . for 810 two different resonances for the trimethylsilyl groups in contrast to this , only one sime3 signal was observed for 16 , which suggests dissociation of thf , which can also be concluded from broadened signals in the respective h spectrum . for hafnocene bis(plumbylene ) complex 15 only h , c , and si nmr data could be obtained . the spectra for these nuclei are very similar to those of 11 and consistent with the proposed structure . as all six phosphine tetrylene complexes ( 510 ) are isostructural , only one example of each group is shown [ figure 1 : 7 and figure 2 : 10 ; depictions of the others ( 5 , 6 , 8 , and 9 ) are available in the supporting information ( figures s1s4 ) ] . compounds 510 feature planar geometries around the tetrel(ii ) center and short transition - metal tetrel(ii ) bond lengths . the sums of bond angles around tin or lead in all six examples match 360 almost exactly with a maximum deviation of 0.5. the group 4e ( e = sn or pb ) bond lengths are significantly shorter than the sum of tabulated covalent radii or respective single bonds in the case of tin ( table 1 ) . for lead this comparison can not be made , as compounds containing ti , zr , or hf bonds to pb have not been reported so far . the x - ray crystallographic study strongly supports the conclusions drawn from the nmr observations with respect to the extent of bond order between the group 4 transition metals and tin or lead . interesting in this respect is also the p transition - metal e ( e = sn or pb ) angle of 90 in all compounds that ensures non - disturbance of the transition - metal interaction by the orthogonally coordinated phosphine ligand . molecular structure of 7 ( thermal ellipsoid plot drawn at the 30% probability level ) . hydrogen atoms omitted for clarity ( bond lengths in , angles in ) . hf(1)p(1 ) 2.663(3 ) , hf(1)sn(1 ) 2.7585(11 ) , p(1)c(13 ) 1.826(9 ) , si(1)si(2 ) 2.356(4 ) , si(1)sn(1 ) 2.626(2 ) , si(2)c(17 ) 1.889(10 ) , si(4)sn(1 ) 2.622(3 ) , p(1)hf(1)sn(1 ) 90.53(7 ) , si(4)sn(1)si(1 ) 99.41(8 ) , si(4)sn(1)hf(1 ) 129.95(6 ) , si(1)sn(1)hf(1 ) 130.25(6 ) . molecular structure of 10 ( thermal ellipsoid plot drawn at the 30% probability level ) . hydrogen atoms omitted for clarity ( bond lengths in , angles in ) . hf(1)p(1 ) 2.670(5 ) , hf(1)pb(1 ) 2.7927(12 ) , p(1)c(11 ) 1.830(19 ) , pb(1)si(4 ) 2.690(5 ) , pb(1)si(1 ) 2.705(4 ) , si(1)si(2 ) 2.362(7 ) , si(2)c(17 ) 1.889(17 ) , c(15)c(16 ) 1.55(3 ) , p(1)hf(1)pb(1 ) 90.13(12 ) , si(4)pb(1)si(1 ) 98.24(14 ) , si(4)pb(1)hf(1 ) 130.62(10 ) , si(1)pb(1)hf(1 ) 130.81(10 ) . the crystals obtained of hafnocene bis(stannylene ) 11 and hafnocene bis(plumbylene ) 15 were not of high enough quality to permit a detailed structural discussion , therefore no metrical data can be given . but these structures ( figures s5 and s6 , supporting information ) still serve as proof for the atom connectivity and therefore validate the assignments made based on nmr spectroscopy . the crystals containing titanocene bis(stannylene ) 13 ( figure 3 ) together with cp2tin(sime3)2 ( 14 , figure 4 ) in a 1:1 ratio were of much better quality . the bond lengths of ti sn in 13 , with 2.71 and 2.72 , reflect the diminished degree of back - bonding compared to 5 ( 2.69 ) . the structure of compound 14 is also interesting as it represents a rare example of a structurally characterized titanocene amide with ti in the oxidation state + 3 . molecular structure of 13 ( thermal ellipsoid plot drawn at the 30% probability level ) . hydrogen atoms omitted for clarity ( bond lengths in , angles in ) . ti(1)sn(1 ) 2.7122(13 ) , ti(1)sn(2 ) 2.7154(14 ) , sn(1)si(4 ) 2.6399(19 ) , sn(1)si(1 ) 2.6570(19 ) , sn(2)si(9 ) 2.639(2 ) , sn(2)si(12 ) 2.6577(19 ) , sn(1)ti(1)sn(2 ) 88.98(4 ) , si(4)sn(1)ti(1 ) 132.35(5 ) , si(1)sn(1)ti(1 ) 126.06(5 ) , si(9)sn(2)si(12 ) 98.36(6 ) , si(9)sn(2)ti(1 ) 132.67(5 ) , si(12)sn(2)ti(1 ) 126.22(5 ) . molecular structure of 14 ( thermal ellipsoid plot drawn at the 30% probability level ) . hydrogen atoms omitted for clarity ( bond lengths in , angles in ) . ti(2)n(1 ) 2.020(6 ) , n(1)si(18 ) 1.710(6 ) , n(1)si(17 ) 1.722(6 ) , si(18)n(1)si(17 ) 122.4(3 ) , si(18)n(1)ti(2 ) 117.8(3 ) , si(17)n(1)ti(2 ) 119.8(3 ) . optimizations of the molecular structures of the free tetrylenes 12 and 18 , the group 4 element mono(tetrylene ) complexes 510 , and the metallocenes bis(tetrylene ) complexes 11 , 13 , 15 , and 1921 at the density functional m06 - 2x / sdd ( sn , pb , ti , zr , hf ) and 6 - 31g(d ) ( p , si , c , h ) level of theory result in structural parameters which are very close to those found by x - ray diffraction methods for some of these compounds . data which are important for the discussion are summarized in table 2 . for the tetrylenes 12 and 18 for which no experimental structural data are available , half - chair conformations of the metallacyclopentasilane rings were predicted with the heavy group 14 element and the two neighboring silicon atoms spanning the central plane . a common feature of all optimized molecular structures of the mono(tetrylene ) ( 510 ) and of the metallocene bis(tetrylene ) complexes 11 , 13 , 15 , and 1921 are trigonal planar coordinated sn or pb atoms ( sum of the bond angles around the element atom , (e ) = 358360 ) embedded in a half - chair metallacyclopentasilane ring of local c2 symmetry . the computational results indicate no significant influence of the complexation on the molecular structure of the tetrylene . the most obvious structural modification is a widening of the endocyclic siesi bond angle (siesi ) by 5.47.4 ( see table 2 ) . in accordance with the available experimental structures , the results of the computation predict that the tetrylene units are oriented mostly perpendicular to the central e m p plane in tetrylene complexes 510 ( dihedral angle = 8284 ) , while in the metallocene bis(tetrylene ) complexes 11 , 13 , 15 , and 1921 , the equivalent dihedral angle is somewhat smaller ( = 6574 ) . this specific arrangement allows for an efficient back - bonding from metal d - orbitals to the formally empty p - orbital at the tetrel atom . e(ii ) bond lengths , which are summarized in table 2 , show the expected trends . m separations are always smaller than the corresponding pb m distances . the calculated m e(ii ) bond lengths as well as those determined experimentally ( see table 2 ) are all smaller than standard values for e m single bonds ( sn m : 276 pm ( ti ) , 294 pm ( zr ) , 292 pm ( hf ) ; pb m : 280 pm ( ti ) , 298 pm ( zr ) , 296 pm ( hf ) ) , in no case , however , the values predicted for e = m double bonds are reached ( sn = m : 247 pm ( ti ) , 257 pm ( zr ) , 258 pm ( hf ) ; pb = m : 252 pm ( ti ) , 262 pm ( zr ) , 263 pm ( hf ) ) . in agreement with these structural criteria also , the results of a natural bond orbital ( nbo ) analysis indicate the multiple - bond character for the m e(ii ) linkage in mono- and bis(tetrylene ) complexes 510 , 11 , 13 , 15 , 1921 ( see table 2 ) . in detail , all calculated wiberg bond indices ( wbis ) are significantly larger than computed for the respective m ditetryl compound ( cp2m(eme3)2)[e = sn : 0.83 ( ti ) , 0.93 ( zr ) , 0.94 ( hf ) ; e = pb : 0.79 ( ti ) , 0.91 ( zr ) , 0.92 ( hf ) ] . as it is expected , the wbis for the m e(ii ) bond for stannylene complexes are always larger than computed for the corresponding plumbylene complex , and the calculated bond orders for the m e(ii ) bond , as expressed by the wbis , increase for a given tetrel element in the order ti < zr hf . in addition the calculated bond order is for each m e pair larger for the mono(tetrylene ) complex than for the metallocene bis(tetrylene ) . these trends are also reflected by the computed bond dissociation energies for the m e(ii ) bond bde(me ) for the tetrylene complexes ( see table 2 ) . zr bonds [ by 97 ( 5/6 ) and 98 kj mol ( 8/9 ) in the case of the mono(tetrylene ) complexes and by 68 ( 13/19 ) and 72 kj mol ( 20/21 ) for the bis(tetrylene ) complexes ] , and there is a second although smaller increase predicted for the bde of the e m bonds are for all calculated metallocene complexes 2936 kj mol weaker than the corresponding sn m linkages . e bonds in mono(tetrylene ) ( 5 , 8) and bis(tetrylene ) complexes 13 , 20 are very similar ( see table 2 ) . the situation differs , however , for the hafnocene and zirconocene complexes for which the computed bdes of the m e(ii ) bond are smaller in bis(tetrylene ) complexes than in their mono(tetrylene ) counterparts by 1831 kj mol . bde of the me bonds calculated using the m06 - 2x functional are given as bde(me ) . for comparison , the bde computed applying the b3lyp functional , here denoted as bde(me ) , is summarized as well . finally , the noncovalent contributions to the bde , bde(me ) , calculated from the difference between bde(me ) and bde(me ) are listed . mean value computed from the dissociation of the complex into a cp2 m fragment and two tetrylenes . the bonding between the zirconium and the tin atoms in the mono(tetrylene ) complex 6 is rationalized by the orbital interaction diagram shown in figure 5 . this orbital interaction diagram is also valid qualitatively for all investigated metallocene mono(tetrylene ) complexes 510 . plots of the surface diagrams for frontier molecular orbitals ( fmos ) of compound 6 can be found in the supporting information . e(ii ) bond in complexes 510 is best described by the conventional -bonding/(d / p ) -back - bonding scheme for carbene complexes . in the framework of perturbation theory , the relative extent of back - bonding in the metallocene tetrylene complexes can be estimated by the evaluation of the calculated orbital stabilization energy e(dxz/ ) and the corresponding destabilization energy e(*/px ) ( see figure 5 and table 2 ) . both energy differences increase for both kinds of metallocene mono(tetrylene ) complexes along the series ti < zr < hf . this suggests that the dxz px -back - bonding is smallest for the titanium complexes ( 5 , 8) and largest for the hafnium compounds ( 7 , 10 ) . fmo interaction scheme for monotetrylene complex 6 , derived from m06 - 2x / sdd ( zr , sn ) , 6 - 31g(d ) ( si , p , c , h ) calculations . this mo scheme is qualitatively valid for all investigated monotetrylene complexes 510 . the analysis also indicates that for each group 4 metallocene , the lowering of the -orbital as expressed by e(dxz/ ) is slightly smaller in the stannylene complexes ( 57 ) than in the corresponding plumbylene complexes ( 810 , by 0.07 ev ( ti ) , 0.02 ev ( zr ) , 0.01 ev ( hf ) ) . on the other hand , the effect of the tetrylene on the * level is more significant , as the destabilization energy e(*/px ) is markedly larger for the stannylene complexes ( 57 ) than for the corresponding plumbylene complexes ( 810 , by 0.25 ev ( ti ) , 0.17 ev ( zr ) , 0.15 ev ( hf ) ) . the poor ability of the titanocene to engage in -bonding is mostly due to the poor spatial and energetic match between the 3dxz orbital of titanium and the 5px orbital of the tin ( e(dxz / px ) = 2.65 vs 2.08 ev for zr or 1.88 ev for hf ) or 6px of lead atom ( ( e(dxz / px ) = 2.80 vs 2.22 ev for zr or 2.03 ev for hf ) . nmr chemical shifts of group 14 carbene analogs are always extremely large due to a dominant paramagnetic contribution , which arises from the efficient interaction of the applied magnetic field with the filled spz - type mo and the orthogonal empty p - type orbital at the dicoordinated tetrel element . the paramagnetic shift is very large for small energy differences e between these two magnetically active orbitals . in the mono(tetrylene ) complexes the spz orbital is transformed to the -orbital of the m e(ii ) bond , and the px orbital can be associated with the *-orbital ( figure 5 ) . therefore , the sn nmr chemical shift of the stannylene complexes 57 and the pb nmr chemical shift of the metallocene plumbylene complexes 810 are determined mainly by the energy difference between these two molecular orbitals , e ( see figure 5 ) . while the energy of the -orbital remains nearly constant for a given tetrel element along the series of group 4 metals ( e( ) = 6.09 ev ( 5 ) , 6.13 ev ( 6 ) , 6.12 ev ( 7 ) ) , the *-level is significantly altered by the different extent of back - bonding ( e( * ) = 0.90 ev ( 5 ) , 0.68 ev ( 6 ) , 0.55 ev ( 7 ) ) . consequently , the changes in e , summarized in table 2 , are mostly due to the different extent of back - bonding in that sense as the larger e , the higher the multiple - bond character of the m therefore the experimental sn nmr chemical shift of stannylene compounds 57 and the experimental pb nmr shift of the metallocene plumbylene complexes 810 can be used as a tool to estimate the degree of multiple bonding in these complexes . e(ii ) bond leads to increasing energy differences e , which becomes manifest in a upfield shift of the element resonance in nmr spectroscopy . these relations become obvious by analyzing the data summarized in table 2 , and although only a very limited set of data is used , by the correlations between the reciprocal calculated e and the experimental chemical shifts sn ( 57 ) and pb ( 810 ) shown in figure 6 . plot of the experimental sn ( ) and pb nmr ( ) chemical shifts versus the reciprocal e for monostannylene complexes 57 and monoplumbylene metallocenes 810 . the bonding in bis(stannylene ) zirconocenes was treated principally already by piers . in agreement with that earlier investigation we found that multicenter interactions are important for the understanding of the bonding in bis(tetrylene ) complexes 11 , 13 , 15 , and 1921 . the fmo interaction diagram of the bis(stannylene ) zirconocene 19 is shown in figure 7 . qualitatively , it is also valid for the investigated bis(tetrylene ) complexes 11 , 13 , 15 , 20 , and 21 . the analysis reveals that the me(ii)2 group is built up by two -type orbitals and the -type homo , each delocalized across all three constituent atoms ( see figures 7 and 8 for surface diagrams of the respective mos ) . the delocalization of the -type homo across all three atoms helps to rationalize the smaller bond order and the weaker m e(ii ) bond in group 4 metallocene bis(tetrylene ) complexes compared to their mono(tetrylene ) counterparts . in addition , inspection of the homo of compound 19 indicates some degree of bonding interaction between the two distant sn atoms , although their separation , d(snsn ) , approaches the sum of the van der waals radii , vdwr ( d(snsn ) 403.8 pm , vdwr 434 pm ) . the computed wbi index between this pair of atoms differs significantly from zero ; it is however only 22% of the bond index computed at the same theoretical level for the central sn sn single bond in ( h3si)6sn2 ( 19 : wbi(snsn ) = 0.20 ; ( h3si)6sn2 : wbi(snsn ) = 0.92 , d(snsn ) = 283.7 pm ) . similar small wbis were computed for the bis-(tetrylene ) complexes 11 , 13 , 15 , 20 , and 21 ( wbis range from 0.15 ( pbpb in complex 20 ) to 0.23 ( snsn in compound 11 ) ) . these computational data suggest that in the continuum of possible bonding modes for group 4 metallocene bis(tetrylene ) complexes , beginning with the ditetren complex a , passing the metallacyclopropane b and ending at the delocalized bis(tetrylene ) structure c , the here investigated complexes 11 , 13 , 15 and , 1921 are best described by canonical structure c with only minor contribution from structure b ( scheme 6 ) . fmo interaction scheme for bis(tetrylene ) complex 19 in c2 symmetry , derived from m06 - 2x / sdd(zr , sn ) , 6 - 31g(d ) ( si , c , h ) calculations . the fmo diagram for cp2zr is derived from that of cp2zr(pet3 ) ( see figure 5 ) by removal of the phosphane ligand ( see the supporting information for further details ) . this mo scheme is qualitatively valid for all investigated bis - tetrylene complexes 11 , 13 , 15 , and 1921 . calculated surface diagrams for pertinent molecular orbitals of bis(stannylene ) complex 19 in c2 symmetry , derived from m06 - 2x / sdd(zr , sn ) , 6 - 31g(d ) ( si , c , h ) calculations ( isodensity value : 0.05 ) . ( a ) 1a orbital ( -eme bonding ( homo-4 ) ) ; ( b ) 1b orbital ( eme bonding ( homo-1 ) ) ; ( c ) 2a orbital ( eme bonding ( homo ) ) ; ( d ) 2b orbital ( eme nonbonding ( lumo ) ) ( color code : light blue : zr , greenish gray : sn ; blue gray : si ; and gray : carbon ) . the large and polarizable substituents which are present in all investigated metallocene complexes suggest that attractive dispersion energy contributions to the overall binding energy of the complexes might be a decisive factor . the here applied m06 - 2x functional properly accounts for dispersion forces , while the most prominent deficit of the popular b3lyp functional is the nearly complete negligence of noncovalent van der waals interactions . therefore , the difference in the calculated bond dissociation energies ( bdes ) using these two functionals allows estimating the contribution of noncovalent bonding in metallocene complexes 511 , 13 , 15 , and 1921 . the contribution of noncovalent interactions , bde , to the overall bde , which is calculated as the difference between the bde obtained at the m06 - 2x level and the reduced bde obtained at the b3lyp level ( see table 2 ) , is substantial in all cases . in the case of the titanium complexes it accounts for 60% of the overall bde and even in the hafnium complexes it amounts to 30% . the present study demonstrates that stannylene and plumbylene complexes of all three group 4 metallocenes are synthetically accessible by magnesium reduction of the corresponding metallocene dichlorides in the presence of the phosphine - stabilized tetrylenes 1 or 2 . significant back - bonding from the transition metal to the heavy main group atom was shown by nmr spectroscopy and confirmed by x - ray structure analyses of all six complexes 510 . the stannylene complexes 5 and 7 are the first stannylene complexes of titanium and hafnium to be reported , whereas 810 are the first compounds to feature group 4lead bonds at all . using the base - free compounds 3 and 4 hafnocene bis(tetrylene ) complexes , 11 and 15 , could be prepared . in these complexes the extent of -back - bonding is decreased because of competition of two -acceptor ligands for only one electron pair . the theoretical analysis of the bonding in metallocene mono- and bis(tetrylene ) complexes 510 , 11 , 13 , 15 , and 1921 revealed for all investigated compounds multiple - bonded character for the m the bonding between the group 4 metal and the group 14 element atom can be rationalized in the case of the mono(tetrylene ) complexes with the classical -donor-acceptor interaction . e(ii ) linkage increases descending the group 4 metals and decreases going from sn to its heavier congener pb . as a consequence , the weakest m e(ii ) bonds are found between ti and pb atoms , while the strongest are predicted by the computation for the pair hf sn . e(ii ) bonds is the significantly reduced ability of the titanium atom for d the theoretical analysis of the bonding in mono(tetrylene ) complexes 510 supports the presumption based on experimental data that the nmr chemical shift of the tetrel atom in the complex is a qualitative measure for the extent of multiple - bonding between the metal and the tetrel atom in these complexes . increased d e(ii ) bond is weaker in the bis(tetrylene ) zirconocene and hafnocene complexes 11 , 15 , 19 , and 21 than in the corresponding mono(tetrylene ) complexes 6 , 7 , 9 , and 10 . clearly , this is a result of the reduced multiple - bond character of the m e(ii ) linkage , due to the competition between two acceptor -type orbitals for one metal d - orbital in the metallocene bis(tetrylene ) complexes . this situation is best described by a multicenter bonding which involves the two tetrel atoms and the central metal atom . e -back - bonding in the titanium complexes 5 and 8 is already small and is not further reduced by the addition of a second tetrylene unit in complexes 13 and 20 . e(ii ) bonds in compounds 5 , 8 , 13 , and 20 are predicted by the calculations . all reactions involving air - sensitive compounds were carried out under an atmosphere of dry nitrogen or argon using either schlenk techniques or a glovebox . h ( 300 mhz ) , c ( 75.4 mhz ) , si ( 59.3 mhz ) , p ( 124.4 mhz ) , sn ( 111.8 mhz ) , and pb ( 62.8 mhz ) nmr spectra were recorded on a varian inova 300 spectrometer . if not noted otherwise for all samples , c6d6 was used as solvent . to compensate for the low isotopic abundance of si for the plumbylene complexes , attempts to obtain elemental analysis data gave consistently too low values for c and h. for x - ray structure analyses the crystals were mounted onto the tip of glass fibers , and data collection was performed with a bruker - axs smart apex ccd diffractometer using graphite - monochromated mo k radiation ( 0.71073 ) . the data were reduced to fo2 and corrected for absorption effects with saint and sadabs , respectively . the structures were solved by direct methods and refined by full - matrix least - squares method ( shelxl97 ) . if not noted otherwise , all non - hydrogen atoms were refined with anisotropic displacement parameter . all hydrogen atoms were located in calculated positions to correspond to standard bond lengths and angles . all diagrams were drawn with 30% probability thermal ellipsoids , and all hydrogen atoms were omitted for clarity . crystallographic data ( excluding structure factors ) for the structures of compounds 511 and 1315 reported in this paper have been deposited with the cambridge crystallographic data center as supplementary publication no . ccdc-831742 ( 5 ) , 831743 ( 6 ) , 831744 ( 7 ) , 831751 ( 8) , 831748 ( 9 ) , 831745 ( 10 ) , 855936 ( 11 ) , 855938 ( 13 and 14 ) , and 855937 ( 15 ) . copies of data can be obtained free of charge at : http://www.ccdc.cam.ac.uk / products / csd / request/. compounds 1,2,3 , and 4(7 ) were prepared according to published procedures . for group 4 metallocene monotetrylene complexes 510 : an equimolar ( 0.5 mmol each ) mixture of group 4 metallocene dichloride , magnesium turnings , and 1 or 2 was stirred in thf ( 5 ml ) for 3 h. during this time deeply colored solutions formed . the thf was removed under reduced pressure , and the remaining solid was extracted with pentane ( 3 , 5 ml each ) . the filtrate was concentrated to 6 ml and stored at 60 c for 16 h. crystals could be isolated by decantation . typical yield : about 80% . dark - blue crystals ( yield : 81% ) : h nmr ( in ppm ) : 5.24 ( s , 10h , cp ) , 1.05 ( m , 9h , p(ch2ch3)3 ) , 0.88 ( m , 6h , p(ch2ch3)3 ) , 0.59 ( s , 18h , sime3 ) , 0.54 ( s , 18h , sime3 ) , 0.48 ( s , 6h , sime2 ) , 0.43 ( s , 6h , sime2 ) . c nmr ( in ppm ) : 96.3 ( cp ) , 22.7 ( p(ch2ch3)3 ) , 9.4 ( p(ch2ch3)3 ) , 5.6 ( sime3 ) , 5.4 ( sime3 ) , 0.3 ( sime2 ) , 0.1 ( sime2 ) . si nmr ( in ppm ) : 4.1 ( sime3 ) , 5.0 ( sime3 ) , 20.1 ( sime2 ) , 101.4 ( quart . p nmr ( in ppm ) : 50.2 ( j117sn/119sn= 232 hz , 266 hz ) . sn nmr( in ppm ) : 1635 ( d , jsnp = 266 hz ) . calcd for c32h73psi8snti ( 880.16 ) : c , 43.67 ; h , 8.36 . found : c , 43.22 ; h , 8.09 . deep - purple crystals ( yield : 85% ) : h nmr ( in ppm ) : 5.41 ( s , 10h , cp ) , 1.05 ( m , 9h , p(ch2ch3)3 ) , 0.86 ( m , 6h , p(ch2ch3)3 ) , 0.60 ( s , 18h , sime3 ) , 0.50 ( s , 18h , sime3 ) , 0.49 ( s , 6h , sime2 ) , 0.36 ( s , 6h , sime2 ) . c nmr ( in ppm ) : 96.7 ( cp ) , 20.2 ( d , jpc = 15.8 hz , p(ch2ch3)3 ) , 8.4 ( p(ch2ch3)3 ) , 5.1 ( sime3 ) , 4.7 ( sime3 ) , 3.4 ( sime2 ) . si nmr ( in ppm ) : 5.0 ( d , jpsi = 2.1 hz , sime3 ) , 5.9 ( d , jpsi = 3.2 hz , sime3 ) , 19.4 ( sime2 ) , 108.2 ( d , jpsi = 1.6 hz , quart . sn nmr ( in ppm ) : 1263 ( d , jpsn = 133 hz ) . calcd for c32h73psi8snzr ( 923.51 ) : c , 41.62 ; h , 7.97 . found : c , 40.96 ; h , 8.05 . deep - purple crystals ( yield : 79% ) : h nmr ( in ppm ) : 5.29 ( s , 10h , cp ) , 1.13 ( td , jhh = 7.6 hz , jph = 13.0 hz , 9h , p(ch2ch3)3 ) , 0.88 ( m , 6h , p(ch2ch3)3)3 , 0.62 ( s , 18h , sime3 ) , 0.60 ( s , 6h , sime2 ) , 0.52 ( s , 6h , sime2 ) , 0.51 ( s , 18h , sime3 ) . c nmr ( in ppm ) : 95.5 ( cp ) , 22.1 ( d , jpc = 18.9 hz , p(ch2ch3)3 ) , 9.3 ( p(ch2ch3)3 ) , 5.5 ( sime3 ) , 5.2 ( sime3 ) , 0.3 ( sime2 ) , 0.4 ( sime2 ) . si nmr ( in ppm ) : 4.4 ( d , jpsi = 2.6 hz , sime3 ) , 5.9 ( d , jpsi = 3.6 hz , sime3 ) , 19.0 ( sime2 ) , 110.8 ( d , jpsi = 3.1 hz , quart . p nmr ( in ppm ) : 34.8 ( no tin satellites observed ) . sn nmr ( in ppm ) : 1079 ( d , jsnp = 92 hz ) . calcd for c32h73hfpsi8sn ( 1010.78 ) : c , 38.02 ; h , 7.28 . found : c , 37.73 ; h , 7.25 . dark - green crystals ( yield : 80% ) : h nmr ( in ppm ) : 5.2 ( s , 10h , cp ) , 1.08 ( m , 9h , p(ch2ch3)3 ) , 0.85 ( m , 6h , p(ch2ch3)3 ) , 0.62 ( s , 18h , sime3 ) , 0.58 ( s , 18h , sime3 ) , 0.52 ( s , 6h , sime2 ) , 0.37 ( s , 6h , sime2 ) . c nmr ( in ppm ) : 96.2 ( cp ) , 23.0 ( d , jpc = 5.4 hz , p(ch2ch3)3 ) , 6.7 ( sime3 ) , 6.6 ( sime3 ) , 4.0 ( sime2 ) , 3.2 ( sime2 ) . si nmr ( in ppm ) : 2.3 ( d , jpsi = 2.7 hz , sime3 ) , 3.7 ( d , jpsi = 3.8 hz , sime3 ) , 6.9 ( sime2 ) , 15.6 ( d , jpsi = 2.5 hz , quart . pb nmr ( in ppm ) : 5299 ( fwhm : 217.8 hz ) . deep - purple crystals ( yield : 77% ) : h nmr ( in ppm ) : 5.36 ( s , 10h , cp ) , 1.08 ( m , 9h , p(ch2ch3)3 ) , 0.85 ( m , 6h , p(ch2ch3)3 ) , 0.65 ( s , 18h , sime3 ) , 0.62 ( s , 6h , sime2 ) , 0.55 ( s , 18h , sime3 ) , 0.53 ( s , 6h , sime2 ) . c nmr ( in ppm ) : 96.5 ( cp ) , 19.9 ( d , jpc = 15.1 hz , p(ch2ch3)3 ) , 8.6 ( p(ch2ch3)3 ) , 6.3 ( sime3 ) , 6.2 ( sime3 ) , 3.2 ( sime2 ) , 2.4 ( sime2 ) . si nmr ( in ppm ) : 3.7 ( d , jpsi = 2.0 hz , sime3 ) , 4.1 ( d , jpsi = 3.1 hz , sime3 ) , 7.1 ( sime2 ) , 42.0 ( d , jpsi = 2.4 hz , quart . pb nmr ( in ppm ) : 4165 ( fwhm : 128.5 hz ) . deep - purple crystals ( yield : 84% ) : h nmr ( in ppm ) : 5.24 ( s , 10h , cp ) , 1.15 ( td , jhh = 7.7 hz , jph = 13.1 hz , 9h , p(ch2ch3)3 ) , 0.87 ( m , 6h , p(ch2ch3)3 ) , 0.67 ( s , 18h , sime3 ) , 0.63 ( s , 6h , sime3 ) , 0.62 ( s , 6h , sime3 ) , 0.54 ( s , 18h , sime3 ) . c nmr ( in ppm ) : 94.6 ( cp ) , 21.3 ( d , jpc = 20.2 hz , p(ch2ch3)3 ) , 9.2 ( p(ch2ch3)3 ) , 6.2 ( sime3 ) , 6.1 ( sime3 ) , 2.9 ( sime2 ) , 2.2 ( sime2 ) . si nmr ( in ppm ) : 2.5 ( d , jpsi = 2.2 hz , sime3 ) , 4.3 ( d , jpsi = 3.2 hz , sime3 ) , 7.7 ( sime2 ) , 53.9 ( d , jpsi = 3.7 hz , quart . p nmr ( in ppm ) : 45.5 ( no pb satellites observed ) . pb nmr ( in ppm ) : 3462 ( fwhm : 172.1 hz ) . a mixture of hafnocene dichloride ( 20 mg , 0.05 mmol ) , magnesium turnings ( 5 mg , 4.8 mmol , excess ) , and 3 ( 61 mg , 0.05 mmol ) was suspended in thf ( 5 ml ) , subjected to ultrasonification , and stirred for 1 h. a black suspension developed . all volatile materials were removed in vacuo , and the residue was extracted with pentane ( 10 ml ) . the dark filtrate was concentrated to 3 ml and stored at 60 c for 60 h. black needle - shaped crystals of 11 ( 40 mg , 0.03 mmol , 54% ) were isolated upon filtration and dried in vacuo . h nmr ( in ppm ) : 4.85 ( s , 10h , cp ) , 0.47 ( s , 36h , sime3 ) , 0.46 ( s , 36h , sime3 ) , 0.29 ( s , 12h , sime2 ) , 0.25 ( s , 12h , sime2 ) . c nmr ( in ppm ) : 96.3 ( cp ) , 13.9 ( sime2 ) , 13.8 ( sime2 ) , 4.9 ( sime3 ) , 4.8 ( sime3 ) . si nmr ( in ppm ) : 6.8 ( sime3 ) , 10.8 ( sime3 ) , 20.1 ( sime2 ) , 85.3 ( quart . calcd for c42h106hfsi16sn2 ( 1476.57 ) : c , 34.16 ; h , 7.24 . found : c , 33.71 ; h , 7.29 . a mixture of titanocene dichloride ( 25 mg , 0.10 mmol ) , magnesium turnings ( 6 mg , 0.25 mmol ) , and distannene 3 ( 117 mg , 0.10 mmol ) was suspended in thf ( 4 ml ) , subjected to ultrasonification for 2 min , and stirred for 1 h. a deep - purple suspension developed . all volatile materials were removed in vacuo , and the blue residue was extracted 3 with pentane ( 2 ml each ) . the dark - blue filtrate was concentrated to 2 ml and stored at 60 c for 16 h. blue crystals of 13 ( 87 mg ( 0.07 mmol , 70% ) were isolated by decantation and dried in vacuo . h nmr ( in ppm ) : 5.36 ( s , 10h , cp ) , 0.46 ( s , 72h , sime3 ) , 0.34 ( s , 24h , sime2 ) . c nmr ( in ppm ) : 94.6 ( cp ) , 4.7 ( sime3 ) , 0.4 ( sime2 ) . si nmr ( in ppm ) : 1.7 ( sime3 ) , 19.4 ( sime2 ) , 79.0 ( quart . the crystals containing cp2tin(sime3)2 were presumably formed by reduction of cp2ticl2 with magnesium in the presence of a batch of 3 which was contaminated with kn(sime3)2 from the synthesis of 3 . the mixed crystals are green from the ti(iii)-compound but were suitable for x - ray crystallography , whereas from pure 13 , no crystals of good quality could be obtained . a mixture of hafnocene dichloride ( 20 mg , 0.05 mmol ) , magnesium turnings ( 4 mg , 0.16 mmol ) , and 4 ( 67 mg , 0.05 mmol ) was suspended in thf ( 5 ml ) , subjected to ultrasonification for 2 min , and stirred for 1 h. the suspension developed a red coloration and was evaporated to dryness . three times extraction with pentane ( 3 ml each ) yielded a black solution which was concentrated to 2 ml and stored at 60 c for 36 h. black needle - shaped crystals of 15 ( 33 mg , 0.02 mmol , 40% ) were isolated by decantation and dried in vacuo . h nmr ( in ppm ) : 6.21 ( 10h , cp ) , 0.81 ( 24h , sime2 ) , 0.74 ( 72h , sime3 ) . c nmr ( in ppm ) : 96.9 ( cp ) , 6.9 ( sime3 ) , 3.6(sime2 ) . si nmr( in ppm ) : 1.0 ( sime3 ) , 4.9 ( sime2 ) , 16.6 ( quart . cp2zrcl2 ( 100 mg , 0.34 mmol ) , 4 ( 114 mg , 0.17 mmol ( considered to be monomeric in solution ) , and magnesium turnings ( 24 mg , 1.0 mmol , excess ) were suspended in thf and subjected to ultrasonification for 5 min . all volatile materials were removed in vacuo , and the black residue was extracted with pentane ( 3 3 ml ) . the red - purple extract was concentrated to 3 ml and stored for 72 h at 60 c . purple crystals of 16 ( 106 mg , 0.11 mmol , 63% ) were isolated by decantation and cautiously dried in vacuo . nmr ( c6d6 , rt , in ppm ) : h : 4.82 ( s , 10h , cp ) , 2.68 ( br , 4h , thf ) , 1.63 ( br , 4h , thf ) , 0.44 ( s , 12h , sime2 ) , 0.29 ( s , 36h , sime3 ) . c : 95.6 ( cp ) , 60.6 ( thf ) , 20.1 ( thf ) , 3.5 ( sime3 ) , 2.0(sime2 ) . si : 10.0 ( sime3 ) , 11.3 ( sime2 ) , 36.7 ( quart . pb : 5770 . a mixture of tacl5 ( 75 mg , 0.21 mmol ) , magnesium turnings ( 12 mg , 0.5 mmol , excess ) , pet3 ( 50 mg , 0.42 mmol ) , and 1 ( 150 mg , 0.21 mmol ) were suspended in thf and subjected to ultrasonification for 2 min , during which time the suspension turned purple . the mixture was stirred for 3 h at rt , and then all volatile materials were removed in vacuo . the purple extract was concentrated ( 4 ml ) and stored at 60 c for 48 h. deep - purple crystals of 17 ( 107 mg , 0.10 mmol , 46% ) were isolated by decantation and dried in vacuo . h nmr ( in ppm ) : 2.17 ( m , 6h , p(ch2ch3)3 ) , 1.05 ( m , 9h , p(ch2ch3)3 ) , 0.51 ( s , 12h , sime2 ) , 0.47 ( s , 36h , sime3 ) . c nmr ( in ppm ) : 24.2 ( p(ch2ch3)3 ) , 9.0 ( p(ch2ch3)3 ) , 4.5 ( sime3 ) , 0.9 ( sime2 ) . si nmr ( in ppm ) : 6.7 ( sime3 ) , 18.6 ( sime2 ) , 105.9 ( quart . p nmr ( in ppm ) : 38.7 ( s , jpsn = 73 hz ) . sn nmr ( in ppm ) : 1985 ( t , jpsn = 73 hz ) . calcd for c28h78cl3p2si8snta ( 1107.57 ) : c , 30.36 ; h , 7.10 . found : c , 30.17 ; h , 7.025 .

reduction of group 4 metallocene dichlorides with magnesium in the presence of cyclic disilylated stannylene or plumbylene phosphine adducts yielded the respective metallocene tetrylene phosphine complexes . under the same conditions the use of the respective dimerized stannylene or plumbylene gave metallocene ditetrylene complexes . a computational analysis of these reactions revealed for all investigated compounds multiple - bonded character for the m e(ii ) linkage , which can be rationalized in the case of the monotetrylene complex with the classical -donor/-acceptor interaction . the strength of the m e(ii ) bond increases descending group 4 and decreases going from sn to its heavier congener pb . the weakness of the ti e(ii ) bonds is caused by the significantly reduced ability of the titanium atom for d p -back - bonding .

Introduction Results and Discussion Conclusion Experimental Section

while the tin compound 3 is the result of a dimerization rearrangement process of a disilylated stannylene and exists as an endocyclic bicyclic distannene , the plumbylene appears as a monomer in solution but crystallizes as a weak donor we report on an extension of these studies to explore the coordination chemistry of disilylated stannylenes and plumbylenes with early transition metals and in particular with group 4 metallocenes . that the heavier tetrylenes can display quite different bonding motifs to transition metals for the synthesis of group 4 metallocene plumbylene and stannylene complexes a new general approach was sought . in the mentioned examples , the zirconocene stannylene complexes were prepared by warming negishi zirconocene cp2zrcl22buli in the presence of 2 equiv of stannylene from 80 c to room temperature , thus limiting the scope of the reaction to zirconium . alternatively , we found that reductions of group 4 metallocene dichlorides with magnesium in the presence of stannylene or plumbylene phosphine adducts ( 1 and 2 ) provided smooth conversion to the desired complexes ( 510 ) ( scheme 1 ) . by reaction of 1,2-dipotassiodisilanes and -digermanes with metallocene dichlorides , we recently succeeded in the synthesis of group 4 disilene and digermene complexes . when this reaction , however , was carried out with the respective 1,2-dipotassiodistannane , instead of the expected distannene complexes , metallacyclotetrastannanes were isolated as the only tin containing products . this behavior of tin was attributed to its greater preference for the divalent state compared to its lighter congeneres . however , when cp2hfcl2 was reduced with magnesium in the presence of an equimolar amount of 3 , the only pentane soluble product was the hafnocene bis(stannylene ) complex 11 ( scheme 2 ) . its formation is apparently facilitated by the reversible rearrangement of 3 to the exocyclic distannene isomer , which is the direct dimerization product of the monomeric stannylene 12 ( table 2 ) . under very similar conditions , employing magnesium reduction of hafnocene dichloride in the presence of 2 equiv of 4 even the hafnocene bis(plumbylene ) complex 15 could be obtained in moderate yield ( scheme 3 ) . the formation of the zirconocene plumbylene complex 16 , where the vacant coordination site at zirconium is occupied by a thf molecule , showed this not to be the case . in order to assess the general applicability of this simple access to group 4 metal tetrylenes , we reacted tacl5 as a group 5 compound with magnesium in the presence of stannylene adduct 1 ( scheme 5 ) . sn bond in solution is the presence of sn satellites in the sn nmr spectrum of 3 . the group 4 metallocene stannylene complexes the chemical shift of titanocene complex 5 was found to be 1635 ppm with the signal being a doublet with jsnp = 276 hz coupling to phosphorus . the same trend can also be observed in the p spectra of these complexes , as the chemical shift ranges from 50.2 ppm in the titanocene complex ( 5 ) down to 38.5 ppm for zirconium ( 6 ) to finally 34.8 ppm in the hafnium case ( 7 ) ( table 1 ) . nmr spectroscopy thus clearly suggests an increasing degree of -back - donation when descending group 4 . however , the different behavior of the latter can be explained by intramolecular donation of electron density into the empty tin p - orbital from the attached amino groups of the parent stannylene . the difference between the sn nmr chemical shifts of 7 and 11 can be rationalized on the basis of -back - donation . for this reason -back - donation to tin is enhanced in this system , whereas in 11 the situation can be described as competition of two -donor -acceptor ligands for the same electron pair . while it is not completely clear how valid a comparison of the sn nmr shift of the tantalum compound 17 with the group 4 metallocene complexes of the same stannylene is , it seems fair to state that the chemical shift of = 1985 ppm is in the same region . a steady decrease in the chemical shift value descending group 4 was found : titanocene plumbylene complex 8 resonates at = 5299 ppm , zirconocene complex 9 at = 4165 ppm , and finally hafnocene plumbylene 10 at = 3462 ppm . the weaker bonds between pb and the respective metal , compared to the analogous stannylene complexes , in addition to the fact that the pb signals are comparably broad [ values for full width at half - maximum ( fwhm ) range from 130 to 220 hz ] seem to impede the observation of the jpbp coupling . the dependence of the m e back - bonding from the additional phosphine ligands can be estimated from a comparison of the pb nmr chemical shifts of the plumbylene zirconocene 9( = 4165 ppm ) with the respective thf adduct 16 ( = 5770 ) . the increasing electronic saturation of the pb atom also can be observed in the si nmr spectra . for 810 two different resonances for the trimethylsilyl groups in contrast to this , only one sime3 signal was observed for 16 , which suggests dissociation of thf , which can also be concluded from broadened signals in the respective h spectrum . as all six phosphine tetrylene complexes ( 510 ) are isostructural , only one example of each group is shown [ figure 1 : 7 and figure 2 : 10 ; depictions of the others ( 5 , 6 , 8 , and 9 ) are available in the supporting information ( figures s1s4 ) ] . the sums of bond angles around tin or lead in all six examples match 360 almost exactly with a maximum deviation of 0.5. the group 4e ( e = sn or pb ) bond lengths are significantly shorter than the sum of tabulated covalent radii or respective single bonds in the case of tin ( table 1 ) . optimizations of the molecular structures of the free tetrylenes 12 and 18 , the group 4 element mono(tetrylene ) complexes 510 , and the metallocenes bis(tetrylene ) complexes 11 , 13 , 15 , and 1921 at the density functional m06 - 2x / sdd ( sn , pb , ti , zr , hf ) and 6 - 31g(d ) ( p , si , c , h ) level of theory result in structural parameters which are very close to those found by x - ray diffraction methods for some of these compounds . for the tetrylenes 12 and 18 for which no experimental structural data are available , half - chair conformations of the metallacyclopentasilane rings were predicted with the heavy group 14 element and the two neighboring silicon atoms spanning the central plane . in accordance with the available experimental structures , the results of the computation predict that the tetrylene units are oriented mostly perpendicular to the central e m p plane in tetrylene complexes 510 ( dihedral angle = 8284 ) , while in the metallocene bis(tetrylene ) complexes 11 , 13 , 15 , and 1921 , the equivalent dihedral angle is somewhat smaller ( = 6574 ) . e(ii ) bond lengths , which are summarized in table 2 , show the expected trends . the calculated m e(ii ) bond lengths as well as those determined experimentally ( see table 2 ) are all smaller than standard values for e m single bonds ( sn m : 276 pm ( ti ) , 294 pm ( zr ) , 292 pm ( hf ) ; pb m : 280 pm ( ti ) , 298 pm ( zr ) , 296 pm ( hf ) ) , in no case , however , the values predicted for e = m double bonds are reached ( sn = m : 247 pm ( ti ) , 257 pm ( zr ) , 258 pm ( hf ) ; pb = m : 252 pm ( ti ) , 262 pm ( zr ) , 263 pm ( hf ) ) . in agreement with these structural criteria also , the results of a natural bond orbital ( nbo ) analysis indicate the multiple - bond character for the m e(ii ) linkage in mono- and bis(tetrylene ) complexes 510 , 11 , 13 , 15 , 1921 ( see table 2 ) . as it is expected , the wbis for the m e(ii ) bond for stannylene complexes are always larger than computed for the corresponding plumbylene complex , and the calculated bond orders for the m e(ii ) bond , as expressed by the wbis , increase for a given tetrel element in the order ti < zr hf . these trends are also reflected by the computed bond dissociation energies for the m e(ii ) bond bde(me ) for the tetrylene complexes ( see table 2 ) . zr bonds [ by 97 ( 5/6 ) and 98 kj mol ( 8/9 ) in the case of the mono(tetrylene ) complexes and by 68 ( 13/19 ) and 72 kj mol ( 20/21 ) for the bis(tetrylene ) complexes ] , and there is a second although smaller increase predicted for the bde of the e m bonds are for all calculated metallocene complexes 2936 kj mol weaker than the corresponding sn m linkages . the situation differs , however , for the hafnocene and zirconocene complexes for which the computed bdes of the m e(ii ) bond are smaller in bis(tetrylene ) complexes than in their mono(tetrylene ) counterparts by 1831 kj mol . the bonding between the zirconium and the tin atoms in the mono(tetrylene ) complex 6 is rationalized by the orbital interaction diagram shown in figure 5 . plots of the surface diagrams for frontier molecular orbitals ( fmos ) of compound 6 can be found in the supporting information . e(ii ) bond in complexes 510 is best described by the conventional -bonding/(d / p ) -back - bonding scheme for carbene complexes . in the framework of perturbation theory , the relative extent of back - bonding in the metallocene tetrylene complexes can be estimated by the evaluation of the calculated orbital stabilization energy e(dxz/ ) and the corresponding destabilization energy e(*/px ) ( see figure 5 and table 2 ) . this suggests that the dxz px -back - bonding is smallest for the titanium complexes ( 5 , 8) and largest for the hafnium compounds ( 7 , 10 ) . the analysis also indicates that for each group 4 metallocene , the lowering of the -orbital as expressed by e(dxz/ ) is slightly smaller in the stannylene complexes ( 57 ) than in the corresponding plumbylene complexes ( 810 , by 0.07 ev ( ti ) , 0.02 ev ( zr ) , 0.01 ev ( hf ) ) . on the other hand , the effect of the tetrylene on the * level is more significant , as the destabilization energy e(*/px ) is markedly larger for the stannylene complexes ( 57 ) than for the corresponding plumbylene complexes ( 810 , by 0.25 ev ( ti ) , 0.17 ev ( zr ) , 0.15 ev ( hf ) ) . nmr chemical shifts of group 14 carbene analogs are always extremely large due to a dominant paramagnetic contribution , which arises from the efficient interaction of the applied magnetic field with the filled spz - type mo and the orthogonal empty p - type orbital at the dicoordinated tetrel element . in the mono(tetrylene ) complexes the spz orbital is transformed to the -orbital of the m e(ii ) bond , and the px orbital can be associated with the *-orbital ( figure 5 ) . therefore , the sn nmr chemical shift of the stannylene complexes 57 and the pb nmr chemical shift of the metallocene plumbylene complexes 810 are determined mainly by the energy difference between these two molecular orbitals , e ( see figure 5 ) . while the energy of the -orbital remains nearly constant for a given tetrel element along the series of group 4 metals ( e( ) = 6.09 ev ( 5 ) , 6.13 ev ( 6 ) , 6.12 ev ( 7 ) ) , the *-level is significantly altered by the different extent of back - bonding ( e( * ) = 0.90 ev ( 5 ) , 0.68 ev ( 6 ) , 0.55 ev ( 7 ) ) . consequently , the changes in e , summarized in table 2 , are mostly due to the different extent of back - bonding in that sense as the larger e , the higher the multiple - bond character of the m therefore the experimental sn nmr chemical shift of stannylene compounds 57 and the experimental pb nmr shift of the metallocene plumbylene complexes 810 can be used as a tool to estimate the degree of multiple bonding in these complexes . e(ii ) bond leads to increasing energy differences e , which becomes manifest in a upfield shift of the element resonance in nmr spectroscopy . the delocalization of the -type homo across all three atoms helps to rationalize the smaller bond order and the weaker m e(ii ) bond in group 4 metallocene bis(tetrylene ) complexes compared to their mono(tetrylene ) counterparts . the computed wbi index between this pair of atoms differs significantly from zero ; it is however only 22% of the bond index computed at the same theoretical level for the central sn sn single bond in ( h3si)6sn2 ( 19 : wbi(snsn ) = 0.20 ; ( h3si)6sn2 : wbi(snsn ) = 0.92 , d(snsn ) = 283.7 pm ) . these computational data suggest that in the continuum of possible bonding modes for group 4 metallocene bis(tetrylene ) complexes , beginning with the ditetren complex a , passing the metallacyclopropane b and ending at the delocalized bis(tetrylene ) structure c , the here investigated complexes 11 , 13 , 15 and , 1921 are best described by canonical structure c with only minor contribution from structure b ( scheme 6 ) . in the case of the titanium complexes it accounts for 60% of the overall bde and even in the hafnium complexes it amounts to 30% . the present study demonstrates that stannylene and plumbylene complexes of all three group 4 metallocenes are synthetically accessible by magnesium reduction of the corresponding metallocene dichlorides in the presence of the phosphine - stabilized tetrylenes 1 or 2 . the theoretical analysis of the bonding in metallocene mono- and bis(tetrylene ) complexes 510 , 11 , 13 , 15 , and 1921 revealed for all investigated compounds multiple - bonded character for the m the bonding between the group 4 metal and the group 14 element atom can be rationalized in the case of the mono(tetrylene ) complexes with the classical -donor-acceptor interaction . e(ii ) linkage increases descending the group 4 metals and decreases going from sn to its heavier congener pb . as a consequence , the weakest m e(ii ) bonds are found between ti and pb atoms , while the strongest are predicted by the computation for the pair hf sn . e(ii ) bonds is the significantly reduced ability of the titanium atom for d the theoretical analysis of the bonding in mono(tetrylene ) complexes 510 supports the presumption based on experimental data that the nmr chemical shift of the tetrel atom in the complex is a qualitative measure for the extent of multiple - bonding between the metal and the tetrel atom in these complexes . increased d e(ii ) bond is weaker in the bis(tetrylene ) zirconocene and hafnocene complexes 11 , 15 , 19 , and 21 than in the corresponding mono(tetrylene ) complexes 6 , 7 , 9 , and 10 . clearly , this is a result of the reduced multiple - bond character of the m e(ii ) linkage , due to the competition between two acceptor -type orbitals for one metal d - orbital in the metallocene bis(tetrylene ) complexes . e -back - bonding in the titanium complexes 5 and 8 is already small and is not further reduced by the addition of a second tetrylene unit in complexes 13 and 20 . e(ii ) bonds in compounds 5 , 8 , 13 , and 20 are predicted by the calculations . for group 4 metallocene monotetrylene complexes 510 : an equimolar ( 0.5 mmol each ) mixture of group 4 metallocene dichloride , magnesium turnings , and 1 or 2 was stirred in thf ( 5 ml ) for 3 h. during this time deeply colored solutions formed . the crystals containing cp2tin(sime3)2 were presumably formed by reduction of cp2ticl2 with magnesium in the presence of a batch of 3 which was contaminated with kn(sime3)2 from the synthesis of 3 .

the coding region of fat-1 cdna was optimized for mammalian cell expression , and the resultant cdna , renamed mfat-1 , was synthesized by geneart . the mfat-1 expression cassette , consisting of the mfat-1 cdna driven by a cytomegalovirus ( cmv ) enhancer and chicken -actin promoter tethered with a muscle creatine kinase ( mck ) enhancer ( supplemental fig . 1 , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0284/dc1 ) , was introduced into c57bl/6 mice by pronuclear microinjection . all animal protocols were approved by the university of pittsburgh institutional animal care and usage committee . the mfat-1 cdna was inserted into an adenoviral shuttle vector under the control of cmv promoter . the generation of ad - mfat-1 viral vector and the large - scale preparation of ad - mfat-1 ( and ad - lacz ) vector were carried out by the viral core facility at the university of pittsburgh . rat insulinoma ins-1 cells cultured in rpmi-1640 medium were infected in the presence of 7.5 mmol / l glucose ( no serum ) with either a control ( ad - lacz ) or ad - mfat1 virus at a multiplicity of infection ( moi ) of 100 for 4 h. isolated islets were cultured overnight after isolation and were then infected for 4 h with ad - mfat1 or ad - lacz at 250500 moi ( 10 cells / islet ) . adenovirus - infected ins-1 cells or islets ( 48 h after ) were exposed to the following cytokines : 5 ng / ml human interleukin-1 ( il-1 ) , 100 ng / ml -interferon ( ifn- ) , and 10 ng / ml tumor necrosis factor- ( tnf- ) ( roche diagnostics ) for 48 h. each experiment was performed in triplicate . the cells or islets were double stained with propidium iodide ( pi ) and hoescst nuclear dye before imaging analysis using a confocal microscope for islets or an inverted fluorescent microscope for ins-1 cells . briefly , the pancreas was injected through the pancreatic duct with hanks ' buffered saline solution ( hbss ) containing collagenase ( type v ) , excised , incubated in hbss for 15 min at 37c , and then filtered through a 500-m wire mesh . the digested pancreas was rinsed with hbss , and islets were separated by ficoll - gradient centrifugation . insulin release from isolated islets or ins-1 cells was measured as previous described ( 11 ) with some modifications . ins-1 cells or groups of 10 islets of similar size were preincubated in krebs - ringer bicarbonate buffer ( krbb ) ( supplemented with 10 mmol / l hepes and 0.5% bsa ) and 2.8 mmol / l glucose for 1 h at 37c , washed once in krbb plus 1% bsa , and then incubated in 1 ml fresh krbb containing either low glucose ( 2.8 mmol / l ) or high glucose ( 11.1 for ins-1 or 22.2 mmol / l for islets ) for 30 min . in a parallel set of experiments , the ins-1 cells or the islets were incubated with krbb containing high glucose plus 10 nmol / l glucagon - like peptide-1 ( glp-1 ) . at the end of incubation , supernatant was collected and the cells or islets were harvested in krbb for elisa determination of secreted or cellular insulin content ( linco research ) . groups of 10 islets of similar size were preincubated in krbb and 1.1 mmol / l glucose for 1 h at 37c , washed once in krbb , and then incubated in 0.5 ml fresh krbb containing 1.1 mmol / l glucose plus 10 mmol / l leucine and 10 mmol / l glutamine for 60 min . at the end of incubation , supernatant was collected and the islets were harvested in krbb for measuring insulin content . groups of six islets of similar size were preincubated in krbb containing 2.8 mmol / l glucose with or without 50 ng / ml pertussis toxin ( ptx ) and 10 mol / l prostaglandin e2 ( pge2 ) for 12 h before the study of 22.2 mmol / l glucose - stimulated insulin secretion . the medium was collected and analyzed for insulin concentration . ins-1 cells were infected with ad - mfat-1 and ad - lacz for 4 h before being switched to the medium without viral particles . after 48 h the cells were treated with rpmi-1640 medium containing with il-1 ( 5 ng / ml ) or tnf- ( 10 ng / ml ) for various lengths of time . analysis of extra cellular signal related kinse 1/2 ( erk1/2 ) phosphorylation and ib- phosphorylation were performed using antibodies against phosphor - erk1/2 and phospo ib- , followed by control blots with antibodies against erk1/2 and i- , respectively . total rna was prepared using trizol reagent ( invitrogen , carlsbad , ca ) , treated with dnase i ( ambion , austin , tx ) , and reverse transcribed with a superscript kit ( invitrogen ) . the real - time pcrs were carried out on an abi7900 taqman machine with the following cycles : 50c for 2 min , one cycle ; 95c for 10 min , one cycle ; and 95c for 15 s and 60c for 1 min , 40 cycles . linearity of each assay was determined based on a co - efficient value of at least 0.99 for the standard curve . the cdna concentrations of all samples were within the linear range of the standard curve . the design of double - labeled probes and the flanking primers was carried out using primer - express software ( applied biosystems , foster city , ca ) . the probes for rat pancreatic duodenal homeobox-1 ( pdx-1 ) , insulin 1 , glucokinase ( gk ) , and 18s rrna were labeled with fam ( 5 ) and bhq-1 ( 3 ) . rat pdx-1 : probe , 5-6fam - atgaaatccaccaaagctcacgcgtg-3 ; forward primer , 5-ccgcgttcatctccctttc-3 ; reverse primer , 5-ctcctgcccactggctttt-3. rat insulin 1 : probe , 5-6fam - cagcacctttgtggtcctcacctgg-3 ; forward primer , 5-ctgcccaggcttttgtcaa-3 ; reverse primer , 5-tccccacacaccaggtacaga-3. rat gk : probe , 5-6fam - ccgtgtacaagctgcacccgagc-3 ; forward primer , 5-catcactgtgggcgtggat-3 , reverse primer , 5-ggcgtgaaaccgctcctt-3. rat 18s rrna : probe , 5-6fam - tggaccggcgcaagacggac-3 ; forward primer , 5-cgccgctagaggtgaaattc-3 ; reverse primer , 5-ttggcaaatgctttcgc tc-3. lipids extraction from tissues was performed according to the general technique of bligh and dyer ( 12 ) . identification of components was by comparison of retention times with those of authentic standards ( sigma ) . all paired comparisons were subject to a two - tailed student t test with p < 0.05 considered statistically significant . the coding region of fat-1 cdna was optimized for mammalian cell expression , and the resultant cdna , renamed mfat-1 , was synthesized by geneart . the mfat-1 expression cassette , consisting of the mfat-1 cdna driven by a cytomegalovirus ( cmv ) enhancer and chicken -actin promoter tethered with a muscle creatine kinase ( mck ) enhancer ( supplemental fig . 1 , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0284/dc1 ) , was introduced into c57bl/6 mice by pronuclear microinjection . all animal protocols were approved by the university of pittsburgh institutional animal care and usage committee . the mfat-1 cdna was inserted into an adenoviral shuttle vector under the control of cmv promoter . the generation of ad - mfat-1 viral vector and the large - scale preparation of ad - mfat-1 ( and ad - lacz ) vector were carried out by the viral core facility at the university of pittsburgh . rat insulinoma ins-1 cells cultured in rpmi-1640 medium were infected in the presence of 7.5 mmol / l glucose ( no serum ) with either a control ( ad - lacz ) or ad - mfat1 virus at a multiplicity of infection ( moi ) of 100 for 4 h. isolated islets were cultured overnight after isolation and were then infected for 4 h with ad - mfat1 or ad - lacz at 250500 moi ( 10 cells / islet ) . adenovirus - infected ins-1 cells or islets ( 48 h after ) were exposed to the following cytokines : 5 ng / ml human interleukin-1 ( il-1 ) , 100 ng / ml -interferon ( ifn- ) , and 10 ng / ml tumor necrosis factor- ( tnf- ) ( roche diagnostics ) for 48 h. each experiment was performed in triplicate . the cells or islets were double stained with propidium iodide ( pi ) and hoescst nuclear dye before imaging analysis using a confocal microscope for islets or an inverted fluorescent microscope for ins-1 cells . briefly , the pancreas was injected through the pancreatic duct with hanks ' buffered saline solution ( hbss ) containing collagenase ( type v ) , excised , incubated in hbss for 15 min at 37c , and then filtered through a 500-m wire mesh . the digested pancreas was rinsed with hbss , and islets were separated by ficoll - gradient centrifugation . insulin release from isolated islets or ins-1 cells was measured as previous described ( 11 ) with some modifications . ins-1 cells or groups of 10 islets of similar size were preincubated in krebs - ringer bicarbonate buffer ( krbb ) ( supplemented with 10 mmol / l hepes and 0.5% bsa ) and 2.8 mmol / l glucose for 1 h at 37c , washed once in krbb plus 1% bsa , and then incubated in 1 ml fresh krbb containing either low glucose ( 2.8 mmol / l ) or high glucose ( 11.1 for ins-1 or 22.2 mmol / l for islets ) for 30 min . in a parallel set of experiments , the ins-1 cells or the islets were incubated with krbb containing high glucose plus 10 nmol / l glucagon - like peptide-1 ( glp-1 ) . at the end of incubation , supernatant was collected and the cells or islets were harvested in krbb for elisa determination of secreted or cellular insulin content ( linco research ) . groups of 10 islets of similar size were preincubated in krbb and 1.1 mmol / l glucose for 1 h at 37c , washed once in krbb , and then incubated in 0.5 ml fresh krbb containing 1.1 mmol / l glucose plus 10 mmol / l leucine and 10 mmol / l glutamine for 60 min . at the end of incubation , groups of six islets of similar size were preincubated in krbb containing 2.8 mmol / l glucose with or without 50 ng / ml pertussis toxin ( ptx ) and 10 mol / l prostaglandin e2 ( pge2 ) for 12 h before the study of 22.2 mmol / l glucose - stimulated insulin secretion . ins-1 cells were infected with ad - mfat-1 and ad - lacz for 4 h before being switched to the medium without viral particles . after 48 h the cells were treated with rpmi-1640 medium containing with il-1 ( 5 ng / ml ) or tnf- ( 10 ng / ml ) for various lengths of time . analysis of extra cellular signal related kinse 1/2 ( erk1/2 ) phosphorylation and ib- phosphorylation were performed using antibodies against phosphor - erk1/2 and phospo ib- , followed by control blots with antibodies against erk1/2 and i- , respectively . total rna was prepared using trizol reagent ( invitrogen , carlsbad , ca ) , treated with dnase i ( ambion , austin , tx ) , and reverse transcribed with a superscript kit ( invitrogen ) . the real - time pcrs were carried out on an abi7900 taqman machine with the following cycles : 50c for 2 min , one cycle ; 95c for 10 min , one cycle ; and 95c for 15 s and 60c for 1 min , 40 cycles . linearity of each assay was determined based on a co - efficient value of at least 0.99 for the standard curve . the cdna concentrations of all samples were within the linear range of the standard curve . the design of double - labeled probes and the flanking primers was carried out using primer - express software ( applied biosystems , foster city , ca ) . the probes for rat pancreatic duodenal homeobox-1 ( pdx-1 ) , insulin 1 , glucokinase ( gk ) , and 18s rrna were labeled with fam ( 5 ) and bhq-1 ( 3 ) . rat pdx-1 : probe , 5-6fam - atgaaatccaccaaagctcacgcgtg-3 ; forward primer , 5-ccgcgttcatctccctttc-3 ; reverse primer , 5-ctcctgcccactggctttt-3. rat insulin 1 : probe , 5-6fam - cagcacctttgtggtcctcacctgg-3 ; forward primer , 5-ctgcccaggcttttgtcaa-3 ; reverse primer , 5-tccccacacaccaggtacaga-3. rat gk : probe , 5-6fam - ccgtgtacaagctgcacccgagc-3 ; forward primer , 5-catcactgtgggcgtggat-3 , reverse primer , 5-ggcgtgaaaccgctcctt-3. rat 18s rrna : probe , 5-6fam - tggaccggcgcaagacggac-3 ; forward primer , 5-cgccgctagaggtgaaattc-3 ; reverse primer , 5-ttggcaaatgctttcgc tc-3. lipids extraction from tissues was performed according to the general technique of bligh and dyer ( 12 ) . identification of components was by comparison of retention times with those of authentic standards ( sigma ) . all paired comparisons were subject to a two - tailed student t test with p < 0.05 considered statistically significant . although the newly generated transgenic mouse model is similar to the one reported earlier ( called fat-1 mice ) ( 13 ) , the codons of fat-1 cdna are further optimized for efficient translation in mammalian systems ( hence renamed the generation of mfat-1 mice was not only verified through genotyping but also confirmed by examining the relative content of n-6 and n-3 pufas in the tails as well as in isolated pancreatic islets ( table 1 ) . in both tissue samples of mfat-1 transgenic mice there was a significant increase in n-3 pufas ( 2.6- and 3.3-fold in tails and islets , respectively ) , accompanied by a prominent decrease in n-6 pufas ( 2.7- and 3.8-fold in tails and islets , respectively ) , which resulted in sharply reduced n-6to n-3 ratios relative to those in the wild - type tissues ( table 1 ) . interestingly , the total percentage of n-3 plus n-6 pufas in mfat-1 transgenic tissues was modestly reduced , which corresponded to a minor percentage increase in some other fatty acid species , primarily reflected in the most dominant peak of oleic acid ( see legend to table 1 ) . similar observations have been made in previously reported fat-1expressing tissues or cells ( 14,15 ) . although the underlying reason for this observation is still not clear , it is likely due to the increased preferential usage of n-3 pufas as substrates by various enzymes in mfat-1expressing tissues or cells , which might have contributed to the reduced inflammatory states described below . analysis of n-6 and n-3 pufa composition data are means sd . the tissues ( 50 mg of tail samples or 200 islets ) were collected from the mfat-1 transgenic or wild - type control mice . fatty acid analysis was also performed on the ins-1 or the wild - type islets infected with adenoviral vectors carrying either mfat-1 or lacz cdna . the compositions of n-6 or n-3 pufas were expressed using relative percentages , i.e. , the distribution areas of n-3 or n-6 pufa peaks divided by the total peak areas of all detectable saturated and unsaturated free fatty acids ( from the same sample ) resolved from the gas chromatography column . in the transgenic tissues , the modest drop in relative percentages of n-3 plus n-6 pufas corresponded to a minor percentage increase in some other detectable fatty acid species . however , the increase was primarily reflected in the most dominant peak of oleic acids ( tails , 26.9% [ wt ] vs. 33.0% [ mfat-1 ] ; islets , 17.6% [ wt ] vs. 22.8% [ mfat-1 ] ) . the specificity of mfat-1 enzymatic activity primarily caused the changes of n-6 and n-3 pufas . p < 0.001 when mfat-1 group compared with corresponding control ( wt or lacz ) group . we investigated the impact of cellular production of n-3 pufas on insulin secretion . in response to high glucose stimulation , the levels of insulin secretion were significantly higher in the islets isolated from the mfat-1 transgenic than in those from the wild type ( fig . addition of glp-1 further stimulated insulin secretion in both wild - type and transgenic islets , but the level was significantly higher in mfat-1 transgenic islets ( fig . the enhanced insulin secretion of mfat-1 transgenic islets was unlikely due to enlarged islet size because confocal microscopy analysis of insulin - stained pancreatic sections did not find significant difference in islet sizes between the transgenic and wild - type groups ( supplemental fig . although the levels of insulin secretion were expressed in absolute concentrations ( nanograms of secreted insulin / islet , fig . 1a ) , these insulin secretion patterns still held up after we expressed them in relative concentrations ( secreted insulin as a percentage of total insulin content , supplemental fig . these data depict the enhanced insulin - secreting action of the -cells that were pre - exposed to a high n-3 pufa environment ( as a result of mfat-1 expression ) even long before birth . we wondered if acute elevation of n-3 pufas via mfat-1 activity in the -cells would also enhance insulin secretion . to this end , we used an adenoviral expression vector to introduce mfat-1 cdna ( or the control bacterial lacz gene ) into the islets originally isolated from wild - type mice and into a rat insulinoma cell line , ins-1 . owing to mfat-1 expression , the cellular levels of n-3 pufas were sharply elevated , and the levels of n-6 pufas and the ratios of n-6 to n-3 were significantly decreased in these virally transduced cells ( table 1 ) . the virally induced expression of mfat-1 in the islets and ins-1 cells caused significant enhancement in glucose- and glp-1stimulated insulin secretion ( fig . interestingly , even at the baseline of 2.8 mmol / l , glucose could evoke significantly higher insulin secretion in mfat-1expressing islets ( though not necessarily in the insulinoma ins-1 cells ) than in control islets ( fig . , the relative fold of stimulation did not differ between the transgenic and wild - type islets ( supplemental fig . 3e and f ) , although this does not seem to be the case for the insulinoma ins-1 cells ( supplemental fig . such observation made us wonder if the high glucose concentrations only amplified an enhanced secretion mechanism in the mfat-1expressing islets and prompted us to inquire if the enhancement of insulin secretion as a result of cell production of n-3 pufas is beyond the scope of high glucose and glp-1 . to this end , we tested whether certain types of amino acid induced insulin secretion could also be elevated in mfat-1 transgenic islets . we adopted a widely established protocol to evaluate this issue by applying leucine and glutamine to the culture of mfat-1 transgenic islets in the presence of low concentrations of glucose ( 1.1 mmol / l ) ( 16 ) . although the amino acid mixtures stimulated insulin secretion on top of the very low secretion levels elicited by 1.1 mmol / l glucose , there was a significant increase of insulin secretion in the mfat-1expressing islets above that in the wild - type islets ( fig . 1d and supplemental fig . 3d ) . in addition , the fold of stimulation of insulin secretion by amino acids was also substantially higher in mfat-1expressing islets ( supplemental fig . it should be noted that elevation of n-3 pufas in islets ( or ins-1 cells ) via the mfat-1 gene ( delivered either transgenically or virally ) did not alter total insulin content ( supplemental fig . the broad positive impact on glucose , amino acids , and glp-1induced insulin release observed in such engineered islets was likely due to the modulation of secretory mechanisms . insulin secretion in response to glucose , amino acids , and glp-1 stimulation in transgenic or adenoviral - driven expression of mfat-1 islets or ins-1 cells . a : islets isolated from the transgenic ( mfat-1 ) or wild - type ( wt ) mice were incubated with different concentrations of glucose in the presence or absence of glp-1 ( 10 nmol / l ) for 30 min . b and c : isolated islets from wild - type mice ( b ) or ins-1 cells ( c ) were preinfected with adenovirus carrying mfat-1 or lacz genes for 24 h before the study of insulin secretion , as outlined in a. d : islets isolated from wild - type and mfat-1 transgenic mice were incubated with 1.1 mmol / l glucose in the presence or absence of 10 mmol / l leucine ( leu ) and 10 mmol / l glutamine ( glu ) for 60 min before the medium was collected for measurement of secreted insulin . in a d , the levels of secreted insulin ( ng / islet ) in each panel were the means + sem of three independent experiments with each condition carried out in five or six replicates and 1020 islets in each replicate . p < 0.01 , * p < 0.05 when compared with the control group . to assess the impact of cellular production of n-3 pufas on the viability of pancreatic islets , we incubated the isolated islets with a mixture of tnf- , il-1 , and ifn- at the concentrations typically applied in previously published studies ( 17 ) . double nuclear staining was applied to the live islets using propidium iodide and hoechst dye for staining dead cells and all the cells , respectively . as expected , cytokine treatment for 48 h led to a sharply elevated level of cell death in the wild - type islets ( 58 vs. 9% in the nontreated , fig . 2a and b ) ; however , the islets derived from the mfat-1 transgenic mice strongly resisted cytokine - induced cell death ( 13 vs. 8% in the nontreated islets , fig . a and b : isolated islets from transgenic or wild type were challenged with the cytokine mix for 48 h before fluorescent confocal imaging . c and d : islets derived from wild - type mice ( c ) or ins-1 cells ( d ) were infected with adenovirus carrying either mfat-1 or lacz gene for 24 h before being challenged with the cytokine mix for 48 h. the live islets or cells were incubated with propidium iodide and hoechst dye before imaging analysis . the images were collected under either a confocal ( for islets ) or regular ( for ins-1 ) fluoresecent microscope . the rates of cell death were calculated based on at least four randomly selected sections or areas of the images . ( a high - quality digital representation of this figure is available in the online issue . ) introduction of mfat-1 cdna into wild - type islets or cultured -cells using an adenoviral vector also provided strong protection against cytokine - induced cell death . after infected with the adenovirus carrying mfat-1 cdna in vitro , the ins-1 cells or pancreatic islets originally derived from the wild - type mice displayed significantly reduced dead cells after cytokine challenge ( fig . 2c and d ) , which was in sharp contrast to the high death rates observed in the control islets or ins-1 cells infected with the lacz - carrying adenovirus ( fig . 2c and d ) . the strong resistance to cytokine - induced cell death led us to investigate the ability of cytokines to signal in mfat-1expressing -cells . to this end , we infected ins-1 cells with adenovirus carrying either mfat-1 or lacz genes and then evaluated the activation of erk1/2 and nf-b , two canonical pathways for il-1 and tnf- , respectively ( 1820 ) . although , as expected , il-1 significantly stimulated the activity of erk1/2 in the lacz - expressing ins-1 cells , it failed to do so in the mfat-1expressing cells ( fig . , tnf- evoked a robust and temporally dependent phosphorylation of ib- , a critical early step for the activation of nf-b signaling pathway , in the lacz - expressing ins-1 cells ( fig . 3c and d ) . however , the phosphorylation of ib- induced by tnf- was sharply attenuated and even delayed in the mfat-1expressing cells ( fig . such differential cytokine signaling pattern is very likely to have contributed to strong resistance of mfat-1expressing -cells to cytokine - induced cell death . activation of erk1/2 and nf-b in ins-1 cells infected with ad - mfat-1 or ad - lacz . ins-1 cells were infected with ad - mfat-1 or ad - lacz for 4 h. approximately 48 h later , the cells were exposed to il-1 ( 5 ng / ml ) ( a and b ) or tnf- ( 10 ng / ml ) ( c and d ) for the indicated time . the cells were then extracted for protein for western blot assays of erk1/2 and ib- phosphorylation . p < 0.01 , * p < 0.05 when compared with the corresponding control group . to gain further mechanistic understanding of the aforementioned phenotypes related to insulin secretion , we focused on the roles of inflammation modulation . elevation of n-3 pufas is known to suppress the production of pge2 ( 21,22 ) , a negative regulator of glucose - stimulated insulin secretion ( 23,24 ) . we found that pge2 concentration was significantly reduced in the medium incubated with the mfat-1expressing ins-1 cells relative to that incubated with the lacz - expressing control cells ( fig . the medium culturing the mfat-1 transgenic islets contained less pge2 than the one culturing the wild - type islets ( fig . 4b ) . as expected , addition of arachidonic acid ( aa , an n-6 pufa ) , a substrate of cycloxygenase-2 ( cox-2 ) for pge2 synthesis , to the incubation medium increased pge2 secretion from ins-1 cells ; however , such an increase was significantly attenuated in mfat-1expressing ins-1 cells ( fig . 4a ) . preincubation with pge2 ( 10 mol / l ) for 12 h attenuated glucose - stimulated insulin secretion in the mfat-1 transgenic and wild - type islets ( fig . 4c ) . importantly , pretreatment with pge2 completely neutralized the mfat-1caused enhancement in insulin secretion ( fig . 4c , no statistical significance when comparing mfat-1+pge2 vs. wild - type without pge2 ) , suggesting that the reduction of pge2 synthesis plays a critical role in n-3 pufa regulated insulin secretion . inhibitory regulatory g - protein ( gi ) has been thought as a major signaling component in mediating the actions of pge2 ( 25,26 ) . preincubation of pancreatic islets with pertussis toxin along with pge2 , a known inhibitor of gi ( 27,28 ) , partially rescued insulin secretion in the wild - type islets and completely neutralized the inhibitory effect of pge2 on insulin secretion ( fig . 4c ) , suggesting that the aforementioned action of pge2 was mediated at least in part through gi . finally , in analyzing gene expression , we further found that elevation of n-3 pufas through mfat-1 activity in the -cells could lead to changes in the expression of certain genes that are critical to the functions and viability of -cells . for example , the mrna levels of pdx-1 , gk , and insulin ( insulin1 ) were significantly increased in the ins-1 cells infected with adenoviruses carrying mfat-1 cdna relative to the lacz - expressing control cells ( fig . pge2 production and the effects of pge2 and pertussis toxin on insulin secretion in mfat-1expressing ins-1 cells and islets . a : measurement pge2 concentrations in the medium incubated with ins-1 cells ( preinfected with ad - mfat-1 or ad - lacz ) in the presence or absence of aa ( 3 or 9 mol / l ) for 30 min . b : the concentrations of pge2 in the medium incubated for 30-min with isolated islets from mfat-1 transgenic or wild - type mice . c : islets isolated from the transgenic or wild - type mice were preincubated with 10 mol / l pge2 in the presence or absence of 50 ng / ml ptx for 12 h before the study of glucose - stimulated insulin secretion . the results are means + sem from three independent repeats with each condition carried out in eight replicates and six islets in each replicate . p < 0.01 , * p < 0.05 when mfat-1 group compared with corresponding control group real - time pcr assays of mrna expression of pdx-1 , gk , and insulin 1 in the ins-1 cells infected with ad - mfat-1 or ad - lacz . p < 0.01 , * p < 0.05 when compared with control ( ad - lacz ) cells . in this study , we have shown that cellular increase of n-3 pufas and reduction of n-6 pufas through transgenic expression of mfat-1 enhances glucose , amino acid , and glp-1stimulated insulin secretion in isolated pancreatic islets and renders the islets strongly resistant to cytokine - induced cell death . acute and stable elevation of n-3 pufas through adenoviral transduction of mfat-1 gene in isolated islets and in ins-1 cells was able to recapitulate the observations in the mfat-1 transgenic islets . although the underlying mechanisms remain to be systematically investigated , initial studies described here offer some mechanistic insights into how mfat-1 expression ( and the subsequent changes in pufa lipid species ) impact insulin secretion as well as the survival of pancreatic -cells . for example , pge2 has been well demonstrated as a negative regulator of insulin secretion ( 23,24 ) . the reduction of pge2 synthesis in the mfat-1expressing cells or islets was the result of decreased availability of aa , an n-6 pufa , to cox-2 enzyme , as mfat-1 activity converted aa to epa ( eicosapentaenoic acid ) . consistent with this concept , aa - induced synthesis of pge2 was also attenuated in mfat-1expressing cells ( fig . though not necessarily the only mechanism , the regulation of pge2 largely contributed to the elevation of insulin secretion in mfat-1expressing islets , since addition of 10 mol / l pge2 to the islet culture almost completely eliminated the increased portion of insulin secretion ( relative to that in the wild - type islets without pge2 treatment ) ( fig . in strengthening this argument , we have revealed that the negative impact of pge2 on insulin secretion is mediated at least in part by gi , as pertussis toxin was able to neutralize the action of pge2 while co - incubated with mfat-1 transgenic and wild - type islets . thus , the reduction of pge2 secretion from the mfat-1expressing islets and -cells is critical for the enhanced insulin secretion , suggesting that the control of inflammation through reduction of n-6 pufas can have a beneficial effect on -cell functions . the strong resistance to cytokine - caused destruction in mfat-1expressing islets and ins-1 cells was also reflected at the level of cytokine signaling . specifically , we have shown that cytokine - induced activation of erk1/2 and nf-b pathways was significantly suppressed , both of which were critical for cytokines ' actions . we should emphasize that the attenuation of tnf-activated nf-b signaling is not necessarily confined to the pancreatic -cells only . our recent studies have found similar observations in fat-1 transgenic hepatocytes ( j.l . and a.z . , unpublished observations ) . thus , the control of cytokine signaling appears to be a common theme that has broad physiological implications . although it is predicted to enhance insulin signaling and sensitivity in the liver tissue , the reduction of tnf-activated nf-b signaling leads to protection from cytokine - inflicted islet destruction . finally , from the perspective of gene expression , mfat-1induced increase in pdx-1 , glucokinase , and insulin 1 expression may have also played important roles in improved insulin secretion and -cell survival . modern diets contain excessive n-6 pufas but very low levels of n-3 pufas , which results in an unhealthy n-6to since the eicosanoid products derived from n-6 pufas are more potent mediators of inflammation than the similar products derived from n-3 pufas , an imbalanced n-6to n-3 ratio in favor of n-6 pufas will be highly proinflammatory , which is believed to have contributed to the pathogenesis of such modern diseases as autoimmune disorders and diabetes ( 1,30 ) . the production of n-3 pufas in the -cells and islets via mfat-1 activity is achieved by using n-6 pufas as the substrates , thus bringing down the ratios of n-6 to n-3 as well as the inflammatory state . thus , although the increase of n-3 pufas is critical , the decrease of n-6 pufas may be just as important in determining the phenotypes of mfat-1expressing islets . in keeping with this concept , we have already shown that the reduction of pge2 secretion from the mfat-1expressing islets and -cells is critical for the enhanced insulin secretion , suggesting that the control of inflammation through reduction of n-6 pufas is a main theme in the regulation of -cell functions by n-3 pufas . recent clinical studies have established that daily compensation of n-3 pufas starting at 1 year of age can have clear benefits against the development of type 1 diabetes and islet autoimmunity ( 6 ) . consistent with these clinical observations , studies in rodents have shown that administration of n-3 pufas could restore palmitate- or linoleic acid impaired insulin secretion ( 8,9 ) . although we can not fully equate our transgenic approach to the dietary means of gaining n-3 pufas , both methods provide the same types of n-3 pufas to the whole body . the results presented here suggest that elevation of cellular n-3 pufas has a direct beneficial impact on the survival of pancreatic -cells from inflammation attack and that they may explain mechanistically why increased intake of n-3 pufas in early childhood can help reduce the incidence of type 1 diabetes . our data may also add n-3 pufas to the growing list of factors that can help improve the functions and viability of -cells . thus , it warrants further investigation whether mfat-1 expression in disease models such as nonobese diabetic ( nod ) mice can mitigate the development of type 1 diabetes . from a therapeutic perspective , the phenotypes of the islets infected with mfat-1containing adenovirus bear strong clinical relevance . engineered islets with enhanced insulin secretion and viability are always sought to overcome inflammatory destruction and the scarcity of available islets ( 31,32 ) . in this context , the mfat-1expressing islets should be urgently tested in transplantation settings and may represent an additional tool for the treatment of type 1 diabetes .

objectiveto evaluate the direct impact of n-3 polyunsaturated fatty acids ( n-3 pufas ) on the functions and viability of pancreatic -cells.research design and methodswe developed an mfat-1 transgenic mouse model in which endogenous production of n-3 pufas was achieved through overexpressing a c. elegans n-3 fatty acid desaturase gene , mfat-1 . the islets and ins-1 cells expressing mfat-1 were analyzed for insulin secretion and viability in response to cytokine treatment.resultsthe transgenic islets contained much higher levels of n-3 pufas and lower levels of n-6 pufas than the wild type . insulin secretion stimulated by glucose , amino acids , and glucagon - like peptide-1 ( glp-1 ) was significantly elevated in the transgenic islets . when challenged with tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and -interferon ( ifn- ) , the transgenic islets completely resisted cytokine - induced cell death . adenoviral transduction of mfat-1 gene in wild - type islets and in ins-1 cells led to acute changes in the cellular levels of n-3- and n-6 pufas and recapitulated the results in the transgenic islets . the expression of mfat-1 led to decreased production of prostaglandin e2 ( pge2 ) , which in turn contributed to the elevation of insulin secretion . we further found that cytokine - induced activation of nf-b and extracellular signal related kinase 1/2 ( erk1/2 ) was significantly attenuated and that the expression of pancreatic duodenal hemeobox-1 ( pdx-1 ) , glucokinase , and insulin-1 was increased as a result of n-3 pufa production.conclusionsstable cellular production of n-3 pufas via mfat-1 can enhance insulin secretion and confers strong resistance to cytokine - induced -cell destruction . the utility of mfat-1 gene in deterring type 1 diabetes should be further explored in vivo .

RESEARCH DESIGN AND METHODS Generation of mfat-1 transgenic mice. Ad- Cytokine treatment and imaging analysis. Islet isolation, glucose-stimulated insulin secretion in islets or INS-1 cells, and quantitative analysis of gene expression levels. Leucine and glutamine-stimulated insulin secretion in isolated islets. Pertussis toxin and prostaglandin E Western blotting. Quantitative analysis of gene expression levels in INS-1 cells. Gas chromatography analysis of fatty acid compositions. Statistical analysis. RESULTS DISCUSSION Supplementary Material

adenovirus - infected ins-1 cells or islets ( 48 h after ) were exposed to the following cytokines : 5 ng / ml human interleukin-1 ( il-1 ) , 100 ng / ml -interferon ( ifn- ) , and 10 ng / ml tumor necrosis factor- ( tnf- ) ( roche diagnostics ) for 48 h. each experiment was performed in triplicate . in a parallel set of experiments , the ins-1 cells or the islets were incubated with krbb containing high glucose plus 10 nmol / l glucagon - like peptide-1 ( glp-1 ) . groups of six islets of similar size were preincubated in krbb containing 2.8 mmol / l glucose with or without 50 ng / ml pertussis toxin ( ptx ) and 10 mol / l prostaglandin e2 ( pge2 ) for 12 h before the study of 22.2 mmol / l glucose - stimulated insulin secretion . analysis of extra cellular signal related kinse 1/2 ( erk1/2 ) phosphorylation and ib- phosphorylation were performed using antibodies against phosphor - erk1/2 and phospo ib- , followed by control blots with antibodies against erk1/2 and i- , respectively . the probes for rat pancreatic duodenal homeobox-1 ( pdx-1 ) , insulin 1 , glucokinase ( gk ) , and 18s rrna were labeled with fam ( 5 ) and bhq-1 ( 3 ) . adenovirus - infected ins-1 cells or islets ( 48 h after ) were exposed to the following cytokines : 5 ng / ml human interleukin-1 ( il-1 ) , 100 ng / ml -interferon ( ifn- ) , and 10 ng / ml tumor necrosis factor- ( tnf- ) ( roche diagnostics ) for 48 h. each experiment was performed in triplicate . in a parallel set of experiments , the ins-1 cells or the islets were incubated with krbb containing high glucose plus 10 nmol / l glucagon - like peptide-1 ( glp-1 ) . at the end of incubation , groups of six islets of similar size were preincubated in krbb containing 2.8 mmol / l glucose with or without 50 ng / ml pertussis toxin ( ptx ) and 10 mol / l prostaglandin e2 ( pge2 ) for 12 h before the study of 22.2 mmol / l glucose - stimulated insulin secretion . analysis of extra cellular signal related kinse 1/2 ( erk1/2 ) phosphorylation and ib- phosphorylation were performed using antibodies against phosphor - erk1/2 and phospo ib- , followed by control blots with antibodies against erk1/2 and i- , respectively . the probes for rat pancreatic duodenal homeobox-1 ( pdx-1 ) , insulin 1 , glucokinase ( gk ) , and 18s rrna were labeled with fam ( 5 ) and bhq-1 ( 3 ) . although the newly generated transgenic mouse model is similar to the one reported earlier ( called fat-1 mice ) ( 13 ) , the codons of fat-1 cdna are further optimized for efficient translation in mammalian systems ( hence renamed the generation of mfat-1 mice was not only verified through genotyping but also confirmed by examining the relative content of n-6 and n-3 pufas in the tails as well as in isolated pancreatic islets ( table 1 ) . in both tissue samples of mfat-1 transgenic mice there was a significant increase in n-3 pufas ( 2.6- and 3.3-fold in tails and islets , respectively ) , accompanied by a prominent decrease in n-6 pufas ( 2.7- and 3.8-fold in tails and islets , respectively ) , which resulted in sharply reduced n-6to n-3 ratios relative to those in the wild - type tissues ( table 1 ) . interestingly , the total percentage of n-3 plus n-6 pufas in mfat-1 transgenic tissues was modestly reduced , which corresponded to a minor percentage increase in some other fatty acid species , primarily reflected in the most dominant peak of oleic acid ( see legend to table 1 ) . although the underlying reason for this observation is still not clear , it is likely due to the increased preferential usage of n-3 pufas as substrates by various enzymes in mfat-1expressing tissues or cells , which might have contributed to the reduced inflammatory states described below . fatty acid analysis was also performed on the ins-1 or the wild - type islets infected with adenoviral vectors carrying either mfat-1 or lacz cdna . , the distribution areas of n-3 or n-6 pufa peaks divided by the total peak areas of all detectable saturated and unsaturated free fatty acids ( from the same sample ) resolved from the gas chromatography column . in the transgenic tissues , the modest drop in relative percentages of n-3 plus n-6 pufas corresponded to a minor percentage increase in some other detectable fatty acid species . we investigated the impact of cellular production of n-3 pufas on insulin secretion . in response to high glucose stimulation , the levels of insulin secretion were significantly higher in the islets isolated from the mfat-1 transgenic than in those from the wild type ( fig . addition of glp-1 further stimulated insulin secretion in both wild - type and transgenic islets , but the level was significantly higher in mfat-1 transgenic islets ( fig . the enhanced insulin secretion of mfat-1 transgenic islets was unlikely due to enlarged islet size because confocal microscopy analysis of insulin - stained pancreatic sections did not find significant difference in islet sizes between the transgenic and wild - type groups ( supplemental fig . these data depict the enhanced insulin - secreting action of the -cells that were pre - exposed to a high n-3 pufa environment ( as a result of mfat-1 expression ) even long before birth . we wondered if acute elevation of n-3 pufas via mfat-1 activity in the -cells would also enhance insulin secretion . owing to mfat-1 expression , the cellular levels of n-3 pufas were sharply elevated , and the levels of n-6 pufas and the ratios of n-6 to n-3 were significantly decreased in these virally transduced cells ( table 1 ) . the virally induced expression of mfat-1 in the islets and ins-1 cells caused significant enhancement in glucose- and glp-1stimulated insulin secretion ( fig . , the relative fold of stimulation did not differ between the transgenic and wild - type islets ( supplemental fig . such observation made us wonder if the high glucose concentrations only amplified an enhanced secretion mechanism in the mfat-1expressing islets and prompted us to inquire if the enhancement of insulin secretion as a result of cell production of n-3 pufas is beyond the scope of high glucose and glp-1 . to this end , we tested whether certain types of amino acid induced insulin secretion could also be elevated in mfat-1 transgenic islets . we adopted a widely established protocol to evaluate this issue by applying leucine and glutamine to the culture of mfat-1 transgenic islets in the presence of low concentrations of glucose ( 1.1 mmol / l ) ( 16 ) . although the amino acid mixtures stimulated insulin secretion on top of the very low secretion levels elicited by 1.1 mmol / l glucose , there was a significant increase of insulin secretion in the mfat-1expressing islets above that in the wild - type islets ( fig . in addition , the fold of stimulation of insulin secretion by amino acids was also substantially higher in mfat-1expressing islets ( supplemental fig . it should be noted that elevation of n-3 pufas in islets ( or ins-1 cells ) via the mfat-1 gene ( delivered either transgenically or virally ) did not alter total insulin content ( supplemental fig . the broad positive impact on glucose , amino acids , and glp-1induced insulin release observed in such engineered islets was likely due to the modulation of secretory mechanisms . insulin secretion in response to glucose , amino acids , and glp-1 stimulation in transgenic or adenoviral - driven expression of mfat-1 islets or ins-1 cells . a : islets isolated from the transgenic ( mfat-1 ) or wild - type ( wt ) mice were incubated with different concentrations of glucose in the presence or absence of glp-1 ( 10 nmol / l ) for 30 min . b and c : isolated islets from wild - type mice ( b ) or ins-1 cells ( c ) were preinfected with adenovirus carrying mfat-1 or lacz genes for 24 h before the study of insulin secretion , as outlined in a. d : islets isolated from wild - type and mfat-1 transgenic mice were incubated with 1.1 mmol / l glucose in the presence or absence of 10 mmol / l leucine ( leu ) and 10 mmol / l glutamine ( glu ) for 60 min before the medium was collected for measurement of secreted insulin . to assess the impact of cellular production of n-3 pufas on the viability of pancreatic islets , we incubated the isolated islets with a mixture of tnf- , il-1 , and ifn- at the concentrations typically applied in previously published studies ( 17 ) . as expected , cytokine treatment for 48 h led to a sharply elevated level of cell death in the wild - type islets ( 58 vs. 9% in the nontreated , fig . 2a and b ) ; however , the islets derived from the mfat-1 transgenic mice strongly resisted cytokine - induced cell death ( 13 vs. 8% in the nontreated islets , fig . c and d : islets derived from wild - type mice ( c ) or ins-1 cells ( d ) were infected with adenovirus carrying either mfat-1 or lacz gene for 24 h before being challenged with the cytokine mix for 48 h. the live islets or cells were incubated with propidium iodide and hoechst dye before imaging analysis . introduction of mfat-1 cdna into wild - type islets or cultured -cells using an adenoviral vector also provided strong protection against cytokine - induced cell death . after infected with the adenovirus carrying mfat-1 cdna in vitro , the ins-1 cells or pancreatic islets originally derived from the wild - type mice displayed significantly reduced dead cells after cytokine challenge ( fig . 2c and d ) , which was in sharp contrast to the high death rates observed in the control islets or ins-1 cells infected with the lacz - carrying adenovirus ( fig . the strong resistance to cytokine - induced cell death led us to investigate the ability of cytokines to signal in mfat-1expressing -cells . , tnf- evoked a robust and temporally dependent phosphorylation of ib- , a critical early step for the activation of nf-b signaling pathway , in the lacz - expressing ins-1 cells ( fig . such differential cytokine signaling pattern is very likely to have contributed to strong resistance of mfat-1expressing -cells to cytokine - induced cell death . elevation of n-3 pufas is known to suppress the production of pge2 ( 21,22 ) , a negative regulator of glucose - stimulated insulin secretion ( 23,24 ) . we found that pge2 concentration was significantly reduced in the medium incubated with the mfat-1expressing ins-1 cells relative to that incubated with the lacz - expressing control cells ( fig . the medium culturing the mfat-1 transgenic islets contained less pge2 than the one culturing the wild - type islets ( fig . as expected , addition of arachidonic acid ( aa , an n-6 pufa ) , a substrate of cycloxygenase-2 ( cox-2 ) for pge2 synthesis , to the incubation medium increased pge2 secretion from ins-1 cells ; however , such an increase was significantly attenuated in mfat-1expressing ins-1 cells ( fig . preincubation with pge2 ( 10 mol / l ) for 12 h attenuated glucose - stimulated insulin secretion in the mfat-1 transgenic and wild - type islets ( fig . 4c , no statistical significance when comparing mfat-1+pge2 vs. wild - type without pge2 ) , suggesting that the reduction of pge2 synthesis plays a critical role in n-3 pufa regulated insulin secretion . preincubation of pancreatic islets with pertussis toxin along with pge2 , a known inhibitor of gi ( 27,28 ) , partially rescued insulin secretion in the wild - type islets and completely neutralized the inhibitory effect of pge2 on insulin secretion ( fig . finally , in analyzing gene expression , we further found that elevation of n-3 pufas through mfat-1 activity in the -cells could lead to changes in the expression of certain genes that are critical to the functions and viability of -cells . for example , the mrna levels of pdx-1 , gk , and insulin ( insulin1 ) were significantly increased in the ins-1 cells infected with adenoviruses carrying mfat-1 cdna relative to the lacz - expressing control cells ( fig . b : the concentrations of pge2 in the medium incubated for 30-min with isolated islets from mfat-1 transgenic or wild - type mice . c : islets isolated from the transgenic or wild - type mice were preincubated with 10 mol / l pge2 in the presence or absence of 50 ng / ml ptx for 12 h before the study of glucose - stimulated insulin secretion . p < 0.01 , * p < 0.05 when mfat-1 group compared with corresponding control group real - time pcr assays of mrna expression of pdx-1 , gk , and insulin 1 in the ins-1 cells infected with ad - mfat-1 or ad - lacz . in this study , we have shown that cellular increase of n-3 pufas and reduction of n-6 pufas through transgenic expression of mfat-1 enhances glucose , amino acid , and glp-1stimulated insulin secretion in isolated pancreatic islets and renders the islets strongly resistant to cytokine - induced cell death . acute and stable elevation of n-3 pufas through adenoviral transduction of mfat-1 gene in isolated islets and in ins-1 cells was able to recapitulate the observations in the mfat-1 transgenic islets . although the underlying mechanisms remain to be systematically investigated , initial studies described here offer some mechanistic insights into how mfat-1 expression ( and the subsequent changes in pufa lipid species ) impact insulin secretion as well as the survival of pancreatic -cells . for example , pge2 has been well demonstrated as a negative regulator of insulin secretion ( 23,24 ) . though not necessarily the only mechanism , the regulation of pge2 largely contributed to the elevation of insulin secretion in mfat-1expressing islets , since addition of 10 mol / l pge2 to the islet culture almost completely eliminated the increased portion of insulin secretion ( relative to that in the wild - type islets without pge2 treatment ) ( fig . in strengthening this argument , we have revealed that the negative impact of pge2 on insulin secretion is mediated at least in part by gi , as pertussis toxin was able to neutralize the action of pge2 while co - incubated with mfat-1 transgenic and wild - type islets . thus , the reduction of pge2 secretion from the mfat-1expressing islets and -cells is critical for the enhanced insulin secretion , suggesting that the control of inflammation through reduction of n-6 pufas can have a beneficial effect on -cell functions . the strong resistance to cytokine - caused destruction in mfat-1expressing islets and ins-1 cells was also reflected at the level of cytokine signaling . specifically , we have shown that cytokine - induced activation of erk1/2 and nf-b pathways was significantly suppressed , both of which were critical for cytokines ' actions . although it is predicted to enhance insulin signaling and sensitivity in the liver tissue , the reduction of tnf-activated nf-b signaling leads to protection from cytokine - inflicted islet destruction . finally , from the perspective of gene expression , mfat-1induced increase in pdx-1 , glucokinase , and insulin 1 expression may have also played important roles in improved insulin secretion and -cell survival . modern diets contain excessive n-6 pufas but very low levels of n-3 pufas , which results in an unhealthy n-6to since the eicosanoid products derived from n-6 pufas are more potent mediators of inflammation than the similar products derived from n-3 pufas , an imbalanced n-6to n-3 ratio in favor of n-6 pufas will be highly proinflammatory , which is believed to have contributed to the pathogenesis of such modern diseases as autoimmune disorders and diabetes ( 1,30 ) . the production of n-3 pufas in the -cells and islets via mfat-1 activity is achieved by using n-6 pufas as the substrates , thus bringing down the ratios of n-6 to n-3 as well as the inflammatory state . thus , although the increase of n-3 pufas is critical , the decrease of n-6 pufas may be just as important in determining the phenotypes of mfat-1expressing islets . in keeping with this concept , we have already shown that the reduction of pge2 secretion from the mfat-1expressing islets and -cells is critical for the enhanced insulin secretion , suggesting that the control of inflammation through reduction of n-6 pufas is a main theme in the regulation of -cell functions by n-3 pufas . recent clinical studies have established that daily compensation of n-3 pufas starting at 1 year of age can have clear benefits against the development of type 1 diabetes and islet autoimmunity ( 6 ) . the results presented here suggest that elevation of cellular n-3 pufas has a direct beneficial impact on the survival of pancreatic -cells from inflammation attack and that they may explain mechanistically why increased intake of n-3 pufas in early childhood can help reduce the incidence of type 1 diabetes . our data may also add n-3 pufas to the growing list of factors that can help improve the functions and viability of -cells . in this context , the mfat-1expressing islets should be urgently tested in transplantation settings and may represent an additional tool for the treatment of type 1 diabetes .

endometriosis is an estrogen - dependent chronic inflammatory condition that affects women in their reproductive period , and is associated with pelvic pain and infertility . this gynecologic condition has a prevalence of 5% , and a peak between 25 and 35 years of age . a 0.1% annual incidence has been reported among women aged 15 to 49 years . despite the high morbidity and healthcare costs associated with this condition , the exact cause of endometriosis remains unknown , as estrogens fuel ectopic endometrial growth , and alterations of estrogen signaling have been associated with the disease , one of the proposed mechanism may include the influence in levels and availability of sex hormones . it has been hypothesized that participation in recreational or occupational physical activity ( pa ) may decrease estrogen levels and , with extreme exercise , reduce the frequency of ovulation . further , pa may increase levels of sex hormone binding globulin ( shbg ) , which would reduce bioavailable estrogens . hyperinsulinemia may increase concentrations of estrogens through decreasing concentrations of shbg , and may increase concentrations of insulin - like growth factor-1 ( igf-1 , that can stimulate endometrial cell proliferation ) through decreasing concentrations of insulin - like growth factor binding protein ( igfbp)-1 . finally , regular pa seems to have protective effects on diseases that involve inflammatory processes and oxidative stress , as it increases systemic levels of antiinflammatory cytokines . on the basis of these biological hypotheses , in the last years most studies suggested that adult pa decreases endometriosis risk , but up to date , to the best of our knowledge , no meta - analysis has been performed to critically evaluate and statistically combine the results of comparable studies . to summarize the currently available information and to provide the estimates of the possible effect size of this association , we conducted a systematic review and a meta - analysis of epidemiological data from case control and cohort studies , on the relation between pa and risk of endometriosis . this study was undertaken in accordance with the preferred reporting items for systematic reviews and meta - analyses ( prisma ) statement . ethical approval and written informed consent from patients were not necessary because our study was based on summaries and analyses of results of published studies . we carried out a literature search of all case control and cohort studies published as original articles in english up to april 2016 . we searched the electronic databases medline ( 1966 to 2016/04/12 ) and embase ( 1985 to 2016/04/12 ) using the medical subject heading ( mesh ) term physical activity or exercise or walking combined with furthermore , we reviewed reference lists of retrieved articles to search for other pertinent studies . two authors reviewed the papers and independently selected the articles eligible for the systematic review . studies were selected for the review if they met all of the following criteria : case control or cohort study reporting original data ; diagnosis of endometriosis was clinically and/or histologically based ; number or percentage of subject with and without endometriosis according to pa were provided ; full - length articles , published in english . if multiple published reports from a same study were available , we included only the one with the most detailed information . data were extracted independently by 2 investigators and discrepancies were resolved by discussion . for each study , the following information was collected on a standard form : first author 's last name ; year of publication ; country of origin ; study design ; number of subjects ; age , if available ; category of pa , if available ; relative risks ( rr ) or odds ratios ( or ) of endometriosis and corresponding 95% confidence intervals ( ci ) ; covariates adjusted for in the statistical analysis . study quality was independently evaluated by 2 reviewers , using the newcastle - ottawa quality assessment scale for case control ( selection , comparability , exposure ) and cohort studies ( selection , comparability , outcome ) . we combined the or and rr estimates from each study , using the adjusted estimates as published . to account for adjustments in the estimates we computed unadjusted ors from the exposure distributions of cases and controls as reported in the publications if the adjusted or was unavailable . ors and 95% cis were calculated by using both fixed - effect and random - effect models . since the studies included in the meta - analysis were different in many aspects , first of all the design and the population included , we chose to present the results of the random - effect model . the i statistic was used as a measure of heterogeneity , categorized , as suggested by higgins et al , in low ( 25% ) , moderate ( 50% ) , and high ( 75% ) . the funnel plot was used to detect small study effect . in these studies , cases and controls when available , the estimates included in the pooled analyses came from the comparison between cases and controls with the same characteristic ( infertile / infertile , fertile / fertile , unselected / unselected ) . if unavailable , we included the estimates from the comparison with fertile controls . to avoid duplication of data , if a study had 2 control groups and did not report an overall estimate , we chose to include the fertile control group in the main analysis . main comparison was pa yes versus no . when the overall result was not published , we calculated the or from the published numbers of cases and controls ( crude or ) . as we were authors of one of the papers , original data were available and we could calculate and include the adjusted or for pa versus not pa . since in the cohort studies the prevalence of endometriosis was comparatively low , we calculated the ors as estimates of rrs . a dose effect analysis was also planned : as the cut - off values were different among studies , we compared the highest category as reported in each paper versus no pa ; if a moderate level of pa was present , we included it in the low moderate versus no pa analysis , else we included the lowest level of pa as reported . in a paper reporting several levels of pa , we calculated the crude rr of endometriosis for low moderate pa adding subjects under and in the intermediate level , and for high pa adding those over the intermediate level . the results of the meta - analysis were also presented through cumulative meta - analysis over time : studies were added one a time according to publication year , and results summarized as each new study was added . in some cases , the study - specific 95% cis might slightly differ from those published in the original publications because of rounding . we performed an analysis in subgroups by type of controls ( fertile , infertile , both or not specified ) . as some studies included both types of controls , we did not compute an overall estimate , to avoid duplication . a subgroup analysis by type of observational study was initially planned , but as we retrieved just one cohort study reporting current pa , we calculated the pooled estimate including and excluding this study , to evaluate if the result was significantly different . review manager ( revman ; computer program , version 5.3 ; the nordic cochrane centre , the cochrane collaboration , copenhagen , denmark , 2014 ) was used to analyze the data . we carried out a literature search of all case control and cohort studies published as original articles in english up to april 2016 . we searched the electronic databases medline ( 1966 to 2016/04/12 ) and embase ( 1985 to 2016/04/12 ) using the medical subject heading ( mesh ) term physical activity or exercise or walking combined with furthermore , we reviewed reference lists of retrieved articles to search for other pertinent studies . two authors reviewed the papers and independently selected the articles eligible for the systematic review . studies were selected for the review if they met all of the following criteria : case control or cohort study reporting original data ; diagnosis of endometriosis was clinically and/or histologically based ; number or percentage of subject with and without endometriosis according to pa were provided ; full - length articles , published in english . if multiple published reports from a same study were available , we included only the one with the most detailed information . data were extracted independently by 2 investigators and discrepancies were resolved by discussion . for each study , the following information was collected on a standard form : first author 's last name ; year of publication ; country of origin ; study design ; number of subjects ; age , if available ; category of pa , if available ; relative risks ( rr ) or odds ratios ( or ) of endometriosis and corresponding 95% confidence intervals ( ci ) ; covariates adjusted for in the statistical analysis . study quality was independently evaluated by 2 reviewers , using the newcastle - ottawa quality assessment scale for case control ( selection , comparability , exposure ) and cohort studies ( selection , comparability , outcome ) . we combined the or and rr estimates from each study , using the adjusted estimates as published . to account for adjustments in the estimates , we used log or transformation ( and corresponding standard error ) . we computed unadjusted ors from the exposure distributions of cases and controls as reported in the publications if the adjusted or was unavailable . ors and 95% cis were calculated by using both fixed - effect and random - effect models . since the studies included in the meta - analysis were different in many aspects , first of all the design and the population included , we chose to present the results of the random - effect model . the i statistic was used as a measure of heterogeneity , categorized , as suggested by higgins et al , in low ( 25% ) , moderate ( 50% ) , and high ( 75% ) . the funnel plot was used to detect small study effect . in these studies , cases and controls when available , the estimates included in the pooled analyses came from the comparison between cases and controls with the same characteristic ( infertile / infertile , fertile / fertile , unselected / unselected ) . if unavailable , we included the estimates from the comparison with fertile controls . to avoid duplication of data , if a study had 2 control groups and did not report an overall estimate , we chose to include the fertile control group in the main analysis . . when the overall result was not published , we calculated the or from the published numbers of cases and controls ( crude or ) . as we were authors of one of the papers , original data were available and we could calculate and include the adjusted or for pa versus not pa . since in the cohort studies the prevalence of endometriosis was comparatively low , we calculated the ors as estimates of rrs . a dose effect analysis was also planned : as the cut - off values were different among studies , we compared the highest category as reported in each paper versus no pa ; if a moderate level of pa was present , we included it in the low moderate versus no pa analysis , else we included the lowest level of pa as reported . in a paper reporting several levels of pa , we calculated the crude rr of endometriosis for low moderate pa adding subjects under and in the intermediate level , and for high pa adding those over the intermediate level . the results of the meta - analysis were also presented through cumulative meta - analysis over time : studies were added one a time according to publication year , and results summarized as each new study was added . in some cases , the study - specific 95% cis might slightly differ from those published in the original publications because of rounding . we performed an analysis in subgroups by type of controls ( fertile , infertile , both or not specified ) . as some studies included both types of controls , we did not compute an overall estimate , to avoid duplication . a subgroup analysis by type of observational study was initially planned , but as we retrieved just one cohort study reporting current pa , we calculated the pooled estimate including and excluding this study , to evaluate if the result was significantly different . other 2 cohort studies were retrieved , but they only considered past pa . review manager ( revman ; computer program , version 5.3 ; the nordic cochrane centre , the cochrane collaboration , copenhagen , denmark , 2014 ) was used to analyze the data . figure 1 shows the flowchart of the selection of publications . from the literature search , combining embase and medline results we found 109 papers . activating english , human , and not review as filters , we identified 60 studies . after reading abstracts , we excluded 8 case reports , 11 studies without controls , 10 reviews / guidelines , 3 papers on malignancies , and 17 on other subjects ( endometriosis and infertility , endometriosis as reason for surgery , prevalence of endometriosis , bone health , etc . ) . eleven papers were extracted and extensively read : 2 were excluded because they were analyses on the same group of women , 1 because information was just recorded on energetic pa during menstruation . flow chart of the selection of studies on physical activity ( pa ) and risk of endometriosis included in the meta - analysis . then , we identified 1 additional publication by reviewing the reference lists of the retrieved papers . thus , in the present systematic review and meta - analysis , we combined data from 9 studies . two studies were on the same sample of women , but they separately analyzed current and past pa . table 1 shows the main characteristics of the studies included in the present meta - analysis . of these , description of studies included in the meta - analysis of endometriosis and recreational physical activity . seven studies investigated recent pa , whereas 5 also or exclusively asked information about pa during adolescence . we performed two separate meta - analyses , including a total of 3355 cases for recent exposure and 4600 cases for past exposure . pa was evaluated in several different ways : hours of pa per week , metabolic equivalents ( met)-h / week , author - defined low or high intensity activity . the main results of the published studies are reported in table 2 . authors who provided adjusted estimates of or found that current pa was protective against endometriosis : results from cramer et al and garavaglia et al were statistically significant . the cohort study of current pa provided , besides overall estimates , rrs according to fertile status : a slight effect of pa was observed in women with no past or concurrent infertility , but no association emerged when considering infertile women . main results of studies included in the meta - analysis of endometriosis and recreational physical activity . the main limitation of all studies regarded the choice of controls , that were not representative of general population , but were extracted by hospital controls , or infertility clinics , or included in cohorts selected for profession . our search strategy did not exclude studies where cases had clinically confirmed endometriosis , but most selected papers enrolled only women with laparoscopically or surgically confirmed endometriosis as cases . the only exception was the paper by kvaskoff et al , reporting data on past pa , that included only women reporting surgically confirmed diagnosis . figure 2 shows the study - specific and summary ors of endometriosis for ever versus never pa . the summary or was 0.85 ( 95% ci : 0.671.07 ) ( heterogeneity chi - square between studies = 11.12 , p = 0.08 ) . excluding the cohort study reporting current pa , the summary or was 0.73 ( 95% ci : 0.580.92 , heterogeneity chi - square = 3.21 , p = 0.67 ) . rerunning the analysis , only including adjusted estimates , we found an overall or of 0.69 ( 95% ci : 0.530.89 , heterogeneity chi - square 1.69 , p = 0.64 ) . considering i statistic , moderate heterogeneity was present in the overall analysis of current pa ( i = 46% ) , whereas the pooled estimates both from case control studies and adjusted ors showed low heterogeneity ( i = 0% ) . study - specific and summary odds ratios of endometriosis for any versus no physical activity ( 95% confidence interval in brackets ) : ( a ) all studies ; ( b ) only adjusted estimates . excluding each study in turn , the highest estimate was observed excluding vitonis et al ( or 0.73 , 95% ci : 0.580.92 ) , the lowest excluding cramer et al ( 1.02 , 95% ci : 0.931.14 ) . a cumulative meta - analysis ( fig . 3 ) showed a steadily increasing estimate over years , from 0.60 ( 95% ci : 0.420.85 , estimate of cramer et al , 1986 ) to 0.85 ( 95% ci : 0.671.07 ) including the last published study . cumulative meta - analysis of endometriosis for any versus no physical activity ( 95% confidence interval in brackets ) . odds ratios are shown ( with the corresponding 95% ci ) by year of publication of subsequent reports . figure 4 shows the study - specific and summary ors of low moderate current pa ( a ) and high current pa ( b ) , 1.00 ( 95% ci : 0.781.28 ) and 0.75 ( 95% ci : 0.531.07 ) , respectively . study - specific and summary odds ratios of endometriosis for low level ( a ) and moderate / high level ( b ) versus no physical activity ( 95% confidence interval in brackets ) . in fact , in some studies cases were selected for infertility , or a subanalysis for infertility was provided . since all cases were laparoscopically or surgically confirmed , it is likely that women without infertility would undergo these procedures only if pelvic pain was severe : thus , they are different from women who underwent the evaluation for infertility problems . the overall estimate was not calculated , since 1 study was considered in both groups . we found that the or for endometriosis in infertile women was 0.76 ( 95% ci : 0.511.12 ) and in fertile ones , more probably undergoing examination for pelvic pain , 1.03 ( 95% ci : 0.931.14 ) . study - specific and summary odds ratios of endometriosis for any versus no physical activity , by selection of cases ( 95% confidence interval in brackets ) . when the cases were infertile and controls were fertile women , pa appeared more protective ( or 0.60 , 95% ci : 0.440.83 ) , whereas when controls were women not selected for fertility , the result was not significant ( or 1.03 , 95% ci : 0.931.14 ) ( figure not shown ) . information about past pa was collected for different age ranges . however , since the information included at least part of adolescence years ( fig . the heterogeneity was significant ( chi - square 18.99 , p = 0.0008 ) ; the pooled estimate was 0.81 ( 95% ci : 0.641.04 ) : the study by cramer et al indicated a significant inverse association between pa , if started at less than 16 years , and endometriosis risk , and a similar finding emerged in garavaglia et al , whereas none of the other studies indicated such a significant effect , the study by dhillon and holt even suggesting the opposite . study - specific and summary odds ratios of endometriosis for any versus no physical activity during adolescent years ( 95% confidence interval in brackets ) . figure 7 shows the funnel plots for current pa versus no current pa ( a ) and past pa versus no past pa ( b ) . funnel plot of studies on physical activity , current ( a ) and past ( b ) , and risk of endometriosis . or = odds ratio , se = standard error . the general results of this analysis suggest that pa may reduce the risk of endometriosis , but does not conclusively support the hypothesis . although the pooled estimate of adjusted ors shows a significant protective effect of recent pa on endometriosis risk , the overall estimate was not statistically significant when including all retrieved articles . studies on the association between pa during adolescence and endometriosis are inconsistent , and our meta - analysis can not reach conclusive findings . a relationship between pa and endometriosis risk is biologically plausible for both the inflammatory and estrogen - dependent nature of the disease . inflammation is a typical feature of endometriosis , as the presence of ectopic tissue in the peritoneal cavity is associated with overproduction of prostaglandins , cytokines , and chemokines . endometriosis is associated with statistically significantly increased levels of peritoneal interleukin ( il)-6 and il-8 . disease - related excessive production of proinflammatory cytokines might predispose to endothelial dysfunction , accelerated atherosclerosis , and metabolic disorders such as insulin resistance . conversely , myokines secreted by skeletal muscle include various cytokines such as leukemia inhibitory factor , il-7 , irisin , able to induce an antiinflammatory response . it has been shown that fewer inflammatory markers are detectable after long - term behavioral changes involving both reduced energy intake and increased physical exercise . moreover , pa reduces insulin resistance , thus avoiding stimulation possibly linked to hyperglycemia and hyperinsulinemia . hyperinsulinemia may increase levels of estrogens through decreasing concentrations of shbg , and may increase levels of igf-1 through decreasing levels of igfbp-1 . both estrogens and igf-1 stimulates endometrial cell proliferation . finally , regular physical exercise is associated with a cumulative effect of reduction of menstrual flow and of estrogen action . in 2014 , a review on endometriosis and pa addressed the issue of the effects of pa on women with endometriosis , in terms of prevalence and possible therapeutic effects . the authors of this review noted that the few existing studies are observational , with little or no statistical significance , but the indication of an inverse relationship between the practice of physical exercise and the risk of endometriosis may be due to the discomfort experienced by women , that prevent the practice of physical exercise . as a narrative review , among 6 reviewed papers , it included 2 that did not meet our criteria . one of them found that strenuous exercise during menstruation but not in other periods was associated with a 2-fold risk of endometriosis ; the other paper focused on effects of pa on endometriosis related pain and interaction between painkillers and pa , concluding that taking painkillers might be less effective among endometriosis patients performing regular daily sport activities , and , thus , it might impose them to an unnecessary burden of possible side - effects . the data considered in that review are inconclusive regarding the benefits of physical exercise as a risk factor for the disease . the authors also noted that no data existed about the potential impact of exercise on the course of the endometriosis . only recently though it focused on sleep quality and pain threshold , nunes et al considered that performing pa regularly can contribute toward relieving sleep disorders and consequently may improve the quality of sleep ; therefore , patients with endometriosis should be encouraged to exercise during treatment . even if it considers pa in women with endometriosis , this study just addresses a general effect on women 's well being , rather than the role of regular exercise in relieving pelvic pain and affecting the disease course . in the interpretation of the association , the protective effect of high - level pa on risk of endometriosis may be at least partially explained by the fact that women affected by pain related to endometriosis may reduce their level of activity . otherwise , it has been showed that pa lowers pain symptoms . several mechanisms primed with aerobic exercise can lead to an important pain decrease . through numerous neuroendocrine modifications and a modulating action on the central and peripheral nervous systems , regular activity has a large series of possible ways of interacting with the course of pain symptoms . the exercise - induced endogenous analgesia is presumed to be due to the release of endogenous opioids and growth factors and activation of ( supra)spinal nociceptive inhibitory mechanisms orchestrated by the brain . the symptoms , in fact , may be reduced by pa to an extent that the women may not present them ; thus , in these women the endometriosis remains largely undiagnosed . along this line , a previous review on endometriosis and pa reported an article on beneficial effects of pa and interaction between painkillers and pa in women with endometriosis : the authors concluded that taking painkillers might be less effective among endometriosis patients performing regular daily sport activities . this diagnostic bias is of course a potential major limitation of epidemiological studies on the association between pa and endometriosis risk . however , up to date , information about the effectiveness of physical exercise to reduce endometriosis - related pain is scanty , and further research is needed . moreover , since elective endometriosis drug treatment , that is hormones , can lead to depression , investigating whether pa can control these symptoms may be an interesting line of research . even if pa may prevent the current symptoms ( mainly pain ) of endometriosis , we are unable to conclude that it prevents the development and progression of the disease . in order to take into account this point , we have analyzed the role of pa during adolescence , thus , probably , before the onset of the disease . unfortunately , studies on the association between pa during adolescence and endometriosis are inconsistent , and our meta - analysis can not reach conclusive findings . further , we have considered separately the role of pa on the risk of endometriosis in women with pain or infertility . also in this case , the analysis was not conclusive , since studies which have analyzed the role of pa in women with pain and endometriosis are few . we can assume that cases with no infertility , who underwent a laparoscopic evaluation , had experienced pelvic pain , a condition potentially preventing them from regular exercise . to test this hypothesis , vitonis et al performed an analysis on the level of activity 4 years before diagnosis : they observed a moderately protective association between pa and endometriosis , similar to that seen in the main analysis ( activity levels 2 years before diagnosis ) . aiming to account for this potential confounder , findings by cramer et al were adjusted for menstrual pain . on the contrary , dhillon and holt did not consider menstrual pain for inclusion as a confounder , because it could be either in the causal pathway between pa and endometrioma , or a marker of disease . information on pelvic pain was collected by some authors , but not used to adjust or estimates . kvaskoff et al , garavaglia et al , and vitonis et al did not report pain ( chronic or menstrual ) . another potential limitation is that information on pa duration and intensity was not homogeneously collected . cramer et al reported the age at which regular exercise began , so that duration depended on woman 's age at enrolment , and if she continued to exercise regularly ( at least 12 h / wk ) over years . if protection was due to pa duration or initiation at younger age dhillon and holt included women in the category of current pa = yes if they were exercising during the 2 years before the reference date , or according to pa between 12 and 21 years of age , for past pa : in this study , exercise was defined regular if performed at least 24 times per year . information on level of pa at different ages was given in garavaglia et al , but no minimum duration was required to define pa as a regular activity . other authors just recorded information on current pa and did not provide details about duration or type . conversely , kvaskoff et al focused on past pa : walking to school or out - of - school activity , in terms of hours per week between 8 and 15 years . finally , vitonis et al ascertained regular exercise as that performed in the last 2 years ; moreover , they also analyzed cumulative pa , to estimate long - term pa effect . among other confounding factors , we have to consider socioeconomic status and body mass . the diagnosis of endometriosis has been shown to be more frequent among higher social class and more educated women , that are also more frequently involved in leisure activities . closer attention to health may favor the diagnosis of endometriosis , thus producing an incorrect estimation of the real association with pa . further , pa is associated with body weight , which in turn is inversely associated with endometriosis . a few studies included adjustment for these covariates , whereas others did not . thus , we can not totally exclude the possibility that confounding may play some role in the observed association . any misclassification should , however , tend to reduce the estimated association , though in a validity study it has been noted a strong correlation between heavy activities reported but weak or no associations for light or moderate activities . then , high - level pa should be consistently and reliably reported , though the categories of pa are not homogeneously classified through studies . lastly , a limitation of this meta - analysis was that we only searched 2 databases . with reference to other sources of bias , the funnel plots did not show any evidence of small study effect , providing further indication of the robustness of our results . two main methodological problems in the epidemiology of endometriosis are the selection of cases and the choice of controls . in this study , comparison between cases not selected for infertility did not show any effect of recent pa , whereas an inverse association between pa and risk of endometriosis was observed in studies comparing infertile cases and fertile or unselected controls . if cases are infertile , the use of fertile controls may have led to bias . for instance , in controls with a laparoscopic evaluation and no infertility , if the possible symptoms influenced the activity levels , then the protective effect may be due not to biological mechanisms but to impairment . on the other hand , the underlying causes of infertility in controls might influence the results . moreover , the only published cohort study showed opposite results as compared to case control ones . however , it must be considered that we have included crude results in the analysis of pa versus no pa , whereas in the original paper the adjusted rrs showed a modestly protective effect of pa on endometriosis risk . thus , it is of interest to understand the role of pa in the adolescents ( i.e. , probably before the onset of endometriosis ) or later in life . few studies have analyzed the association between pa early in life and subsequent endometriosis risk . the large previously reported french cohort study has suggested that walking 5 or more hours a week at age 8 to 15 years slightly increases the risk of endometriosis later in life . in that study , however , no association emerged with out - of - school pa . the nurses health study ii found an increased risk of endometriosis among women reporting strenuous pa at 12 to 13 years of age . however , also in this study , other types of activity and activity at any other adolescent age were unrelated to the risk . finally , no association emerged between any pa and risk of endometrioma at 12 to 21 years of age in a case control study including 77 cases of endometriosis , conducted in the united states ; in this study even high pa was not related with endometriosis risk . conversely , in an old study conducted by cramer et al , strenuous exercises started at age 15 years decreased endometriosis risk . a recent study showed a protective effect of pa at 15 to 19 years , but it did not suggest a dose effect relation between hours per week and risk reduction . in our estimate , pa in adolescent years was inversely related to endometriosis risk , though the ci includes 1 . whereas single studies showed inconsistent results , in our analysis pa level showed a modest dose effect relation , with no risk reduction for low moderate level of pa and 20% reduction ( although not significant ) for high - level pa . an inverse dose effect relation was retrieved in some studies ( in the comparison with fertile controls ) , but in the nurses health study ii , the rrs of endometriosis declined with increasing pa and then showed an irregular trend for the 2 highest levels : similar trends emerged in the comparison with fertile and infertile controls . it has been suggested that the risk factors ( as well as the pathogenesis ) of pelvic and deep endometriosis may differ . we could not analyze separately the effect of pa on the risk of endometriosis in different sites , given the small number of studies reporting this information . however , in the study by heilier et al , peritoneal endometriosis and deep endometriotic nodules were separately analyzed : the authors concluded that both forms of the disease share similar patterns of risk and protective factors . the general results of this analysis suggest that pa may reduce the risk of endometriosis , but does not conclusively support the hypothesis . although the pooled estimate of adjusted ors shows a significant protective effect of recent pa on endometriosis risk , the overall estimate was not statistically significant when including all retrieved articles . studies on the association between pa during adolescence and endometriosis are inconsistent , and our meta - analysis can not reach conclusive findings . a relationship between pa and endometriosis risk is biologically plausible for both the inflammatory and estrogen - dependent nature of the disease . inflammation is a typical feature of endometriosis , as the presence of ectopic tissue in the peritoneal cavity is associated with overproduction of prostaglandins , cytokines , and chemokines . endometriosis is associated with statistically significantly increased levels of peritoneal interleukin ( il)-6 and il-8 . disease - related excessive production of proinflammatory cytokines might predispose to endothelial dysfunction , accelerated atherosclerosis , and metabolic disorders such as insulin resistance . conversely , myokines secreted by skeletal muscle include various cytokines such as leukemia inhibitory factor , il-7 , irisin , able to induce an antiinflammatory response . it has been shown that fewer inflammatory markers are detectable after long - term behavioral changes involving both reduced energy intake and increased physical exercise . moreover , pa reduces insulin resistance , thus avoiding stimulation possibly linked to hyperglycemia and hyperinsulinemia . hyperinsulinemia may increase levels of estrogens through decreasing concentrations of shbg , and may increase levels of igf-1 through decreasing levels of igfbp-1 . both estrogens and igf-1 stimulates endometrial cell proliferation . finally , regular physical exercise is associated with a cumulative effect of reduction of menstrual flow and of estrogen action . in 2014 , a review on endometriosis and pa addressed the issue of the effects of pa on women with endometriosis , in terms of prevalence and possible therapeutic effects . the authors of this review noted that the few existing studies are observational , with little or no statistical significance , but the indication of an inverse relationship between the practice of physical exercise and the risk of endometriosis may be due to the discomfort experienced by women , that prevent the practice of physical exercise . as a narrative review , among 6 reviewed papers , it included 2 that did not meet our criteria . one of them found that strenuous exercise during menstruation but not in other periods was associated with a 2-fold risk of endometriosis ; the other paper focused on effects of pa on endometriosis related pain and interaction between painkillers and pa , concluding that taking painkillers might be less effective among endometriosis patients performing regular daily sport activities , and , thus , it might impose them to an unnecessary burden of possible side - effects . the data considered in that review are inconclusive regarding the benefits of physical exercise as a risk factor for the disease . the authors also noted that no data existed about the potential impact of exercise on the course of the endometriosis . only recently though it focused on sleep quality and pain threshold , nunes et al considered that performing pa regularly can contribute toward relieving sleep disorders and consequently may improve the quality of sleep ; therefore , patients with endometriosis should be encouraged to exercise during treatment . even if it considers pa in women with endometriosis , this study just addresses a general effect on women 's well being , rather than the role of regular exercise in relieving pelvic pain and affecting the disease course . . the protective effect of high - level pa on risk of endometriosis may be at least partially explained by the fact that women affected by pain related to endometriosis may reduce their level of activity . otherwise , it has been showed that pa lowers pain symptoms . several mechanisms primed with aerobic exercise can lead to an important pain decrease . through numerous neuroendocrine modifications and a modulating action on the central and peripheral nervous systems , regular activity has a large series of possible ways of interacting with the course of pain symptoms . the exercise - induced endogenous analgesia is presumed to be due to the release of endogenous opioids and growth factors and activation of ( supra)spinal nociceptive inhibitory mechanisms orchestrated by the brain . further , pa may keep the pain symptoms of endometriosis at bay . the symptoms , in fact , may be reduced by pa to an extent that the women may not present them ; thus , in these women the endometriosis remains largely undiagnosed . along this line , a previous review on endometriosis and pa reported an article on beneficial effects of pa and interaction between painkillers and pa in women with endometriosis : the authors concluded that taking painkillers might be less effective among endometriosis patients performing regular daily sport activities . this diagnostic bias is of course a potential major limitation of epidemiological studies on the association between pa and endometriosis risk . however , up to date , information about the effectiveness of physical exercise to reduce endometriosis - related pain is scanty , and further research is needed . moreover , since elective endometriosis drug treatment , that is hormones , can lead to depression , investigating whether pa can control these symptoms may be an interesting line of research . even if pa may prevent the current symptoms ( mainly pain ) of endometriosis , we are unable to conclude that it prevents the development and progression of the disease . in order to take into account this point , we have analyzed the role of pa during adolescence , thus , probably , before the onset of the disease . unfortunately , studies on the association between pa during adolescence and endometriosis are inconsistent , and our meta - analysis can not reach conclusive findings . further , we have considered separately the role of pa on the risk of endometriosis in women with pain or infertility . also in this case , the analysis was not conclusive , since studies which have analyzed the role of pa in women with pain and endometriosis are few . we can assume that cases with no infertility , who underwent a laparoscopic evaluation , had experienced pelvic pain , a condition potentially preventing them from regular exercise . to test this hypothesis , vitonis et al performed an analysis on the level of activity 4 years before diagnosis : they observed a moderately protective association between pa and endometriosis , similar to that seen in the main analysis ( activity levels 2 years before diagnosis ) . aiming to account for this potential confounder , findings by cramer et al were adjusted for menstrual pain . on the contrary , dhillon and holt did not consider menstrual pain for inclusion as a confounder , because it could be either in the causal pathway between pa and endometrioma , or a marker of disease . information on pelvic pain was collected by some authors , but not used to adjust or estimates . kvaskoff et al , garavaglia et al , and vitonis et al did not report pain ( chronic or menstrual ) . another potential limitation is that information on pa duration and intensity was not homogeneously collected . cramer et al reported the age at which regular exercise began , so that duration depended on woman 's age at enrolment , and if she continued to exercise regularly ( at least 12 h / wk ) over years . if protection was due to pa duration or initiation at younger age dhillon and holt included women in the category of current pa = yes if they were exercising during the 2 years before the reference date , or according to pa between 12 and 21 years of age , for past pa : in this study , exercise was defined regular if performed at least 24 times per year . information on level of pa at different ages was given in garavaglia et al , but no minimum duration was required to define pa as a regular activity . other authors just recorded information on current pa and did not provide details about duration or type . conversely , kvaskoff et al focused on past pa : walking to school or out - of - school activity , in terms of hours per week between 8 and 15 years . finally , vitonis et al ascertained regular exercise as that performed in the last 2 years ; moreover , they also analyzed cumulative pa , to estimate long - term pa effect . among other confounding factors the diagnosis of endometriosis has been shown to be more frequent among higher social class and more educated women , that are also more frequently involved in leisure activities . closer attention to health may favor the diagnosis of endometriosis , thus producing an incorrect estimation of the real association with pa . further , pa is associated with body weight , which in turn is inversely associated with endometriosis . a few studies included adjustment for these covariates , whereas others did not . thus , we can not totally exclude the possibility that confounding may play some role in the observed association . any misclassification should , however , tend to reduce the estimated association , though in a validity study it has been noted a strong correlation between heavy activities reported but weak or no associations for light or moderate activities . then , high - level pa should be consistently and reliably reported , though the categories of pa are not homogeneously classified through studies . lastly , a limitation of this meta - analysis was that we only searched 2 databases . with reference to other sources of bias , the funnel plots did not show any evidence of small study effect , providing further indication of the robustness of our results . two main methodological problems in the epidemiology of endometriosis are the selection of cases and the choice of controls . in this study , comparison between cases not selected for infertility did not show any effect of recent pa , whereas an inverse association between pa and risk of endometriosis was observed in studies comparing infertile cases and fertile or unselected controls . if cases are infertile , the use of fertile controls may have led to bias . for instance , in controls with a laparoscopic evaluation and no infertility , if the possible symptoms influenced the activity levels , then the protective effect may be due not to biological mechanisms but to impairment . on the other hand , the underlying causes of infertility in controls might influence the results . however , it must be considered that we have included crude results in the analysis of pa versus no pa , whereas in the original paper the adjusted rrs showed a modestly protective effect of pa on endometriosis risk . thus , it is of interest to understand the role of pa in the adolescents ( i.e. , probably before the onset of endometriosis ) or later in life . few studies have analyzed the association between pa early in life and subsequent endometriosis risk . the large previously reported french cohort study has suggested that walking 5 or more hours a week at age 8 to 15 years slightly increases the risk of endometriosis later in life . in that study , however , no association emerged with out - of - school pa . the nurses health study ii found an increased risk of endometriosis among women reporting strenuous pa at 12 to 13 years of age . however , also in this study , other types of activity and activity at any other adolescent age were unrelated to the risk . finally , no association emerged between any pa and risk of endometrioma at 12 to 21 years of age in a case control study including 77 cases of endometriosis , conducted in the united states ; in this study even high pa was not related with endometriosis risk . conversely , in an old study conducted by cramer et al , strenuous exercises started at age 15 years decreased endometriosis risk . a recent study showed a protective effect of pa at 15 to 19 years , but it did not suggest a dose effect relation between hours per week and risk reduction . in our estimate , pa in adolescent years was inversely related to endometriosis risk , though the ci includes 1 . whereas single studies showed inconsistent results , in our analysis pa level showed a modest dose effect relation , with no risk reduction for low moderate level of pa and 20% reduction ( although not significant ) for high - level pa . an inverse dose effect relation was retrieved in some studies ( in the comparison with fertile controls ) , but in the nurses health study ii , the rrs of endometriosis declined with increasing pa and then showed an irregular trend for the 2 highest levels : similar trends emerged in the comparison with fertile and infertile controls . it has been suggested that the risk factors ( as well as the pathogenesis ) of pelvic and deep endometriosis may differ . we could not analyze separately the effect of pa on the risk of endometriosis in different sites , given the small number of studies reporting this information . however , in the study by heilier et al , peritoneal endometriosis and deep endometriotic nodules were separately analyzed : the authors concluded that both forms of the disease share similar patterns of risk and protective factors . supporting healthy behavior is the main goal of health promotion , and healthy behavior is a result of a multidimensional approach that is influenced by several factors . healthy behavior is not only pa , but also a construct of mental , educational , and environmental situations that enables us to live a healthy life . in the situation of endometriosis and pain , or perception of pain , social factors and environment play an important role ; thus , the social and psychological support might be of further relevance . sedentary behavior and physical inactivity are risk factors for cancer , diabetes , and ischemic coronary heart disease . on the contrary , pa has been described as the real polypill : regular exercise reduces the risk of cardiovascular events and all - cause mortality , cardiovascular disease , type ii diabetes , stroke , metabolic syndrome . regular exercise has been compared to drug intervention ( hypoglycemic , lipid - lowering , antihypertensive , antithrombotic drugs ) , concluding that the overall risk reduction due to pa goes beyond reducing traditional cardiovascular risk factors . moreover , exercise , and especially the contracting muscle , is a source of several drug - like molecules with beneficial effects across all ages . in general , regular pa has preventive / therapeutic effects against most prevalent chronic diseases , and a dose - dependent effect is usually observed in the general population . as regards endometriosis , though the present meta - analysis suggests that pa may reduce the risk , it does not conclusively support the hypothesis . exercise probably has pleiotropic positive effects in almost every organ system potentially having myokine - mediated direct and indirect antiinflammatory effects in chronic inflammatory diseases . however , the results of this analysis seem also to depend on the study design and the choice of controls . whether these findings are really explained by the benefit of exercise at molecular and endocrine level or related to confounding mechanisms , including the fact that pa may improve pain , needs to be further investigated . in consideration of the fact that if we wish to analyze the role of pa in the development of the endometriosis , the exposure ( i.e. , moderate or intense pa ) should act in the early phases of the disease , interventional studies on endometriosis prevention are substantially not feasible . control studies on risk factors for endometriosis should focus on collecting a detailed history of pa at all ages , and investigating potential changes of lifestyle habits due to pelvic pain . on the contrary , randomized studies identifying whether regular exercise prevents the progression of the endometriosis are feasible . multicountry studies are further requested , in consideration of the fact that occupational or leisure pa is strongly related with other determinants of endometriosis , such as economic status , reproductive pattern , and menstrual pattern .

abstractbackground : the potential association between endometriosis and physical activity ( pa ) has been suggested in several epidemiological studies.we aimed to establish whether pa influences endometriosis risk.methods:medline and embase were searched using physical activity or exercise combined with endometriosis , in medical subject headings and free text . we selected original articles in english , published up to april 2016 , evaluating the association between endometriosis and recent or past pa ( case control or cohort studies ) . references of retrieved papers were reviewed . we computed summary odds ratios ( ors ) of endometriosis for recent and past pa.results:six case control and 3 cohort studies included 3355 cases for recent pa and 4600 cases for past pa . the summary or for endometriosis according to pa level , calculated by the random - effect model , was 0.85 [ 95% confidence interval ( ci ) 0.671.07 ] for any recent versus no pa . as compared to no recent pa , ors for low and moderate / high pa were 1.00 ( 95% ci : 0.681.28 ) and 0.75 ( 95% ci : 0.531.07 ) , respectively.conclusions:though it suggests that pa may reduce the risk of endometriosis , this meta - analysis does not conclusively support the hypothesis . whether our findings are really explained by the benefit of exercise at molecular and endocrine level , or related to confounding mechanisms , such as study design , choice of controls , and pa potentially improving pain , needs to be further investigated .

Introduction Methods Study eligibility Data sources Study selection Data abstraction Risk of bias assessment Data synthesis Subgroup analyses Results Discussion Main findings Strengths and limitations Conclusions

it has been hypothesized that participation in recreational or occupational physical activity ( pa ) may decrease estrogen levels and , with extreme exercise , reduce the frequency of ovulation . on the basis of these biological hypotheses , in the last years most studies suggested that adult pa decreases endometriosis risk , but up to date , to the best of our knowledge , no meta - analysis has been performed to critically evaluate and statistically combine the results of comparable studies . to summarize the currently available information and to provide the estimates of the possible effect size of this association , we conducted a systematic review and a meta - analysis of epidemiological data from case control and cohort studies , on the relation between pa and risk of endometriosis . we carried out a literature search of all case control and cohort studies published as original articles in english up to april 2016 . we searched the electronic databases medline ( 1966 to 2016/04/12 ) and embase ( 1985 to 2016/04/12 ) using the medical subject heading ( mesh ) term physical activity or exercise or walking combined with furthermore , we reviewed reference lists of retrieved articles to search for other pertinent studies . studies were selected for the review if they met all of the following criteria : case control or cohort study reporting original data ; diagnosis of endometriosis was clinically and/or histologically based ; number or percentage of subject with and without endometriosis according to pa were provided ; full - length articles , published in english . for each study , the following information was collected on a standard form : first author 's last name ; year of publication ; country of origin ; study design ; number of subjects ; age , if available ; category of pa , if available ; relative risks ( rr ) or odds ratios ( or ) of endometriosis and corresponding 95% confidence intervals ( ci ) ; covariates adjusted for in the statistical analysis . ors and 95% cis were calculated by using both fixed - effect and random - effect models . since the studies included in the meta - analysis were different in many aspects , first of all the design and the population included , we chose to present the results of the random - effect model . the i statistic was used as a measure of heterogeneity , categorized , as suggested by higgins et al , in low ( 25% ) , moderate ( 50% ) , and high ( 75% ) . in a paper reporting several levels of pa , we calculated the crude rr of endometriosis for low moderate pa adding subjects under and in the intermediate level , and for high pa adding those over the intermediate level . the results of the meta - analysis were also presented through cumulative meta - analysis over time : studies were added one a time according to publication year , and results summarized as each new study was added . we carried out a literature search of all case control and cohort studies published as original articles in english up to april 2016 . we searched the electronic databases medline ( 1966 to 2016/04/12 ) and embase ( 1985 to 2016/04/12 ) using the medical subject heading ( mesh ) term physical activity or exercise or walking combined with furthermore , we reviewed reference lists of retrieved articles to search for other pertinent studies . studies were selected for the review if they met all of the following criteria : case control or cohort study reporting original data ; diagnosis of endometriosis was clinically and/or histologically based ; number or percentage of subject with and without endometriosis according to pa were provided ; full - length articles , published in english . for each study , the following information was collected on a standard form : first author 's last name ; year of publication ; country of origin ; study design ; number of subjects ; age , if available ; category of pa , if available ; relative risks ( rr ) or odds ratios ( or ) of endometriosis and corresponding 95% confidence intervals ( ci ) ; covariates adjusted for in the statistical analysis . since the studies included in the meta - analysis were different in many aspects , first of all the design and the population included , we chose to present the results of the random - effect model . a dose effect analysis was also planned : as the cut - off values were different among studies , we compared the highest category as reported in each paper versus no pa ; if a moderate level of pa was present , we included it in the low moderate versus no pa analysis , else we included the lowest level of pa as reported . in a paper reporting several levels of pa , we calculated the crude rr of endometriosis for low moderate pa adding subjects under and in the intermediate level , and for high pa adding those over the intermediate level . the results of the meta - analysis were also presented through cumulative meta - analysis over time : studies were added one a time according to publication year , and results summarized as each new study was added . as some studies included both types of controls , we did not compute an overall estimate , to avoid duplication . after reading abstracts , we excluded 8 case reports , 11 studies without controls , 10 reviews / guidelines , 3 papers on malignancies , and 17 on other subjects ( endometriosis and infertility , endometriosis as reason for surgery , prevalence of endometriosis , bone health , etc . ) flow chart of the selection of studies on physical activity ( pa ) and risk of endometriosis included in the meta - analysis . thus , in the present systematic review and meta - analysis , we combined data from 9 studies . of these , description of studies included in the meta - analysis of endometriosis and recreational physical activity . we performed two separate meta - analyses , including a total of 3355 cases for recent exposure and 4600 cases for past exposure . main results of studies included in the meta - analysis of endometriosis and recreational physical activity . the main limitation of all studies regarded the choice of controls , that were not representative of general population , but were extracted by hospital controls , or infertility clinics , or included in cohorts selected for profession . the summary or was 0.85 ( 95% ci : 0.671.07 ) ( heterogeneity chi - square between studies = 11.12 , p = 0.08 ) . excluding the cohort study reporting current pa , the summary or was 0.73 ( 95% ci : 0.580.92 , heterogeneity chi - square = 3.21 , p = 0.67 ) . rerunning the analysis , only including adjusted estimates , we found an overall or of 0.69 ( 95% ci : 0.530.89 , heterogeneity chi - square 1.69 , p = 0.64 ) . considering i statistic , moderate heterogeneity was present in the overall analysis of current pa ( i = 46% ) , whereas the pooled estimates both from case control studies and adjusted ors showed low heterogeneity ( i = 0% ) . study - specific and summary odds ratios of endometriosis for any versus no physical activity ( 95% confidence interval in brackets ) : ( a ) all studies ; ( b ) only adjusted estimates . excluding each study in turn , the highest estimate was observed excluding vitonis et al ( or 0.73 , 95% ci : 0.580.92 ) , the lowest excluding cramer et al ( 1.02 , 95% ci : 0.931.14 ) . 3 ) showed a steadily increasing estimate over years , from 0.60 ( 95% ci : 0.420.85 , estimate of cramer et al , 1986 ) to 0.85 ( 95% ci : 0.671.07 ) including the last published study . cumulative meta - analysis of endometriosis for any versus no physical activity ( 95% confidence interval in brackets ) . figure 4 shows the study - specific and summary ors of low moderate current pa ( a ) and high current pa ( b ) , 1.00 ( 95% ci : 0.781.28 ) and 0.75 ( 95% ci : 0.531.07 ) , respectively . study - specific and summary odds ratios of endometriosis for low level ( a ) and moderate / high level ( b ) versus no physical activity ( 95% confidence interval in brackets ) . we found that the or for endometriosis in infertile women was 0.76 ( 95% ci : 0.511.12 ) and in fertile ones , more probably undergoing examination for pelvic pain , 1.03 ( 95% ci : 0.931.14 ) . study - specific and summary odds ratios of endometriosis for any versus no physical activity , by selection of cases ( 95% confidence interval in brackets ) . when the cases were infertile and controls were fertile women , pa appeared more protective ( or 0.60 , 95% ci : 0.440.83 ) , whereas when controls were women not selected for fertility , the result was not significant ( or 1.03 , 95% ci : 0.931.14 ) ( figure not shown ) . the heterogeneity was significant ( chi - square 18.99 , p = 0.0008 ) ; the pooled estimate was 0.81 ( 95% ci : 0.641.04 ) : the study by cramer et al indicated a significant inverse association between pa , if started at less than 16 years , and endometriosis risk , and a similar finding emerged in garavaglia et al , whereas none of the other studies indicated such a significant effect , the study by dhillon and holt even suggesting the opposite . study - specific and summary odds ratios of endometriosis for any versus no physical activity during adolescent years ( 95% confidence interval in brackets ) . figure 7 shows the funnel plots for current pa versus no current pa ( a ) and past pa versus no past pa ( b ) . funnel plot of studies on physical activity , current ( a ) and past ( b ) , and risk of endometriosis . the general results of this analysis suggest that pa may reduce the risk of endometriosis , but does not conclusively support the hypothesis . studies on the association between pa during adolescence and endometriosis are inconsistent , and our meta - analysis can not reach conclusive findings . in 2014 , a review on endometriosis and pa addressed the issue of the effects of pa on women with endometriosis , in terms of prevalence and possible therapeutic effects . one of them found that strenuous exercise during menstruation but not in other periods was associated with a 2-fold risk of endometriosis ; the other paper focused on effects of pa on endometriosis related pain and interaction between painkillers and pa , concluding that taking painkillers might be less effective among endometriosis patients performing regular daily sport activities , and , thus , it might impose them to an unnecessary burden of possible side - effects . in the interpretation of the association , the protective effect of high - level pa on risk of endometriosis may be at least partially explained by the fact that women affected by pain related to endometriosis may reduce their level of activity . along this line , a previous review on endometriosis and pa reported an article on beneficial effects of pa and interaction between painkillers and pa in women with endometriosis : the authors concluded that taking painkillers might be less effective among endometriosis patients performing regular daily sport activities . this diagnostic bias is of course a potential major limitation of epidemiological studies on the association between pa and endometriosis risk . even if pa may prevent the current symptoms ( mainly pain ) of endometriosis , we are unable to conclude that it prevents the development and progression of the disease . unfortunately , studies on the association between pa during adolescence and endometriosis are inconsistent , and our meta - analysis can not reach conclusive findings . if protection was due to pa duration or initiation at younger age dhillon and holt included women in the category of current pa = yes if they were exercising during the 2 years before the reference date , or according to pa between 12 and 21 years of age , for past pa : in this study , exercise was defined regular if performed at least 24 times per year . the diagnosis of endometriosis has been shown to be more frequent among higher social class and more educated women , that are also more frequently involved in leisure activities . lastly , a limitation of this meta - analysis was that we only searched 2 databases . two main methodological problems in the epidemiology of endometriosis are the selection of cases and the choice of controls . in this study , comparison between cases not selected for infertility did not show any effect of recent pa , whereas an inverse association between pa and risk of endometriosis was observed in studies comparing infertile cases and fertile or unselected controls . moreover , the only published cohort study showed opposite results as compared to case control ones . however , it must be considered that we have included crude results in the analysis of pa versus no pa , whereas in the original paper the adjusted rrs showed a modestly protective effect of pa on endometriosis risk . few studies have analyzed the association between pa early in life and subsequent endometriosis risk . finally , no association emerged between any pa and risk of endometrioma at 12 to 21 years of age in a case control study including 77 cases of endometriosis , conducted in the united states ; in this study even high pa was not related with endometriosis risk . whereas single studies showed inconsistent results , in our analysis pa level showed a modest dose effect relation , with no risk reduction for low moderate level of pa and 20% reduction ( although not significant ) for high - level pa . an inverse dose effect relation was retrieved in some studies ( in the comparison with fertile controls ) , but in the nurses health study ii , the rrs of endometriosis declined with increasing pa and then showed an irregular trend for the 2 highest levels : similar trends emerged in the comparison with fertile and infertile controls . it has been suggested that the risk factors ( as well as the pathogenesis ) of pelvic and deep endometriosis may differ . the general results of this analysis suggest that pa may reduce the risk of endometriosis , but does not conclusively support the hypothesis . studies on the association between pa during adolescence and endometriosis are inconsistent , and our meta - analysis can not reach conclusive findings . in 2014 , a review on endometriosis and pa addressed the issue of the effects of pa on women with endometriosis , in terms of prevalence and possible therapeutic effects . the authors of this review noted that the few existing studies are observational , with little or no statistical significance , but the indication of an inverse relationship between the practice of physical exercise and the risk of endometriosis may be due to the discomfort experienced by women , that prevent the practice of physical exercise . one of them found that strenuous exercise during menstruation but not in other periods was associated with a 2-fold risk of endometriosis ; the other paper focused on effects of pa on endometriosis related pain and interaction between painkillers and pa , concluding that taking painkillers might be less effective among endometriosis patients performing regular daily sport activities , and , thus , it might impose them to an unnecessary burden of possible side - effects . the protective effect of high - level pa on risk of endometriosis may be at least partially explained by the fact that women affected by pain related to endometriosis may reduce their level of activity . along this line , a previous review on endometriosis and pa reported an article on beneficial effects of pa and interaction between painkillers and pa in women with endometriosis : the authors concluded that taking painkillers might be less effective among endometriosis patients performing regular daily sport activities . this diagnostic bias is of course a potential major limitation of epidemiological studies on the association between pa and endometriosis risk . even if pa may prevent the current symptoms ( mainly pain ) of endometriosis , we are unable to conclude that it prevents the development and progression of the disease . unfortunately , studies on the association between pa during adolescence and endometriosis are inconsistent , and our meta - analysis can not reach conclusive findings . to test this hypothesis , vitonis et al performed an analysis on the level of activity 4 years before diagnosis : they observed a moderately protective association between pa and endometriosis , similar to that seen in the main analysis ( activity levels 2 years before diagnosis ) . if protection was due to pa duration or initiation at younger age dhillon and holt included women in the category of current pa = yes if they were exercising during the 2 years before the reference date , or according to pa between 12 and 21 years of age , for past pa : in this study , exercise was defined regular if performed at least 24 times per year . in this study , comparison between cases not selected for infertility did not show any effect of recent pa , whereas an inverse association between pa and risk of endometriosis was observed in studies comparing infertile cases and fertile or unselected controls . however , it must be considered that we have included crude results in the analysis of pa versus no pa , whereas in the original paper the adjusted rrs showed a modestly protective effect of pa on endometriosis risk . the large previously reported french cohort study has suggested that walking 5 or more hours a week at age 8 to 15 years slightly increases the risk of endometriosis later in life . finally , no association emerged between any pa and risk of endometrioma at 12 to 21 years of age in a case control study including 77 cases of endometriosis , conducted in the united states ; in this study even high pa was not related with endometriosis risk . whereas single studies showed inconsistent results , in our analysis pa level showed a modest dose effect relation , with no risk reduction for low moderate level of pa and 20% reduction ( although not significant ) for high - level pa . it has been suggested that the risk factors ( as well as the pathogenesis ) of pelvic and deep endometriosis may differ . we could not analyze separately the effect of pa on the risk of endometriosis in different sites , given the small number of studies reporting this information . however , in the study by heilier et al , peritoneal endometriosis and deep endometriotic nodules were separately analyzed : the authors concluded that both forms of the disease share similar patterns of risk and protective factors . in the situation of endometriosis and pain , or perception of pain , social factors and environment play an important role ; thus , the social and psychological support might be of further relevance . on the contrary , pa has been described as the real polypill : regular exercise reduces the risk of cardiovascular events and all - cause mortality , cardiovascular disease , type ii diabetes , stroke , metabolic syndrome . regular exercise has been compared to drug intervention ( hypoglycemic , lipid - lowering , antihypertensive , antithrombotic drugs ) , concluding that the overall risk reduction due to pa goes beyond reducing traditional cardiovascular risk factors . as regards endometriosis , though the present meta - analysis suggests that pa may reduce the risk , it does not conclusively support the hypothesis . however , the results of this analysis seem also to depend on the study design and the choice of controls . whether these findings are really explained by the benefit of exercise at molecular and endocrine level or related to confounding mechanisms , including the fact that pa may improve pain , needs to be further investigated . multicountry studies are further requested , in consideration of the fact that occupational or leisure pa is strongly related with other determinants of endometriosis , such as economic status , reproductive pattern , and menstrual pattern .

the human cerebral cortex is generally considered the most complex structure of the human body . the cortical expansion and the enlarged surface area following the highly organized folding into gyri and sulci ( cf . sun and hevner , 2014 ) , enable extraordinary human cognitive abilities . although the human brain is not the largest and not the most convoluted brain in the animal kingdom , it has the largest number of neurons . a detailed characterization of the human embryonic and fetal proliferative ventricular and subventricular zones during corticogenesis may aid in defining the properties of human cortical development . the neural progenitor cells underlying the cortical expansion comprise initially neuroepithelial cells , which transform into radial glial cells ( rgcs ) at the onset of neurogenesis ( gtz and huttner , 2005 ; kriegstein and alvarezbuylla , 2009 ) . these neural progenitors , often referred to as neural stem cells ( nscs ) ( kriegstein and alvarezbuylla , 2009 ) , give rise to the enormous diversity of neurons and glial cells in the cerebral cortex . a thorough analysis of the developing monkey cortex showed that the subventricular zone ( svz ) can be further subdivided into an inner svz ( isvz ) and outer svz ( osvz ) by an inner fibrous layer ( smart et al . , 2002 ) . the seminal work of these authors also demonstrated a striking rostral to caudal gradient in differentiation of the cytoarchitectonic compartments of the primate subventricular zone from frontal to occipital cortices ( see discussion ) . the neocortical svz expands even more in the human developing brain ( bayatti et al . , 2008 ; zecevic et al . , 2005 ) , but to a lesser extent in rodents and ferrets ( fietz et al . , 2010 ; lui et al . , 2011 ; reillo et al . , has shown intermediate progenitor cells ( ipcs ) and outer radial glial cells ( orgs ) in both compartments ( betizeau et al . , 2013 ; fietz et al . , 2010 ; hansen et al . , 2010 ; reillo et al . , they share the expression of classical ventricular rg ( vrg ) cell markers such as brain lipidbinding protein ( blbp ) , glial fibrillary acidic protein ( gfap ) , vimentin , pax6 , and sox2 , but not ipc markers such as tbr2 ( fietz et al . , 2010 ; fietz and huttner , 2011 ; hansen et al . , 2010 ; reillo et al . , the diversification of rgc progeny in both spatial and temporal aspects is a matter of intense research , and evidence from human and rodent studies point toward distinct subpopulations of rgcs that may be purely gliogenic , purely neurogenic or multipotent ( hartfuss et al . , 2001 ; howard et al . , 2006 ; li et al . , 2004 ; pinto et al . , the globoseries glycosphingolipid stagespecific embryonic antigen 4 ( ssea4 ) is widely used in characterization of human embryonic stem cell ( hesc ) lines ( adewumi et al . , 2007 ) , and has been shown in a population of differentiating cells in an intermediate stage between pluripotent hescs and neural progenitor cells ( noisa et al . , 2012 ) . in a previous study of human embryonic central nervous system , ssea4 was found in neural progenitor cells in forebrains of human embryos and early fetuses ( barraud et al . , 2007 ) . in spontaneously differentiating hescs , the highly conserved and secreted glycoprotein ykl40 ( bussink et al . , 2007 ) , also known as chitinase 3like 1 ( chi3l1 ) was shown to be upregulated when undifferentiated escs differentiated , with particular expression in neuroectodermal cells ( brchner et al . , 2012 ) . in the developing human forebrain , ykl40 has recently been associated with brain barrier sites such as the radial glial end feet layer at the subpial marginal zone , pericytes of the intermediate and subventricular zones and in the choroid plexus ( chp ) epithelium of the lateral ventricles . furthermore , intriguing small rounded ykl40 positive cells in close relation to and occasionally overlapping with gfappositive radial glial fibers in the svz was identified ( bjrnbak et al . , 2014 ) . the overall distribution of ykl40 in the developing human forebrain was noted to be very similar to a study of ssea4 based on the same material collection ( barraud et al . , 2007 ) . a wide array of studies ykl40 is overexpressed in glioblastoma multiforme compared to normal tissue , lowgrade gliomas ( nigro et al . , 2005 ; 2002 ) and highgrade oligodendrogliomas ( nutt et al . , 2005 ; rousseau et al . , furthermore , high ykl40 expression in glioblastomas is an independent negative prognostic factor ( iwamoto and hormigo , 2014 ; nigro et al . , 2005 ; pelloski et al . , 2005 many neurological and neurodevelopmental diseases are associated with ykl40 ( bonnehbarkay et al . , 2010 ; craigschapiro et al . , 2010 ) , however , as to how ykl40 mediates its biological effects and its role in normal human developmental biology still lacks some clarification . in this study , we aimed to investigate the recently described neural progenitor cell marker ssea4 in relation to the newly discovered ykl40 positive cell population in the svz of developing human forebrain , and examine its proposed astrogenic lineage potential . to this end , we studied human forebrain samples from human embryos and fetuses ( 8th21st weeks post conception ) by immunohistochemistry and confocal microscopy , with antibodies against ssea4 , ykl40 , and against specific cell types known to reside within the developing human forebrain , for example , rgcs , ipcs , neurons , interneurons , and glial cells including microglial cells . forebrains from one late human embryo , 31 mm crownrump length ( crl ) and 12 fetuses ( 38200 mm crl ) corresponding to 8th21st weeks post conception ( wpc ) were examined . the embryo and fetuses were obtained from legal abortions . according to the helsinki declaration ii oral and written information was given and informed consent was obtained from all contributing women , according to and approved by the research ethics committee of the capital region ( kf v.100.1735/90 ) . immediately following operation , the samples were dissected into blocks and fixed for 1224 h at 4c in either 10% neutral buffered formalin , 4% formolcalcium , lillie 's or bouin 's fixatives . serial sections , 310 m thick , were cut in transverse , sagittal , or horizontal planes , placed on silanized glass slides , and used for single and double immunohistochemical experiments . for bright field light microscopy analysis endogenous peroxidase was quenched using a 0.5% solution of hydrogen peroxide in methanol for 15 min . following rinses with tris buffered saline ( tbs , 5 mm trishcl , 146 mm nacl , ph 7.6 ) , nonspecific binding was inhibited by incubation for 30 min with blocking buffer ( chemmate antibody diluent s2022 , dakocytomation , glostrup , denmark ) or 0.2% casein ( sigma , c7078 ) at room temperature . the sections were incubated overnight at 4c with primary antibodies diluted in blocking buffer and washed with tbs . the real envision detection system ( peroxidase / dab+ rabbit / mouse , code k5007 , dakocytomation , glostrup , denmark ) was used for detecting mouse and rabbit primaries . the sections were washed with tbs , followed by incubation for 10 min with 3,3diaminobenzidine chromogen solution . the sections were counterstained with mayers hematoxylin and dehydrated in graded alcohols followed by xylene and coverslipped with dpx mounting media . for immunofluorescence , sections were prepared as for bright field light microscopy including incubation of the first primary antibody overnight at 4c . sections were then incubated for 30 min at room temperature with labeled polymer hrp antimouse ( dakocytomation , envision+ system / hrp k4007 ) followed by tyramid signal amplification ( tsa ) with alexa fluor 488 tyramide ( invitrogen , molecular probes , t20912 ) for 7 min at room temperature . subsequently , the sections were incubated for 30 min at room temperature with biotinspconjugated f(ab')2 fragment donkey antirabbit antibodies ( jackson immunoresearch , 711066152 , 1:200 ) followed by streptavidinconjugated dylight 594 ( vector laboratories , sa5594 , 1:200 ) . finally , a nuclear counterstain with dapi ( 4,6diamidino2phenylindole , invitrogen , molecular probes , d1306 , 1:1,000 ) was added for 3 min , before sections were coverslipped . for double labeling of ykl40 and ssea4 , nonspecific binding was inhibited by incubation in blocking reagent ( invitrogen , molecular probes , t20912 ) for 1 h at room temperature . the sections were then incubated with ykl40 diluted in blocking reagent for 48 h at 4c and washed with tbs . following this , the secondary antibody was added ( jackson biotin antirabbit 1:100 ) , then hrpconjugated streptavidin ( perkin elmer , nel 700 , 711066152 , 1:100 ) followed by tyramid signal amplification for 10 min at room temperature . endogenous peroxidase was quenched with hydrogen peroxide for 30 min prior to the second primary antibody , ssea4 , overnight at 4c followed by labeled polymer hrp antimouse ( dakocytomation , envision+ system / hrp k4007 ) followed by tyramid signal amplification ( tsa ) with dylight 594 for 7 min at room temperature . monoclonal antibodies against ykl40 and ssea4 were used for identification and characterization of the cell population in question ; a polyclonal antiykl40/antichi3l1 antibody was used for double labeling with ssea4 . ipcs , neurons , and interneurons were identified using antibodies against tbr2 , the neuronalspecific neun and doublecortin ( dcx ) and the calciumbinding protein calbindin , respectively . glial cells were labeled with gfap , s100 , olig2 , and the proteoglycan ng2 antibodies , and microglial cells were distinguished with antibodies against iba1 and cd68 . details of the primary antibodies including dilutions and suppliers are listed in table 1 . staining specificity of ykl40 was tested on the same material in a recent study ( see fig . 1 in bjrnbak et al . , 2014 ) , and microscopy a carl zeiss lsm 780 was used . during image acquisition , a sequential scan procedure through the zaxis of the doublelabeled sections was performed when appropriate , covering in total 911 m in depth . confocal images were acquired and analyzed , and individual optical sections were stored as tiff files using zeiss zen vision v10 . representative images for figure editing were chosen from the analyzed samples and processed in adobe photoshop cs6 . distribution of ssea4 immunoreactivity in a coronal section of developing forebrain at the level of foramen interventriculare ( fi ) from a late 8th wpc human embryo ( crl : 31 mm ) . note the reactivity of leptomeninges and in particular the choroid plexus ( chp ) epithelium compared with that of the telencephalic wall . immunoreactivity was absent from the ganglionic eminence ( ge ) , antihem ( ah ) and ventricular zone at this stage . a strikingly similar pattern of ykl40 immunoreactivity in a parallel section from the same embryo has recently been demonstrated ( fig.2b in bjrnbak et al . 2014 ) . abbreviations : ah : antihem ; chp : choroid plexus ; cp : cortical plate ; fi : foramen interventriculare ; ge : ganglionic eminence ; ha : hippocampal anlage ; h : hem ; iz : intermediate zone ; lv : lateral ventricle ; vz : ventricular zone ; 3v : third ventricle . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] a total of 16 different sections from occipital cortices of 21 wpc fetuses were doublelabeled with fluorophorelabeled antibodies against ssea4 or ykl40 and blbp or tbr2 , respectively , and used for counting total and labeled cells of the subventricular zone from occipital cortex . the images were captured with a carl zeiss lsm 780 confocal microscope with a 20 objective and imported into the open source software fiji . the cell counter plugin was used for counting ssea4 and ykl40 immunopositive cells out of total number of nuclear counterstained dapipositive cells , blbpimmunoreactive cells in relation to doublelabeled blbp and ssea4 or ykl40 immunopositive cells and the number of ykl40 or ssea4 positive cells that were also tbr2immunolabeled . forebrains from one late human embryo , 31 mm crownrump length ( crl ) and 12 fetuses ( 38200 mm crl ) corresponding to 8th21st weeks post conception ( wpc ) were examined . the embryo and fetuses were obtained from legal abortions . according to the helsinki declaration ii oral and written information was given and informed consent was obtained from all contributing women , according to and approved by the research ethics committee of the capital region ( kf v.100.1735/90 ) . immediately following operation , the samples were dissected into blocks and fixed for 1224 h at 4c in either 10% neutral buffered formalin , 4% formolcalcium , lillie 's or bouin 's fixatives . serial sections , 310 m thick , were cut in transverse , sagittal , or horizontal planes , placed on silanized glass slides , and used for single and double immunohistochemical experiments . for bright field light microscopy analysis , sections were deparaffinized and rehydrated in xylene following standard protocols . endogenous peroxidase was quenched using a 0.5% solution of hydrogen peroxide in methanol for 15 min . following rinses with tris buffered saline ( tbs , 5 mm trishcl , 146 mm nacl , ph 7.6 ) , nonspecific binding was inhibited by incubation for 30 min with blocking buffer ( chemmate antibody diluent s2022 , dakocytomation , glostrup , denmark ) or 0.2% casein ( sigma , c7078 ) at room temperature . the sections were incubated overnight at 4c with primary antibodies diluted in blocking buffer and washed with tbs . the real envision detection system ( peroxidase / dab+ rabbit / mouse , code k5007 , dakocytomation , glostrup , denmark ) was used for detecting mouse and rabbit primaries . the sections were washed with tbs , followed by incubation for 10 min with 3,3diaminobenzidine chromogen solution . the sections were counterstained with mayers hematoxylin and dehydrated in graded alcohols followed by xylene and coverslipped with dpx mounting media . for immunofluorescence , sections were prepared as for bright field light microscopy including incubation of the first primary antibody overnight at 4c . sections were then incubated for 30 min at room temperature with labeled polymer hrp antimouse ( dakocytomation , envision+ system / hrp k4007 ) followed by tyramid signal amplification ( tsa ) with alexa fluor 488 tyramide ( invitrogen , molecular probes , t20912 ) for 7 min at room temperature . subsequently , the sections were incubated for 30 min at room temperature with biotinspconjugated f(ab')2 fragment donkey antirabbit antibodies ( jackson immunoresearch , 711066152 , 1:200 ) followed by streptavidinconjugated dylight 594 ( vector laboratories , sa5594 , 1:200 ) . finally , a nuclear counterstain with dapi ( 4,6diamidino2phenylindole , invitrogen , molecular probes , d1306 , 1:1,000 ) was added for 3 min , before sections were coverslipped . for double labeling of ykl40 and ssea4 , nonspecific binding was inhibited by incubation in blocking reagent ( invitrogen , molecular probes , t20912 ) for 1 h at room temperature . the sections were then incubated with ykl40 diluted in blocking reagent for 48 h at 4c and washed with tbs . following this , the secondary antibody was added ( jackson biotin antirabbit 1:100 ) , then hrpconjugated streptavidin ( perkin elmer , nel 700 , 711066152 , 1:100 ) followed by tyramid signal amplification for 10 min at room temperature . endogenous peroxidase was quenched with hydrogen peroxide for 30 min prior to the second primary antibody , ssea4 , overnight at 4c followed by labeled polymer hrp antimouse ( dakocytomation , envision+ system / hrp k4007 ) followed by tyramid signal amplification ( tsa ) with dylight 594 for 7 min at room temperature . monoclonal antibodies against ykl40 and ssea4 were used for identification and characterization of the cell population in question ; a polyclonal antiykl40/antichi3l1 antibody was used for double labeling with ssea4 . ipcs , neurons , and interneurons were identified using antibodies against tbr2 , the neuronalspecific neun and doublecortin ( dcx ) and the calciumbinding protein calbindin , respectively . glial cells were labeled with gfap , s100 , olig2 , and the proteoglycan ng2 antibodies , and microglial cells were distinguished with antibodies against iba1 and cd68 . details of the primary antibodies including dilutions and suppliers are listed in table 1 . staining specificity of ykl40 was tested on the same material in a recent study ( see fig . 1 in bjrnbak et al . , 2014 ) , and further controls were performed by omitting primary antibodies . for laser scanning confocal microscopy a carl zeiss lsm 780 was used . during image acquisition , a sequential scan procedure through the zaxis of the doublelabeled sections was performed when appropriate , covering in total 911 m in depth . confocal images were acquired and analyzed , and individual optical sections were stored as tiff files using zeiss zen vision v10 . representative images for figure editing were chosen from the analyzed samples and processed in adobe photoshop cs6 . distribution of ssea4 immunoreactivity in a coronal section of developing forebrain at the level of foramen interventriculare ( fi ) from a late 8th wpc human embryo ( crl : 31 mm ) . note the reactivity of leptomeninges and in particular the choroid plexus ( chp ) epithelium compared with that of the telencephalic wall . immunoreactivity was absent from the ganglionic eminence ( ge ) , antihem ( ah ) and ventricular zone at this stage . a strikingly similar pattern of ykl40 immunoreactivity in a parallel section from the same embryo has recently been demonstrated ( fig.2b in bjrnbak et al . abbreviations : ah : antihem ; chp : choroid plexus ; cp : cortical plate ; fi : foramen interventriculare ; ge : ganglionic eminence ; ha : hippocampal anlage ; h : hem ; iz : intermediate zone ; lv : lateral ventricle ; vz : ventricular zone ; 3v : third ventricle . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] a total of 16 different sections from occipital cortices of 21 wpc fetuses were doublelabeled with fluorophorelabeled antibodies against ssea4 or ykl40 and blbp or tbr2 , respectively , and used for counting total and labeled cells of the subventricular zone from occipital cortex . the images were captured with a carl zeiss lsm 780 confocal microscope with a 20 objective and imported into the open source software fiji . the cell counter plugin was used for counting ssea4 and ykl40 immunopositive cells out of total number of nuclear counterstained dapipositive cells , blbpimmunoreactive cells in relation to doublelabeled blbp and ssea4 or ykl40 immunopositive cells and the number of ykl40 or ssea4 positive cells that were also tbr2immunolabeled . the characterization of the recently described ykl40 positive cell population within the svz of developing human forebrain was based on immunohistochemistry and confocal microscopy . we used antibodies against known cell types , which have been associated with the svz , such as rgcs ( blbp , gfap , nestin ) , ipcs ( tbr2 ) , neurons and migrating interneurons and neurons ( dcx , calbindin , neun ) , glial cells ( gfap , s100 , ng2 , olig2 ) , and microglial cells ( cd68 , iba1 ) . by immunostaining and doublelabeling adjacent sections with antibodies against ssea4 and ykl40 we investigated the apparently similar distribution of ykl40 and ssea4 in the developing human forebrain , supplied by counting ssea4 and ykl40 immunolabeled cells in adjacent sections the choroid plexus showed a uniform strong ssea4 reactivity toward the end of 8th wpc and the subpial layer of radial glial end feet was clearly immunoreactive with the strongest reactivity corresponding to the outer surface of the hippocampal anlage and the lateral part of the dorsolateral wall ( fig . leptomeningeal cells in the piaarachnoid and many of the small vessels in the meninges also showed a strong ssea4 immunostaining . these brain barrierrelated recently published findings ( brchner et al . , 2015 ) will not be dealt with here where the focus is on the subpopulation of ssea4 and ykl40 positive cells in svz . no positive staining reactions were seen corresponding to the ganglionic eminence , the antihem or the early developing subventricular zone ( svz ) . the first indication of cell bodies immunoreactive for ssea4 and ykl40 were found in the antihem adjacent to the lateral ganglionic eminence in 12th wpc fetuses . at 15th wpc 2 ) but scattered positive cells particularly in more rostral parts of the svz were also observed . the ssea4 and ykl40 positive cell populations in the antihem were not stained with markers for ng2 or calbindin ( fig . immunoreactivity for the interneuron marker calbindin was particularly prominent in the lateral ganglionic eminence ( lge ) adjacent to the antihem , whereas blbp staining was characteristic for rgcs . many of the small ssea4 positive cells in the intermediate zone ( iz ) were pericytes and thus not a part of the proposed astrogenic subpopulation . the entire end feet layer which was strongly ssea4 and blbp positive was indicative of all rgcs , which terminate at the subpial basement membrane , and ssea4 ( a ) , ng2 ( b ) , blbp ( c ) , ykl40 ( d ) , and calbindin ( e ) immunoreactivity in coronal sections of frontal cortex from a 15th wpc human fetus ( crl : 111 mm ) . a distinct population of small rounded cells in the antihem ( ah ) is stained for ssea4 ( a ) and ykl40 ( d ) . ng2 positive cells are not associated with the proliferative zones but predominantly found in the lower part of the subplate ( sp ) indicated by arrows in ( b ) . immunoreactivity for the interneuron marker calbindin is particularly prominent in the lateral ganglionic eminence ( lge ) adjacent to the antihem , whereas blbp staining is characteristic for all rgcs ( c ) . many of the small ssea4 positive cells in the intermediate zone ( iz ) in ( a ) are pericytes and thus not a part of the proposed astrogenic subpopulation , and the entire end feet layer ( efl ) which is also strongly ssea4 positive and blbp positive ( not shown ) is indicative of all rgcs which terminate at the subpial basement membrane and therefore not specifically associated with the astrogenic subpopulation . abbreviations : ah : antihem ; cp : cortical plate ; efl : end feet layer ; lge : lateral ganglionic eminence ; iz : intermediate zone ; sp : subplate . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] through thorough examination of embryos and fetuses up until midgestation , it was evident that the population of cells in question was most prominent at later stages . to avoid the major interneuronproducing parts of the svz the ganglionic eminences we chose to focus our investigations from this point onward to midterm occipital cortices , also a major focus for many developmental studies ( see e.g. , smart et al . , 2002 ) . in a 21st wpc fetus , the ykl40 positive cells were distributed within the inner subventricular zone , and few had migrated through the inner fibrous layer to the outer subventricular zone ( fig . no reactivity was seen in corresponding cells in intermediate zone or cortical plate , apart from pericytic reactivity . the many unstained cells in the svz probably belonged to groups of other progenitor cells , interneurons or microglia , harbored in the subventricular zone . ykl40 immunoreactivity in a coronal section of visual cortex from a 21 wpc fetus ( crl : 200 mm ) . a low power overview is shown in ( a ) where the calcarine sulcus ( cs ) is indicated with an arrow . the boxed area is shown in higher magnification in ( b ) which provides an overview of the distribution of the ykl40 positive cells in the inner and outer subventricular zones separated by the inner fibrous layer with migrating positive cells . the boxed area in ( b ) is shown in higher magnification in ( c ) where it is obvious that the ykl40immunoreactive population is not present in the ventricular zone whereas immunostained cells occupy a substantial part of the inner subventricular zone of the visual cortex . the many unstained cells might belong to groups of other progenitor cells , interneurons or microglia . scale bars : a : 2,000 m ; b : 500 m ; c : 100 m . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] several previous studies have shown that no antibody specifically labels all rgcs throughout development ( noctor et al . , 2002 ; pinto and gtz , 2007 ) . the population is very heterogeneous , and may change expression profile in a spatiotemporal manner . to further characterize the cell population , we used the radial glial cell marker blbp double labeled with ssea4 or ykl40 . in all midterm cases investigated 4 ) . both ssea4 and ykl40 positive cells were present in the svz often distributed in small clusters along with or in close vicinity to radial glial cell fibers , rendering the impression that they were migrating along these fibers . some cells in the innermost part of the svz seemed to be only blbppositive and others seemingly only ssea4 or ykl40positive . a proportion of blbp immunoreactive cells colocalized with ssea4 or ykl40 immunopositive cells ( ranging from 11.6 to 49.6% , averaging 30.8% ) and the colocalization of ssea4 and blbp was accordingly similar to that of ykl40 and blbp ( fig . 4 ) . the proportion of ssea4 or ykl40 positive cells that were not blbp positive out of all ssea4 or ykl40 labeled cells were ranging from 36.3 to 93.2% , averaging 61.1% . adjacent sections to that shown in fig . 3 from the same fetus ( 21st wpc , crl : 200 mm ) double immunostained for the radial glial cell marker blbp and ykl40 or ssea4 . the ventricular zone is lined with blbpreactivity , but shows no ykl40 or ssea4 staining . both ykl40 and ssea4 positive cells are apparently migrating along radial glial cell fibers [ arrowheads in ( a ) and ( c ) ] . some cells in the innermost part of the svz seem to be only blbppositive and others seemingly only ykl40positive . the ykl40 and ssea4 positive cells are found in small clusters close to their sites of migration ( arrows in ( a ) and ( c ) ) . the distribution of ssea4 positive cells is apparently similar to that of ykl40 described in ( a ) , and the colocalization of ssea4 and blbp is similar to that of ykl40 and blbp . higher magnification through the zaxis of the sections are shown in ( b ) and ( d ) . ( b ) a threedimensional view of the svz , stained for blbp and ykl40 , shows overlap between a subset of blbp positive rgc fibers and ykl40 throughout the zaxis of the section . ( d ) a maximum projection intensity image of the zaxis of the isvz shows ssea4 positive cells colocalized with blbppositive cells ( arrows ) . scale bars : a , c : 50 m ; b : 10 m ; d : 20 m . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] to examine the distribution in the svz of ssea4 and ykl40 immunoreactive cells , sections were counted with respect to either ssea4 or ykl40 and dapi as a denominator of all cell nuclei or total cells . within the given area of occipital cortex at 21 wpc , the proportions were strikingly similar with an average of 11.99% of ykl40 immunopositive cells out of all nuclei compared to an average of 10.68% of ssea4 immunopositive cells out of all nuclei ( fig . 5 ) . using a different , polyclonal antibody against ykl40/chi3l1 we performed double immunolabeling of the same occipital cortical area of a 21st wpc fetus , and found colocalization of ssea4 and ykl40 in the svz cell population ( fig . adjacent sections of occipital cortex subventricular zone from a 21st wpc human fetus stained for ssea4 or ykl40 and processed with zstacks of equivalent representative areas . maximum intensity projections are applied , and using fiji cell counter , ssea4 , ykl40 , and dapipositive cells are manually counted on 8 adjacent representative sections . a total of 4,584 and 4,121 dapipositive nuclei are counted from 4 ykl40 and 4 ssea4 labeled sections , respectively . of all the counted cells , in the four ykl40 stained sections 11,99% of total cells mean total values of ykl40 and ssea4 out of total cells are shown in a and b , respectively . the inner subventricular zone ( isvz ) of occipital cortex from a 21st wpc human fetus ( crl : 200 mm ) . double immunolabeling with antibodies against ssea4 ( red ) and ykl40 ( green ) , with nuclei labeled with dapi ( blue ) . ( a ) the ssea4 and ykl40 positive cells are oriented in the same direction on a migratory trajectory . merged images of doubleimmunolabeled cells are boxed in ( a ) and shown at higher magnification in ( d ) and ( g ) , with individual channels in ( b ) , ( c ) and ( e ) , ( f ) , respectively . the doublelabeled cells show immunoreactivity within the same cell compartments ( er / golgi apparatus ) in close apposition to the nucleus [ ( d ) , ( g ) ] . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] the previously briefly described relation between this ykl40 positive population and gfap ( bjrnbak et al . , 2014 ) led us to a closer examination of gfap and ykl40 . it has been stated , that only a fraction of rgcs express gfap in humans ( and none in mice during development ) . in occipital cortex , in a 21st wpc fetus , gfap positive processes from rgcs were situated apically , as a ventricular surface lining much like blbp ( fig . 7 ) . in contrast , however , hardly any cells were coexpressing gfap and ykl40 , and when they did the cells were situated in the apical part of the inner subventricular zone . a different area of occipital cortex from a 21st wpc human fetus ( crl : 200 mm ) outside the calcarine sulcus . the entire section stained for ykl40 is shown in ( a ) at low magnification . the boxed area corresponds to a similar area of a neighboring section used for double immunolabeling . ( b ) examination of ykl40 immunoreactivity with rgcs labeled with antibodies against gfap shows a gfaplined ventricular zone and an ykl40 positive population in the svz . the boxed area , seen in ( c ) at higher magnification , shows a differential pattern of gfap positive cells compared to ykl40 positive cells . the ventricular zone ( i ) contains gfap positive radial fibers anchoring at the ventricular surface . the basal part of the vz ( ii ) is a gfap / ykl40 positive zone with hardly any doublelabeled cells , while the deeper layer of the inner svz only contains ykl40 positive and gfap negative cells ( iii ) . scale bars : a : 2,000 m ; b : 100 m ; c : 20 m . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] in an attempt to narrow down the lineage of these ssea4/ykl40 and blbp positive cells , we used antibodies against astrocytes ( s100 ) and the panneuronal marker neun . the s100 positive astrocytes were scattered through the entire cortical wall although somewhat more abundant in the lower cortical plate / upper subplate and in the inner subventricular zone . the density and distribution of the s100 positive cells were clearly different from those of ykl40 stained cells . double immunolabeling of neun together with ykl40 demonstrated a lack of colocalization in the svz and in the cortical plate . to elucidate whether the cell population was related to ipcs , we used the ipcmarker tbr2 , and doublelabeled sections for ssea4 and ykl40 with tbr2 , respectively . . however , in a very few cases , ssea4 and ykl40 did evidently colocalize with tbr2 . in four counted sections of 21st wpc occipital cortex , we found 171 ssea4 or ykl40 positive cells and only 13 of these were colabeled with tbr2 . these cells might be involved in the transition from newborn ipc to fully mature ipc in the subventricular zone . ssea4 and ykl40 positive cells possessed a basal process , and we noted that most cells appeared to migrate in the same radial direction , though a minor fraction appeared to be migrating tangentially . apart from their role in phagocytosis , microglial cells also function as important modulators of neurogenesis ( cunningham et al . , 2013 ) . we showed , however , that iba1 positive cells did not colocalize with ykl40 in the proliferative subventricular zone ( fig . 8) . distribution of s100 ( a , b ) and iba1 ( c ) immunoreactivity in occipital cortex from a 21st wpc fetus ( crl : 200 mm ) , same fetus as in figs . 3 and 4 . s100 positive astrocytes are scattered through the entire cortical wall although somewhat more abundant in the lower cortical plate / upper subplate and in the inner subventricular zone ( a ) which is shown in higher magnification in ( b ) . note that the density and distribution of the s100 positive cells in ( b ) is different from that of ykl40 stained cells in ( d ) . iba1 stained microglial cells are present in both ventricular and inner subventricular zones in ( c ) but do not colocalize with ykl40 positive cells ( see e ) . the panneuronal marker neun together with ykl40 ( d ) demonstrate no ykl40 immunoreactive neurons in the svz . the ykl40 positive population seems to cluster in small groups ( arrows ) prior to migration on a fiber scaffold ( arrowheads ) . note the simultaneous migration of neunpositive cells ( open arrowheads ) and ykl40 positive cells on the same radial glial fiber ( arrowheads ) . in ( e ) , double immunolabeling for regulatory microglia in the inner svz with the microglia marker iba1 and ykl40 ( open arrow ) , showed no crossreactivity , indicating that the ykl40 positive cell population is not of microglial origin . in adjacent sections ( f , g ) , staining for ykl40 and ssea4 and intermediate progenitor cells with tbr2 , respectively , depicts distinct populations in the osvz . virtually no colocalization is observed , however in a very few cases , ykl40 and ssea4 do colocalize with tbr2 ( dashed arrows ) . note that most cells appear to migrate in the same radial direction though tangential migration is also observed . abbreviations : isvz : inner subventricular zone ; osvz : outer subventricular zone ; vz : ventricular zone . scale bars : a : 500 m ; b , c : 100 m ; e : 10 m ; d , f , g : 50 m . [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] finally we performed double immunolabeling and compared the distribution of the colocalized ssea4 and ykl40 immunoreactive cell population in basal vz and svz with cell populations immunostained for the progenitor marker nestin , the oligodendrocyte progenitor marker olig2 and the marker for migrating neuronal precursors dcx . these markers have all been described in human midgestation ventricular and subventricular zones ( e.g. , messam et al . , 2002 ; jakovcevski and zecevic , 2005a , b ; meyer et al . , 2002 ) . most of the vz cells but very few cells in isvz at midgestation of occipital cortex showed immunoreactivity for nestin in marked contrast to the abundant ssea4 immunopositive cells in isvz and lack of ssea4 staining in the vz ( fig . 9a ) . olig2 and ykl40 immunoreactivity in midgestation parietal cortex showed a pattern of distribution similar to that of nestin and ssea4 double immunolabeling . very few cell bodies in the outer vz were positively stained for ykl40 and the merged images showed no overlap ( fig . double immunolabeling of medial temporal cortex with antibodies against dcx and ssea4 demonstrated many ssea4 immunopositive cell bodies in isvz and many dcx positive fibers in both vz and isvz but without overlap in merged images ( fig . nestin , olig2 and dcx positive cells were as expected present in many developmental regions where we found no trace of ssea4/ykl40 reactivity . distribution of nestin ( a ) , olig2 ( b ) , and dcx ( c ) in relation to ssea4/ykl40 shown in merged dapi stained images . the inner subventricular zone ( isvz ) ( a1 ) and ventricular zone ( vz ) ( a2 ) of occipital cortex from a 21st wpc human fetus ( crl : 200 mm ) , doubleimmunolabeled with antibodies against nestin ( red ) and ssea4 ( green ) , and nuclei labeled with dapi ( blue ) . the outer vz which showed no immunoreactivity was left out . note the abundant ssea4 immunopositive cells in isvz ( arrows ) and the very few nestinpositive cells ( arrowheads ) . in vz adjacent to the lateral ventricle the majority of the cells are nestinpositive whereas no ssea4 positive cells are present ( a1 ) . the few yellow strokes in ( a1 ) probably represent closely apposed fibers belonging to ssea4 and nestin positive cells in the merged image . in ( b ) many nuclei of cells in the inner vz of parietal cortex from a 15th wpc human fetus ( crl : 110 mm ) , are positive for olig2 ( arrows ) whereas very few cell bodies are immunoreactive for ykl40 ( arrowhead ) in the outer vz . the isvz and outer vz in the medial wall of the temporal cortex from the same fetus are double immunolabeled with antibodies against dcx ( red ) and ssea4 ( green ) . note the abundant ssea4 immunopositive cells in isvz ( arrows ) and the dcx labeled fibers in both vz and isvz ( arrowheads ) . abbreviations : dcx : doublecortin ; isvz : inner subventricular zone ; svz : subventricular zone ; vz : ventricular zone . a [ color figure can be viewed in the online issue , which is available at wileyonlinelibrary.com . ] so far ykl40 has been associated with various pathological conditions and promoted as a factor with profound implications for both diagnostic and therapeutic applications ( prakash et al . , 2013 ) , whereas the general role of ykl40 in developmental biology has been largely ignored with a few exceptions ( brchner et al . , 2012 ; johansen et al . , 2007 ) . a very recent investigation of ykl40 in the developing human forebrain showed marked ykl40 immunoreactivity in neuroepithelial cells , radial glial end feet , leptomeninges and choroid plexus epithelial cells in early stages of development . later developmental features included strong ykl40 immunoreactivity in astrocyteresembling cells in the developing hippocampus , and a so far unknown population of small rounded ykl40 positive cells in close relation to and occasionally overlapping with gfappositive radial glial fibers in the svz at midgestation ( bjrnbak et al . , 2014 ) . in an earlier study partly based on the same material , ssea4 was depicted as a marker for some nscs and showed immunoreactivity very similar to ykl40 in the early human forebrain ( barraud et al . , 2007 ) . furthermore ssea4 has been proposed as a potential therapeutic target in glioblastoma multiforme ( lou et al . , 2014 ) , much in the same way as ykl40 has . an important part of the present study is a novel characterization of ssea4 in the late embryonic human forebrain where we found a marked ssea4 immunoreactivity in radial glial end feet , leptomeninges , and choroid plexus epithelial cells in early stages of development . later developmental features included a population of small rounded ssea4 positive cells , first in the antihem and later scattered in different parts of the svz , with the same spatiotemporal distribution as the ykl40 positive cells . rgcs show a characteristic apicobasal polarity , with an apical process lining the ventricle and a basal process spanning the neuroepithelium and anchoring at the pial border . the cell bodies occupy the ventricular zone ( vz ) , and through rounds of asymmetric divisions , they give rise to ipcs , which in turn produce two postmitotic neurons or in some cases an additional pair of progenitors ( englund et al . , 2005 ; molnr et al . , 2014 ; noctor et al . , 2004 ; pontious et al . the ipcs create the second proliferative layer just basal to the vz , namely the subventricular zone ( svz ) ( bystron et al . , 2008 ; mllgrd and jacobsen , 1984 ) , which subdivides into an inner and outer subventricular zone , separated by a fibrous layer ( bayatti et al . , 2008 ; smart et al . , 2002 ; zecevic et al . , 2005 ) as mentioned in the introduction . ( 2002 ) clearly distinguished between cytoarchitectonic compartments ( vz , isvz , and osvz ) and their rostrocaudal gradient of histogenesis showing a decreasing depth of all layers except the vz from prefrontal to occipital cortex . the isvz is composed of cells oriented in various directions whereas the osvz exhibits a columnar structure composed of radially oriented nuclei ( smart et al . , although the osvz cells ( or outer radial gliallike cells orgcs ) were first thought to be a unique cell type in fetal primate neocortex , counterparts have also been observed in species such as ferrets ( fietz et al . , 2010 ) and mice ( shitamukai et al . , 2011 ; wang et al . , 2011 ) . thus , although mice do not possess the same cytoarchitectonic compartments as primates they have the same progenitor populations but in different proportions . as summarized very recently by hoerdersuabedissen and molnr ( 2015 ) : to date , all proliferative regions and modes of cell division identified in primate brains have also been documented in rodent brains and other large and small , lissencephalic and gyrencephalic brains ( garciamoreno et al . , 2012 ) , although the svz is disproportionately smaller in mice than in large primates ( cheung et al . , 2010 ) . thus , not only are orgs not primate specific , but their presence in large numbers is not the cause of brain folding as it was originally suggested ( garciamoreno et al . , 2012 ) . the recent seminal work by several groups that led to the identification of orgcs within the osvz has primarily focused on the neurogenic potential of these cells ( betizeau et al . , 2013 ; fietz et al . , 2010 ; hansen et al . , 2010 ; kelava et al . , 2012 ; the diversification of rgc progeny in both spatial and temporal aspects is a matter of intense research , and evidence from human and rodent studies point toward distinct subpopulations of rgcs that may be purely gliogenic , purely neurogenic or multipotent ( hartfuss et al . this suggestion is very much supported by our findings that tbr2 is not expressed in the majority of these ssea4/ykl40 immunopositive progenitors . a tbr2 expressing lineage has been recently studied in the developing mouse brain by vasistha et al . ( 2014 ) who found no glial cell lineage arising from tbr2positive intermediate progenitors using the clone method . in humans , so far no solid setup exists , that enables the segregation of orgcs into neuronal , multipotent , or gliogenic subpopulations . the orgcs differ in morphology from ventricular rgcs in that they are delaminated from the adherens junctions of the rgc epithelium , and they lack an apical process , but , however , retain a basal anchor ( betizeau et al . , 2013 ; 2010 ) . when examining for rgc markers ( blbp , gfap , nestin ) , neuronal and known glial markers , a subgroup of ssea4 and ykl40 positive cells only colocalize with blbp within the isvz and not with the other markers used . interestingly , cell culture studies of human midgestation dissociated vz / svz showed many diverse subtypes of rgcs among which a subtype of dividing cells not labeled with 4a4 or neuronal markers were suggested to represent a multipotent precursor or restricted progenitor line identified by a another yet unused antibody ( howard et al . , 2006 ) . the microtubuleassociated protein dcx , that is expressed by migrating neuronal precursors show no overlap with the investigated cell population . based on cell counts of immunoreactive ssea4 and ykl40 positive cells in adjacent sections and in equal regions of a 21 wpc occipital cortical area , we show that cell numbers between these two are strikingly equivalent within the svz . on a morphological basis , the cells express the same characteristics , with approximately the same nuclear size and a migrating profile , and colocalization from double labeling studies of ssea4 and ykl40 confirms that the cell populations marked by ssea4 or ykl40 do correspond to one another . both gfap and nestin are structural filaments found in various cell types , and also in subsets of glial cells and rgcs . double labeling with gfap and ykl40 and with nestin and ssea4 revealed that hardly any cells colabeled in the basal part of the isvz . a very recent analysis of the clonal dispersion of astrocytes in mouse cerebral cortex indicates that heterogeneous astroglial populations arise from specialized progenitor cells ( garcamarqus and lpezmascaraque , 2013 ) . in a study by singh et al . ( singh et al . , 2011 ) , the expression of ykl40 during in vitro differentiation of human neural progenitors into astrocytes was dramatically increased during astrocyte differentiation . moreover , ykl40 was easily detected in primary cultures of human embryonic astrocytes . in mice , the stem cell marker ssea1 , the ssea4 counterpart in hesc lines , has interestingly been associated with a subpopulation of astrocytes in the adult svz progenitor cells and developmental studies of rat brain showed ssea1 in telencephalic germinal zones ( capela and temple , 2002 ) . it should be noted that ssea4 is associated with human pluripotent stem cells of the inner cell mass , while the murine counterpart associated with pluripotent stem cells is ssea1 ( henderson et al . , 2002 ) . a commonly used ipc marker is the tbox transcription factor tbr2 , and in our study we found that the majority of tbr2positive ipcs did not colocalize with either ssea4 or ykl40 . however , a few ssea4 or ykl40 positive cells did coexpress tbr2 , and these doublelabeled cells possessed a leading process uncharacteristic for ipcs . they might depict an intermediate differentiated stage , as the ipcs migrate to the final location in the isvz and osvz . as gliogenesis progresses particularly from midgestation , appearing astrocytes are appreciated as an ultimately very heterogeneous population of cells , with distinct progenitors and diverse important functions in both normal and diseased brain . we examined glial markers such as s100 ( astrocytes ) and ng2 and olig2 ( oligodendrocytes ) . the sequence of oligodendrocyte development in human fetal forebrain from early oligodendrocyte progenitor cells to mature oligodendrocytes was described by jakovcevski and zecevic ( 2005a ) and the distribution of olig2 from second trimester ( 15th gestational week ) was elucidated in a following paper ( jakovcevski and zecevic , 2005b ) . at midgestation olig2 positive nuclei were mainly positioned close to the vz surface , see also fig . 1 in mo and zecevic ( 2009 ) . no cells were found to express these glial markers in combination with ssea4 or ykl40 . however , this does not rule out that the identified population is in fact part of the astroglial lineage , as the panel of astrocyte markers is by now not sufficiently extensive . blood monocytes are known to enter the early human forebrain via the cortical plate and meninges to become amoeboid microglial cells ( aguzzi et al . , microglial cells have been shown to be important modulators of neurogenesis , and during early human development they are localized to the isvz , subplate , lower cortical plate , and restricted laminar bands at the axonal crossroads in the white matter ( cunningham et al . studies indicate that microglia do not show ykl40 staining in vivo ( bonnehbarkay et al . , 2010 ; , the abundant population of iba1 positive cells within the isvz in 21st wpc fetuses did not colocalize with ykl40 , indicating that the ssea4 and ykl40 positive population is not of microglial origin . the nscs and neural progenitor cells , that is , neuroepithelial cells and rgcs , are as previously described characterized by a panel of molecular markers as well as ultrastructural and morphological features . ssea4 and ykl40 have so far not been a part of this panel , but our results suggest that these may be future important markers for a particular fraction of rgcs with astrogenic potential . in summary , we characterized this ssea4 and ykl40 positive cell population , by means of a range of molecular markers for cell types found within the subventricular zone . colocalization for both ssea4 and ykl40 was only evident for a subset of blbp positive rgcs , and not for the other markers used . the late appearance of this intriguing population of ssea4 and ykl40 positive cells in the svz corresponds to initiation of gliogenesis ( rakic , 1988 ) , and several studies have linked ykl40 expression to astrocytes in vitro and in vivo ( bonnehbarkay et al . the previous description based on the same material , of late appearing ykl40 positive astrocyteresembling cells within the fimbria , a neuronfree region of the hippocampus , strongly supports this notion ( see fig . 10 in bjrnbak et al . , 2014 ) . in line with these observations , we suggest that our newly identified subgroup of ssea4 and ykl40 positive cells constitute a fraction of astroglial progenitors , and suggest the name sypap ( ssea4 and ykl40 positive astroglial progenitors ) for this particular population . immunoreactivity of sypaps depends critically on the stage of development and the region of the forebrain studied and follows the rostrocaudal gradient of histogenesis described by smart et al . we hypothesize that this population has a developmental important role and may prove to be important for understanding human cortical development and glial functioning . identification of an astroglial progenitor population based on the same molecular markers including ssea4 and ykl40 in a rodent model has to our knowledge not yet been performed . the subventricular zone at midgestation harbors many different cell types including interneurons , microglia and combinations of neurons , glial , and rgcs . subclassification of svz progenitors based on single cell gene analysis may be an ultimate way to shed more light onto the diverse behavior of progenitors within the germinal zones , and thus aiding our understanding of late corticogenesis . whether these cells also play a role in origin or maintenance of glioblastoma multiforme , or if they contribute to the population of adult human nscs in the subependymal zone ( alvarezbuylla et al . , 2002 ) , warrants further examination .

the glycosphingolipid ssea4 and the glycoprotein ykl40 have both been associated with human embryonic and neural stem cell differentiation . we investigated the distribution of ssea4 and ykl40 positive cells in proliferative zones of human fetal forebrain using immunohistochemistry and doublelabeling immunofluorescence . a few small rounded ssea4 and ykl40 labeled cells were present in the radial glial blbp positive proliferative zones adjacent to the lateral ganglionic eminence from 12th week post conception . with increasing age , a similarly stained cell population appeared more widespread in the subventricular zone . at midgestation , the entire subventricular zone showed patches of ssea4 , ykl40 , and blbp positive cells . colabeling with markers for radial glial cells ( rgcs ) and neuronal , glial , and microglial markers tested the lineage identity of this subpopulation of radial glial descendants . adjacent to the ventricular zone , a minor fraction showed overlap with gfap but not with nestin , olig2 , ng2 , or s100 . no colocalization was found with neuronal markers neun , calbindin , dcx or with markers for microglial cells ( iba1 , cd68 ) . moreover , the ssea4 and ykl40 positive cell population in subventricular zone was largely devoid of tbr2 , a marker for intermediate neuronal progenitor cells descending from rgcs . ykl40 has recently been found in astrocytes in the neuronfree fimbria , and both ssea4 and ykl40 are present in malignant astroglial brain tumors . we suggest that the population of cells characterized by immunohistochemical combination of antibodies against ssea4 and ykl40 and devoid of neuronal and microglial markers represent a yet unexplored astrogenic lineage illustrating the complexity of astroglial development . glia 2016;64:90104

Introduction Materials and Methods Tissue Samples Immunohistochemistry Cell Counts/Quantification and Qualification Results Discussion

the neural progenitor cells underlying the cortical expansion comprise initially neuroepithelial cells , which transform into radial glial cells ( rgcs ) at the onset of neurogenesis ( gtz and huttner , 2005 ; kriegstein and alvarezbuylla , 2009 ) . these neural progenitors , often referred to as neural stem cells ( nscs ) ( kriegstein and alvarezbuylla , 2009 ) , give rise to the enormous diversity of neurons and glial cells in the cerebral cortex . , has shown intermediate progenitor cells ( ipcs ) and outer radial glial cells ( orgs ) in both compartments ( betizeau et al . , the globoseries glycosphingolipid stagespecific embryonic antigen 4 ( ssea4 ) is widely used in characterization of human embryonic stem cell ( hesc ) lines ( adewumi et al . , 2007 ) , and has been shown in a population of differentiating cells in an intermediate stage between pluripotent hescs and neural progenitor cells ( noisa et al . in a previous study of human embryonic central nervous system , ssea4 was found in neural progenitor cells in forebrains of human embryos and early fetuses ( barraud et al . in the developing human forebrain , ykl40 has recently been associated with brain barrier sites such as the radial glial end feet layer at the subpial marginal zone , pericytes of the intermediate and subventricular zones and in the choroid plexus ( chp ) epithelium of the lateral ventricles . furthermore , intriguing small rounded ykl40 positive cells in close relation to and occasionally overlapping with gfappositive radial glial fibers in the svz was identified ( bjrnbak et al . in this study , we aimed to investigate the recently described neural progenitor cell marker ssea4 in relation to the newly discovered ykl40 positive cell population in the svz of developing human forebrain , and examine its proposed astrogenic lineage potential . to this end , we studied human forebrain samples from human embryos and fetuses ( 8th21st weeks post conception ) by immunohistochemistry and confocal microscopy , with antibodies against ssea4 , ykl40 , and against specific cell types known to reside within the developing human forebrain , for example , rgcs , ipcs , neurons , interneurons , and glial cells including microglial cells . ipcs , neurons , and interneurons were identified using antibodies against tbr2 , the neuronalspecific neun and doublecortin ( dcx ) and the calciumbinding protein calbindin , respectively . glial cells were labeled with gfap , s100 , olig2 , and the proteoglycan ng2 antibodies , and microglial cells were distinguished with antibodies against iba1 and cd68 . a total of 16 different sections from occipital cortices of 21 wpc fetuses were doublelabeled with fluorophorelabeled antibodies against ssea4 or ykl40 and blbp or tbr2 , respectively , and used for counting total and labeled cells of the subventricular zone from occipital cortex . the cell counter plugin was used for counting ssea4 and ykl40 immunopositive cells out of total number of nuclear counterstained dapipositive cells , blbpimmunoreactive cells in relation to doublelabeled blbp and ssea4 or ykl40 immunopositive cells and the number of ykl40 or ssea4 positive cells that were also tbr2immunolabeled . monoclonal antibodies against ykl40 and ssea4 were used for identification and characterization of the cell population in question ; a polyclonal antiykl40/antichi3l1 antibody was used for double labeling with ssea4 . ipcs , neurons , and interneurons were identified using antibodies against tbr2 , the neuronalspecific neun and doublecortin ( dcx ) and the calciumbinding protein calbindin , respectively . glial cells were labeled with gfap , s100 , olig2 , and the proteoglycan ng2 antibodies , and microglial cells were distinguished with antibodies against iba1 and cd68 . immunoreactivity was absent from the ganglionic eminence ( ge ) , antihem ( ah ) and ventricular zone at this stage . a total of 16 different sections from occipital cortices of 21 wpc fetuses were doublelabeled with fluorophorelabeled antibodies against ssea4 or ykl40 and blbp or tbr2 , respectively , and used for counting total and labeled cells of the subventricular zone from occipital cortex . the cell counter plugin was used for counting ssea4 and ykl40 immunopositive cells out of total number of nuclear counterstained dapipositive cells , blbpimmunoreactive cells in relation to doublelabeled blbp and ssea4 or ykl40 immunopositive cells and the number of ykl40 or ssea4 positive cells that were also tbr2immunolabeled . we used antibodies against known cell types , which have been associated with the svz , such as rgcs ( blbp , gfap , nestin ) , ipcs ( tbr2 ) , neurons and migrating interneurons and neurons ( dcx , calbindin , neun ) , glial cells ( gfap , s100 , ng2 , olig2 ) , and microglial cells ( cd68 , iba1 ) . by immunostaining and doublelabeling adjacent sections with antibodies against ssea4 and ykl40 we investigated the apparently similar distribution of ykl40 and ssea4 in the developing human forebrain , supplied by counting ssea4 and ykl40 immunolabeled cells in adjacent sections the choroid plexus showed a uniform strong ssea4 reactivity toward the end of 8th wpc and the subpial layer of radial glial end feet was clearly immunoreactive with the strongest reactivity corresponding to the outer surface of the hippocampal anlage and the lateral part of the dorsolateral wall ( fig . , 2015 ) will not be dealt with here where the focus is on the subpopulation of ssea4 and ykl40 positive cells in svz . no positive staining reactions were seen corresponding to the ganglionic eminence , the antihem or the early developing subventricular zone ( svz ) . the first indication of cell bodies immunoreactive for ssea4 and ykl40 were found in the antihem adjacent to the lateral ganglionic eminence in 12th wpc fetuses . the ssea4 and ykl40 positive cell populations in the antihem were not stained with markers for ng2 or calbindin ( fig . immunoreactivity for the interneuron marker calbindin was particularly prominent in the lateral ganglionic eminence ( lge ) adjacent to the antihem , whereas blbp staining was characteristic for rgcs . the entire end feet layer which was strongly ssea4 and blbp positive was indicative of all rgcs , which terminate at the subpial basement membrane , and ssea4 ( a ) , ng2 ( b ) , blbp ( c ) , ykl40 ( d ) , and calbindin ( e ) immunoreactivity in coronal sections of frontal cortex from a 15th wpc human fetus ( crl : 111 mm ) . a distinct population of small rounded cells in the antihem ( ah ) is stained for ssea4 ( a ) and ykl40 ( d ) . ng2 positive cells are not associated with the proliferative zones but predominantly found in the lower part of the subplate ( sp ) indicated by arrows in ( b ) . immunoreactivity for the interneuron marker calbindin is particularly prominent in the lateral ganglionic eminence ( lge ) adjacent to the antihem , whereas blbp staining is characteristic for all rgcs ( c ) . many of the small ssea4 positive cells in the intermediate zone ( iz ) in ( a ) are pericytes and thus not a part of the proposed astrogenic subpopulation , and the entire end feet layer ( efl ) which is also strongly ssea4 positive and blbp positive ( not shown ) is indicative of all rgcs which terminate at the subpial basement membrane and therefore not specifically associated with the astrogenic subpopulation . through thorough examination of embryos and fetuses up until midgestation , it was evident that the population of cells in question was most prominent at later stages . in a 21st wpc fetus , the ykl40 positive cells were distributed within the inner subventricular zone , and few had migrated through the inner fibrous layer to the outer subventricular zone ( fig . the many unstained cells in the svz probably belonged to groups of other progenitor cells , interneurons or microglia , harbored in the subventricular zone . the boxed area is shown in higher magnification in ( b ) which provides an overview of the distribution of the ykl40 positive cells in the inner and outer subventricular zones separated by the inner fibrous layer with migrating positive cells . the boxed area in ( b ) is shown in higher magnification in ( c ) where it is obvious that the ykl40immunoreactive population is not present in the ventricular zone whereas immunostained cells occupy a substantial part of the inner subventricular zone of the visual cortex . both ssea4 and ykl40 positive cells were present in the svz often distributed in small clusters along with or in close vicinity to radial glial cell fibers , rendering the impression that they were migrating along these fibers . a proportion of blbp immunoreactive cells colocalized with ssea4 or ykl40 immunopositive cells ( ranging from 11.6 to 49.6% , averaging 30.8% ) and the colocalization of ssea4 and blbp was accordingly similar to that of ykl40 and blbp ( fig . the proportion of ssea4 or ykl40 positive cells that were not blbp positive out of all ssea4 or ykl40 labeled cells were ranging from 36.3 to 93.2% , averaging 61.1% . the distribution of ssea4 positive cells is apparently similar to that of ykl40 described in ( a ) , and the colocalization of ssea4 and blbp is similar to that of ykl40 and blbp . to examine the distribution in the svz of ssea4 and ykl40 immunoreactive cells , sections were counted with respect to either ssea4 or ykl40 and dapi as a denominator of all cell nuclei or total cells . using a different , polyclonal antibody against ykl40/chi3l1 we performed double immunolabeling of the same occipital cortical area of a 21st wpc fetus , and found colocalization of ssea4 and ykl40 in the svz cell population ( fig . ( a ) the ssea4 and ykl40 positive cells are oriented in the same direction on a migratory trajectory . in contrast , however , hardly any cells were coexpressing gfap and ykl40 , and when they did the cells were situated in the apical part of the inner subventricular zone . ( b ) examination of ykl40 immunoreactivity with rgcs labeled with antibodies against gfap shows a gfaplined ventricular zone and an ykl40 positive population in the svz . in an attempt to narrow down the lineage of these ssea4/ykl40 and blbp positive cells , we used antibodies against astrocytes ( s100 ) and the panneuronal marker neun . to elucidate whether the cell population was related to ipcs , we used the ipcmarker tbr2 , and doublelabeled sections for ssea4 and ykl40 with tbr2 , respectively . ssea4 and ykl40 positive cells possessed a basal process , and we noted that most cells appeared to migrate in the same radial direction , though a minor fraction appeared to be migrating tangentially . note that the density and distribution of the s100 positive cells in ( b ) is different from that of ykl40 stained cells in ( d ) . iba1 stained microglial cells are present in both ventricular and inner subventricular zones in ( c ) but do not colocalize with ykl40 positive cells ( see e ) . note the simultaneous migration of neunpositive cells ( open arrowheads ) and ykl40 positive cells on the same radial glial fiber ( arrowheads ) . in ( e ) , double immunolabeling for regulatory microglia in the inner svz with the microglia marker iba1 and ykl40 ( open arrow ) , showed no crossreactivity , indicating that the ykl40 positive cell population is not of microglial origin . in adjacent sections ( f , g ) , staining for ykl40 and ssea4 and intermediate progenitor cells with tbr2 , respectively , depicts distinct populations in the osvz . finally we performed double immunolabeling and compared the distribution of the colocalized ssea4 and ykl40 immunoreactive cell population in basal vz and svz with cell populations immunostained for the progenitor marker nestin , the oligodendrocyte progenitor marker olig2 and the marker for migrating neuronal precursors dcx . most of the vz cells but very few cells in isvz at midgestation of occipital cortex showed immunoreactivity for nestin in marked contrast to the abundant ssea4 immunopositive cells in isvz and lack of ssea4 staining in the vz ( fig . nestin , olig2 and dcx positive cells were as expected present in many developmental regions where we found no trace of ssea4/ykl40 reactivity . the inner subventricular zone ( isvz ) ( a1 ) and ventricular zone ( vz ) ( a2 ) of occipital cortex from a 21st wpc human fetus ( crl : 200 mm ) , doubleimmunolabeled with antibodies against nestin ( red ) and ssea4 ( green ) , and nuclei labeled with dapi ( blue ) . note the abundant ssea4 immunopositive cells in isvz ( arrows ) and the very few nestinpositive cells ( arrowheads ) . in vz adjacent to the lateral ventricle the majority of the cells are nestinpositive whereas no ssea4 positive cells are present ( a1 ) . the few yellow strokes in ( a1 ) probably represent closely apposed fibers belonging to ssea4 and nestin positive cells in the merged image . later developmental features included strong ykl40 immunoreactivity in astrocyteresembling cells in the developing hippocampus , and a so far unknown population of small rounded ykl40 positive cells in close relation to and occasionally overlapping with gfappositive radial glial fibers in the svz at midgestation ( bjrnbak et al . an important part of the present study is a novel characterization of ssea4 in the late embryonic human forebrain where we found a marked ssea4 immunoreactivity in radial glial end feet , leptomeninges , and choroid plexus epithelial cells in early stages of development . later developmental features included a population of small rounded ssea4 positive cells , first in the antihem and later scattered in different parts of the svz , with the same spatiotemporal distribution as the ykl40 positive cells . when examining for rgc markers ( blbp , gfap , nestin ) , neuronal and known glial markers , a subgroup of ssea4 and ykl40 positive cells only colocalize with blbp within the isvz and not with the other markers used . based on cell counts of immunoreactive ssea4 and ykl40 positive cells in adjacent sections and in equal regions of a 21 wpc occipital cortical area , we show that cell numbers between these two are strikingly equivalent within the svz . on a morphological basis , the cells express the same characteristics , with approximately the same nuclear size and a migrating profile , and colocalization from double labeling studies of ssea4 and ykl40 confirms that the cell populations marked by ssea4 or ykl40 do correspond to one another . double labeling with gfap and ykl40 and with nestin and ssea4 revealed that hardly any cells colabeled in the basal part of the isvz . moreover , ykl40 was easily detected in primary cultures of human embryonic astrocytes . in mice , the stem cell marker ssea1 , the ssea4 counterpart in hesc lines , has interestingly been associated with a subpopulation of astrocytes in the adult svz progenitor cells and developmental studies of rat brain showed ssea1 in telencephalic germinal zones ( capela and temple , 2002 ) . however , a few ssea4 or ykl40 positive cells did coexpress tbr2 , and these doublelabeled cells possessed a leading process uncharacteristic for ipcs . the sequence of oligodendrocyte development in human fetal forebrain from early oligodendrocyte progenitor cells to mature oligodendrocytes was described by jakovcevski and zecevic ( 2005a ) and the distribution of olig2 from second trimester ( 15th gestational week ) was elucidated in a following paper ( jakovcevski and zecevic , 2005b ) . , 2010 ; , the abundant population of iba1 positive cells within the isvz in 21st wpc fetuses did not colocalize with ykl40 , indicating that the ssea4 and ykl40 positive population is not of microglial origin . ssea4 and ykl40 have so far not been a part of this panel , but our results suggest that these may be future important markers for a particular fraction of rgcs with astrogenic potential . in summary , we characterized this ssea4 and ykl40 positive cell population , by means of a range of molecular markers for cell types found within the subventricular zone . colocalization for both ssea4 and ykl40 was only evident for a subset of blbp positive rgcs , and not for the other markers used . the late appearance of this intriguing population of ssea4 and ykl40 positive cells in the svz corresponds to initiation of gliogenesis ( rakic , 1988 ) , and several studies have linked ykl40 expression to astrocytes in vitro and in vivo ( bonnehbarkay et al . in line with these observations , we suggest that our newly identified subgroup of ssea4 and ykl40 positive cells constitute a fraction of astroglial progenitors , and suggest the name sypap ( ssea4 and ykl40 positive astroglial progenitors ) for this particular population . the subventricular zone at midgestation harbors many different cell types including interneurons , microglia and combinations of neurons , glial , and rgcs . whether these cells also play a role in origin or maintenance of glioblastoma multiforme , or if they contribute to the population of adult human nscs in the subependymal zone ( alvarezbuylla et al .

shinrin - yoku , has received widespread attention as a novel form of therapy . indeed , numerous studies examining the effects of forest environments , including bathing and walking in forest environments , on psychological states , such as emotions and mood1,2,3,4 , and on physiological factors , such as pulse rate and blood pressure ( bp)1,2,3,4 , and salivary amylase ( samy ) activity5 have shown positive outcomes . from the viewpoint of clinical implications , one of these positive effects , that is , reductions of bp , may be particularly relevant for hypertensive patients and/or elderly people who generally have higher bps , since hypertension is a major risk factor for cardiovascular diseases and is also associated with the urban environment6 . horiuchi et al.7 recently investigated the effects of forest walking on bp and other variables in young and elderly people , and reported a reduction of bp in both groups . furthermore , they also reported that the effects were greater in elderly people , suggesting that forest walking may be a useful strategy for improving quality of life in this population7 . however , a limitation of their study was that its protocol could not rule out the influence of exercise per se , that is , walking , meaning that there is a lack of discrimination between the effect of forest environments and the effect of exercise walking . thus , it remains unclear which , the forest environment or exercise walking , has the predominant influence in reducing bp . previous studies demonstrated that a greater magnitude and longer duration of postexercise hypotension ( peh ) was observed after a higher exercise intensity8,9,10 and/or longer exercise9 , suggesting that enhanced energy expenditure ( ee ) elicits a greater reduction in bp . in addition , a previous study demonstrated that a single bout of aerobic exercise caused 24 h bp reductions in middle - aged and aged people with type 2 diabetes and prehypertension , and this reduction may depend on a higher exercise intensity11 . forest walking is carried out at a slower and self - regulated pace , it is possible that ee may be related to changes in the bp of individuals . unfortunately , it remains unclear whether greater ee can produce a greater reduction in bp during forest walking . therefore , it is necessary to clarify a better way to reduce bp after forest walking , because forest bathing/ forest therapy has been reported as a novel form of therapy1,2,3,4 . in this study , we hypothesized that greater ee would lead to a greater reduction in bp following forest walking in middle - aged and elderly people . to test this hypothesis , we used a uniaxial accelerometer to monitor ee , because the device is easy to use and its validity and reliability have been confirmed in a previous study12 . as secondary evaluations , we also measured samy and the profile of mood states ( poms ) , which have been used to assess the impact of forest walking in previous studies1,2,3,4 . fifty - four volunteers ( 19 males and 35 females ) with a mean age of 63.29.4 years ( mean sd ) were recruited from kofu city , and the surrounding community using flyers and the web - page of yamanashi prefecture . some participants were taking medications for hypertension , diabetes , hyperlipidemia , hyperuricemia , and osteoporosis . in addition , the menstrual cycle was surveyed for females on an individual basis . this study was performed on 4 different days , and each participant took part on 1 day . after receiving a detailed description and explanation of the study procedures and the possible risks and benefits of participation , each participant signed an informed consent form . all participants were nonsmokers , and they were asked to abstain from consuming caffeinated beverages for 12 h and to refrain from engaging in strenuous exercise or consuming alcohol for a minimum of 24 h before the experiment . all procedures used in the present study were approved by the ethics committee of the mt . fuji research institute and were performed in accordance with the guidelines of the declaration of helsinki . takeda - no - mori , which was newly registered as a forest therapy home base in japan in 2014 , at 9:00 am each day , and all studies ended at ~12:00 pm . this site is located in kofu city , yamanashi prefecture , which is approximately 100 km west of tokyo . first , the body weight , including clothes , baggage , and shoes , of the participant was measured to calculate energy expenditure . each individual s bp variables , samy , and poms scores were evaluated before and after forest walking . during the pre - forest - walking measurements , each participant sat on a chair in an upright position for 20 min and then completed the poms questionnaire , which took for about 10 min . their saliva was then collected , and bp was measured with the participant still in this position . thus saliva collection and bp measurement were performed after 30 min of resting in a sitting position . the framework of the forest walking was as follows : 1 ) 5 min slow walking , 2 ) 5 min stretching , 3 ) 2530 min self - paced comfortable walking , 4 ) 20 min forest viewing in a supine position , and 5 ) 3035 min self - paced comfortable walking . briefly , after a 10 min recovery period in a sitting position , each subject again completed the poms questionnaire in the same manner as before forest walking . their saliva was then collected , and bp was measured with the participant still in this position . thus both measurements were carried out after a 20 min recovery period in a sitting position . systolic and diastolic bp ( sbp and dbp ) values were measured by an oscillometric method using a digital bp monitor ( hem-7420 , omron , tokyo , japan ) using the upper left arm of each participant while they sat on a chair . bp was measured at least twice , with a 30-s interval between replicates , both before and after forest - walking . if the difference between the measurements of either sbp or dbp was > 5 mmhg , the measurements were repeated . the average bp values of pairs of measurements were taken as the bp values , excluding those that were > 5mmhg values . the samy concentration , which was previously established as a marker of stress13 , 14 , was measured using a handheld samy monitor ( cm-2.1 , nipro corporation , osaka , japan ) . since it has been reported that increased sympathetic nerve activity is a major stimulator of amylase secretion , increases in samy the abovementioned handheld monitor consists of a disposable test strip and an optical analyzer that contains an automatic saliva transcription device . a volume of about 30 l of saliva was collected by the collection paper , which was placed under the tongue of the participant . the time allocated to saliva collection was 30 s for each participant , and it took 30 s to transfer and analyze the saliva using the handheld monitor . the validity of this device for measurement of samy has been confirmed in a previous study14 . ee was measured using a commercial activity monitor based on a uniaxial accelerometry sensor ( lifecorder ex or lifecorder gs , suzuken co. , ltd . , nagoya , japan ) . due to a number of device limitations , we used two kinds of activity monitor ; however , according to the technical details provided by the manufacturer ( suzuken co. , ltd . ) the validity of these devices for assessment of ee using a unique algorithm has been confirmed in a previous study12 . briefly , when the sensor detects three acceleration pulses or more for 4 consecutive s , activity ee ( aee ) is calculated and recorded every 4 seconds using the individual s body weight . the aee data can then be downloaded using special software via a personal computer . the poms is a well - established , factor - based , analytically derived measure of psychological distress , and its reliability and validity have been well documented15 . it measures six mood states : tension and anxiety ( t - a ) , depression ( d ) , anger and hostility ( a - h ) , vigor ( v ) , fatigue ( f ) , and confusion ( c ) . the short japanese version of the poms , which covers 30 items16 , was used , and its raw score was used for our statistical analysis . responders were classified as those participants who showed a reduction in mean arterial pressure ( map ) of > 5% between before and after forest walking . map was used in the classification of responders ( rs ) and nonresponders ( nrs ) as it fully represents the perfusion pressure observed in organs . the percentage of responses ( % response ) in map was calculated using the following equation : ( post - forest - walking map pre - forest - walking map ) ( 100/pre - forest - walking map ) . table 1table 1.physical characteristics , medications and menstrual cycle statuses of the responder and nonresponder groupsrespondernonrespondergender ( males / females , n)11/168/19age ( years)63.91.661.62.1height ( cm)159.31.6159.21.4body weight ( kg)64.82.461.01.6body mass index ( kg m)25.40.724.10.5response of mean arterial pressure ( % ) 9.90.81.70.4medications ( n)hypertension65diabetes24hyperuricemia32hyperlipidemia43osteoporosis22menstrual cycle ( n)menstrual period10follicular phase 11ovulation period 01luteal phase01menopause1416hormone replace treatment00values are shown as the meanstandard error of the mean ( sem ) . * p<0.05 vs. nonresponder group shows the physical characteristics , current medications , and menstrual cycle details of the r and nr groups . p<0.05 vs. nonresponder group map was calculated as [ ( sbpdbp)/3+dbp ] . due to greater variability of the samy values between and within the participants , logarithmic transformation was carried out , and the data for samy were presented as a natural logarithm ( log e ) . the data are expressed as mean values the standard error of the mean ( sem ) . an unpaired t test was used to compare the physical characteristics between the two groups . a test was also conducted to compare gender variability , medication status , menstrual cycle , number of participants in each group on the 4 different days , and classification of bp . a two - way repeated analysis of variance ( anova ) 3.5 , hulinks , chicago , il , usa ) , and a bonferroni post hoc test was employed . a pearson correlation coefficient between percentage ( % ) changes in map and aee during forest walking was obtained , and a p - value of < 0.05 was considered statistically significant . physical characteristics were similar between groups , while , % change in map was significantly greater in rs than in nrs ( table 1 ) . the test revealed no significant differences in gender ( (1)=0.325 , p=0.569 ) , medication ( (4)=1.071 , p=0.899 ) , and menstrual cycle status ( (4)=2.897 , p=0.575 ) . classification of bp according to the guidelines for the management of hypertension in japan17 , is shown for the two groups in table 2table 2.classification of blood pressure ( bp ) values in the two groups , according to the japanese society of hypertensionresponder ( n)nonresponder ( n)optimum bpsbp<120 and dbp < 8058normal bpsbp<130 and dbp < 8562prehypertensionsbp 130139 or dbp 858916hypertension isbp 140159 or dbp 9099107hypertension iisbp 160179 or dbp 909923hypertension iiisbp180 or dbp11031sbp : systolic bp ; dbp : diastolic bp . approximately 60% of the participants in each group were classifies as prehypertensive and hypertensive . the test showed no significant difference in bp classifications between the two groups ( (5)=7.993 , p = 0.157 ) . sbp : systolic bp ; dbp : diastolic bp a comparison of physiological variables between the two groups for before and after forest walking , established pre and post forest - walking , is shown in table 3table 3.comparison of the bp variables and salivary amylase before ( pre ) and after ( post ) forest walking between the two groupsrespondernonrespondertwo - way anova f and p values ( df=1 , 52)pre post pre postgrouptimeinteractionfpfpfpsbp ( mmhg)14141254 * 13441274 * 0.1690.686117.098<0.00119.339<0.001dbp ( mmhg)832762 * 7727721.0520.31032.600<0.00132.928<0.001map ( mmhg)1033923 * 962942 * 0.5820.449129.792<0.00153.939<0.001log e ( samy)3.420.223.150.213.040.223.270.230.2490.6200.0220.8842.7720.102values are shown as the mean sem . no significant differences were observed in pre - forest - walking values of sbp and map between groups ( p>0.05 , respectively ) , and after forest walking , both bp variables were significantly decreased ( p < 0.05 respectively ) . in contrast , pre - forest - walking dbp values were significantly higher in the r group compared with the nr group ( p = 0.027 ) ; moreover , they significantly decreased only in rs ( p < 0.001 ) , while they remained unchanged in nrs ( p = 0.984 ) . two - way repeated anova failed to find significant differences in samy changes with logarithmic transformation between the two groups ( p > 0.05 ) . the aee in the r group during forest walking was significantly greater compared to with that of the nr group ( 1426 vs. 1175 kcal , p = 0.001 ) . p<0.05 vs. nonresponder group within pre - forest - walking values table 4table 4.comparison of the subscales of the profile of mood states before ( pre ) and after ( post ) forest walking between the two groupsrespondernonrespondertwo - way anova f and p values ( df=1 , 52)pre post pre postgrouptimeinteractionfpfpfptension - anxiety3.50.60.70.23.40.50.60.30.0140.90860.684<0.0010.0030.960depression1.90.50.40.21.30.40.50.20.6570.42119.265<0.0011.8810.176anger - hostility1.40.40.10.10.70.20.20.11.9580.16812.418<0.0013.1040.084vigor8.30.810.21.09.70.812.60.93.0470.08719.049<0.0010.8570.359fatigue2.70.71.20.42.20.70.60.20.8980.3489.4470.0030.0050.942confusion4.90.53.00.34.90.62.80.30.0770.78230.282<0.0010.1300.720values are shown as the mean sem . p<0.05 vs. nonresponder group within pre - forest - walking values shows changes in the subscales of the poms between the two groups . there were no significant differences between groups in the pre - forest - walking values of the subscales of the poms ( p>0.05 , respectively ) , with the exception of a - h ( p<0.05 ) . forest walking significantly improved the subscales of the poms in both groups ( p<0.05 , respectively ) , with no statistical differences between the two groups ( p>0.05 , respectively ) . only the a - h subscale of the poms in the nr group did not improve after forest walking ( p>0.05 ) . p<0.05 vs. nonresponder group within pre - forest - walking values aee was correlated with map changes in rs ( f [ 1 , 25]=9.858 , r=0.532 , p=0.004 , n=27 ) , but this was not observed in nrs ( f [ 1 , 25]=1.311 , r=0.223 , p=0.263 , n=27 ) . the major findings of the present study were that forest walking may have a health benefit that reduces map and improves the subscales of the poms in middle - aged and elderly people . in addition , enhanced aee during forest walking was associated with changes in map , but enhanced aee did not affect the poms . collectively , these results indicated that forest walking in itself may improve health ; however , greater - walking - related aee might be required to accentuate physiological benefits . previous studies have demonstrated that the decreases in sbp and/or dbp following forest walking are significantly greater than the sbp and/or dbp decreases observed after walking in urban areas2 , 4 . forest walking - induced reductions in bp can be explained by changes in sympathetic nerve activity . a previous study showed that sympathetic nerve activity , which was assessed through direct measurements of sympathetic nerve activity using microneurography , i.e. , muscle sympathetic nerve activity , may help regulate bp18 . in addition , indirect assessments of sympathetic nerve activity through heart rate variability ( hrv ) were associated with bp control in prehypertensive humans19 . although the precise mechanisms and interactions between changes in bp and hrv are still unclear in this study , there is a possibility that forest environments may increase parasympathetic nerve activity , resulting in bp decreases . horiuchi et al.7 recently found that both sbp and dbp were reduced in elderly people , but not in younger people , after forest walking . however , their study could not discriminate between the effects of walking and forest environments per se7 . therefore , we should be cautious in interpreting results regarding the impact of forest walking on bp changes . it has been suggested that a higher intensity and longer duration provoke a greater magnitude , and longer duration of , bp reductions after a single bout of exercise8,9,10 . in addition , one interesting study revealed that peh was similar following a short period of high- intensity exercise and a longer period of moderate - intensity exercise that were matched for total work rate , suggesting that total energy expenditure may affect peh20 . these previous studies may partially support our results showing that enhanced aee led to a greater map reduction in forest - walking rs . another potential explanation may be related to different initial values in bp between groups . in this study , map was calculated as [ ( sbpdbp)/3+dbp ] , indicating that dbp values may be related to map values . indeed , the initial dbp values before forest walking in the r group were significantly higher than those in the nr group . moreover , the initial map values in the r group were also higher in the nrs with no statistical differences between groups . since it has been demonstrated that people with higher resting bp values may show greater hypotension after exercise21 , even slight differences in map values between groups may have caused greater reductions in the r group . however , the lack of correlation between aee and map in nrs still can not be fully explained . although we can only speculate , the aee in nrs may not be sufficient to reduce map ; it is also possible that less variance in map resulted in no visible relationship . it has been reported that samy levels in individuals exposed to a forest environment were lower than those in individuals in urban environments when both sets of individuals walked in and observed the surrounding landscape5 . similarly , a recent field study also revealed that , in a very natural environment , samy was significantly lower than in a built - up setting22 . our results failed to reveal a marked improvement of these stress markers in both groups . however , it should be noted that these previous studies compared samy between urban and forest environments , and found a significant difference between the two conditions . therefore , they did not investigate whether the forest environment per se reduced salivary stress markers . in addition , beil and hanes22 found that samy increased slightly , even in a very natural environment , despite a significant decrease in the feeling of stress . these findings indicate that our results , which showed no difference between before and after forest - walking , may not be unnatural . previous field studies have found that the subscales of the poms are improved through forest viewing and walking1,2,3 , 7 . these results appear to be consistent with our findings , in that the forest environment , including walking in the forest environments , improved the participants poms subscale scores , and it is unlikely that differential ee and/or map changes are related to individuals poms scores . however , one different result was the significant improvement observed in the a - h subscale of the poms in the r group , with it remaining unchanged in the nr group . as the pre- forest - walking values of a - h were significantly lower in the nr group , which showed no a - h feelings , it might be reasonable to expect no further improvement in this subscale . another possibility is the relationship between these negative feelings and risk of cardiovascular disease . previous studies have found that feelings of great anger or hostility are closely related to hypertension23 . moreover , psychosocial stress has been implicated in the pathogenesis of essential hypertension24 , and elevated stress has also been associated with an increased risk of atherosclerosis25 and acute cardiovascular events26 . the mechanisms underlying these links are complex , because it should be considered that effect of exercise per se on improvements of the poms in both groups . however , it was reported that a health promotion program consisting of easy and simple exercise improved psychological attitudes including a subscale of the poms , indicating that consecutive exercise per se may be useful for improvement of psychological stress27 . thus , although future studies are warranted , our results may indicate that forest walking can improve the poms , irrespective of map changes , suggesting that forest walking may be a potential strategy to reduce psychological stress in middle - aged and elderly people . first , we did not perform a control study , e.g. , a study of city walking . indeed , previous studies have examined effects of forest walking and bathing on bp compared with city walking , and their results showed a greater reduction in bp with forest walking than with city walking2,3,4 . however , it is very difficult to control energy expenditure under different conditions , i.e. , the forest and city , because numerous factors , such as walking speed , gradients , and environmental conditions , can change the activity energy expenditure . accordingly , we did not want to compel subjects to control their walking speed , etc , because it is likely that walking at a comfortable pace is a more important issue in evaluating effects of forest walking . more importantly , our main aim was to examine effects of energy expenditure on bp during forest walking ; furthermore , our study attempted to suggest a way to walk , even in forest environments for human health . in this regard , our results clearly demonstrated that greater aee may have more positive effects , but mental health assessed by the poms was not affected by differences in aee . although future studies are required to elucidate the underlying mechanisms of forest walking for human heath , the current results can be applied to people who would like to try forest walking and can be used to propose an appropriate walking method under forest conditions . second , our participants were both males and females , some participants were taking medications , and the menstrual cycle in middle - aged women was different between groups . it could not be completely ruled out that these variables has possible effects ; however , our statistical analysis clearly demonstrated no differences between the two groups according to gender , medication , and menstrual cycle status . also only one study has reported that beta - blocker treatment influenced resting bp levels ; however , the exercise - induced reduction in bp was unaffected compared with a placebo28 . in addition , a previous study found that gender difference and menstrual cycle29 have little effect on peh . moreover , the numbers of middle - aged women who had a normal menstrual cycle were relatively small in each group in the present study ( n=2 in the r group and n=3 in the nr group ) . thus , it is unlikely that our primary conclusions were affected by these influences . in summary , greater ee during forest walking was associated with a greater reduction in map , but greater ee did not strongly affect changes in the poms . these results suggest that greater - walking - related ee might be required to accentuate physiological beneficial effects on health in middle - aged and aged people , in particular , those who are prehypertensive and hypertensive . furthermore , the forest environment per se can attenuate psychological stress irrespective of aee during walking . with regard to the strategy of forest walking , our results may , therefore , provide useful information for prehypertensive and hypertensive people who can perform a kind of brisk walking and/or people with mental stress who can not .

[ purpose ] forest walking may be effective for human health , but little information is available about effects of energy expenditure on blood pressure responses after forest walking . the aim of this study was to investigate the relationship between the activity energy expenditure and changes in blood pressure in individuals after forest walking . [ subjects ] the subjects were 54 middle - aged and elderly people . [ methods ] all subjects walked in the forest for approximately 90 min . blood pressure , salivary amylase , and the profile of mood states were evaluated before and after forest walking , and activity energy expenditure was monitored throughout forest walking . subjects were divided into two groups according to mean arterial pressure changes : a responder group ( > 5% decreases ) and a nonresponder group ( < 5% ) . [ results ] forest walking significantly reduced the mean arterial pressure and improved the profile of mood states in both groups . activity energy expenditure was related to changes in mean arterial pressure in the responder group , while this relation was not observed in the nonresponder group . differential activity energy expenditure did not strongly affect improvement of the profile of mood states . [ conclusion ] greater walking - related greater activity energy expenditure might be required to accentuate physiological beneficial effects on in middle - aged and aged people . furthermore , the forest environment per se can attenuate psychological stress .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

indeed , numerous studies examining the effects of forest environments , including bathing and walking in forest environments , on psychological states , such as emotions and mood1,2,3,4 , and on physiological factors , such as pulse rate and blood pressure ( bp)1,2,3,4 , and salivary amylase ( samy ) activity5 have shown positive outcomes . from the viewpoint of clinical implications , one of these positive effects , that is , reductions of bp , may be particularly relevant for hypertensive patients and/or elderly people who generally have higher bps , since hypertension is a major risk factor for cardiovascular diseases and is also associated with the urban environment6 . horiuchi et al.7 recently investigated the effects of forest walking on bp and other variables in young and elderly people , and reported a reduction of bp in both groups . furthermore , they also reported that the effects were greater in elderly people , suggesting that forest walking may be a useful strategy for improving quality of life in this population7 . however , a limitation of their study was that its protocol could not rule out the influence of exercise per se , that is , walking , meaning that there is a lack of discrimination between the effect of forest environments and the effect of exercise walking . thus , it remains unclear which , the forest environment or exercise walking , has the predominant influence in reducing bp . in addition , a previous study demonstrated that a single bout of aerobic exercise caused 24 h bp reductions in middle - aged and aged people with type 2 diabetes and prehypertension , and this reduction may depend on a higher exercise intensity11 . forest walking is carried out at a slower and self - regulated pace , it is possible that ee may be related to changes in the bp of individuals . therefore , it is necessary to clarify a better way to reduce bp after forest walking , because forest bathing/ forest therapy has been reported as a novel form of therapy1,2,3,4 . in this study , we hypothesized that greater ee would lead to a greater reduction in bp following forest walking in middle - aged and elderly people . as secondary evaluations , we also measured samy and the profile of mood states ( poms ) , which have been used to assess the impact of forest walking in previous studies1,2,3,4 . fifty - four volunteers ( 19 males and 35 females ) with a mean age of 63.29.4 years ( mean sd ) were recruited from kofu city , and the surrounding community using flyers and the web - page of yamanashi prefecture . this study was performed on 4 different days , and each participant took part on 1 day . after receiving a detailed description and explanation of the study procedures and the possible risks and benefits of participation , each participant signed an informed consent form . all procedures used in the present study were approved by the ethics committee of the mt . first , the body weight , including clothes , baggage , and shoes , of the participant was measured to calculate energy expenditure . each individual s bp variables , samy , and poms scores were evaluated before and after forest walking . the framework of the forest walking was as follows : 1 ) 5 min slow walking , 2 ) 5 min stretching , 3 ) 2530 min self - paced comfortable walking , 4 ) 20 min forest viewing in a supine position , and 5 ) 3035 min self - paced comfortable walking . briefly , after a 10 min recovery period in a sitting position , each subject again completed the poms questionnaire in the same manner as before forest walking . bp was measured at least twice , with a 30-s interval between replicates , both before and after forest - walking . if the difference between the measurements of either sbp or dbp was > 5 mmhg , the measurements were repeated . it measures six mood states : tension and anxiety ( t - a ) , depression ( d ) , anger and hostility ( a - h ) , vigor ( v ) , fatigue ( f ) , and confusion ( c ) . the short japanese version of the poms , which covers 30 items16 , was used , and its raw score was used for our statistical analysis . responders were classified as those participants who showed a reduction in mean arterial pressure ( map ) of > 5% between before and after forest walking . map was used in the classification of responders ( rs ) and nonresponders ( nrs ) as it fully represents the perfusion pressure observed in organs . table 1table 1.physical characteristics , medications and menstrual cycle statuses of the responder and nonresponder groupsrespondernonrespondergender ( males / females , n)11/168/19age ( years)63.91.661.62.1height ( cm)159.31.6159.21.4body weight ( kg)64.82.461.01.6body mass index ( kg m)25.40.724.10.5response of mean arterial pressure ( % ) 9.90.81.70.4medications ( n)hypertension65diabetes24hyperuricemia32hyperlipidemia43osteoporosis22menstrual cycle ( n)menstrual period10follicular phase 11ovulation period 01luteal phase01menopause1416hormone replace treatment00values are shown as the meanstandard error of the mean ( sem ) . * p<0.05 vs. nonresponder group shows the physical characteristics , current medications , and menstrual cycle details of the r and nr groups . due to greater variability of the samy values between and within the participants , logarithmic transformation was carried out , and the data for samy were presented as a natural logarithm ( log e ) . the data are expressed as mean values the standard error of the mean ( sem ) . an unpaired t test was used to compare the physical characteristics between the two groups . a two - way repeated analysis of variance ( anova ) 3.5 , hulinks , chicago , il , usa ) , and a bonferroni post hoc test was employed . a pearson correlation coefficient between percentage ( % ) changes in map and aee during forest walking was obtained , and a p - value of < 0.05 was considered statistically significant . classification of bp according to the guidelines for the management of hypertension in japan17 , is shown for the two groups in table 2table 2.classification of blood pressure ( bp ) values in the two groups , according to the japanese society of hypertensionresponder ( n)nonresponder ( n)optimum bpsbp<120 and dbp < 8058normal bpsbp<130 and dbp < 8562prehypertensionsbp 130139 or dbp 858916hypertension isbp 140159 or dbp 9099107hypertension iisbp 160179 or dbp 909923hypertension iiisbp180 or dbp11031sbp : systolic bp ; dbp : diastolic bp . the test showed no significant difference in bp classifications between the two groups ( (5)=7.993 , p = 0.157 ) . sbp : systolic bp ; dbp : diastolic bp a comparison of physiological variables between the two groups for before and after forest walking , established pre and post forest - walking , is shown in table 3table 3.comparison of the bp variables and salivary amylase before ( pre ) and after ( post ) forest walking between the two groupsrespondernonrespondertwo - way anova f and p values ( df=1 , 52)pre post pre postgrouptimeinteractionfpfpfpsbp ( mmhg)14141254 * 13441274 * 0.1690.686117.098<0.00119.339<0.001dbp ( mmhg)832762 * 7727721.0520.31032.600<0.00132.928<0.001map ( mmhg)1033923 * 962942 * 0.5820.449129.792<0.00153.939<0.001log e ( samy)3.420.223.150.213.040.223.270.230.2490.6200.0220.8842.7720.102values are shown as the mean sem . no significant differences were observed in pre - forest - walking values of sbp and map between groups ( p>0.05 , respectively ) , and after forest walking , both bp variables were significantly decreased ( p < 0.05 respectively ) . in contrast , pre - forest - walking dbp values were significantly higher in the r group compared with the nr group ( p = 0.027 ) ; moreover , they significantly decreased only in rs ( p < 0.001 ) , while they remained unchanged in nrs ( p = 0.984 ) . two - way repeated anova failed to find significant differences in samy changes with logarithmic transformation between the two groups ( p > 0.05 ) . the aee in the r group during forest walking was significantly greater compared to with that of the nr group ( 1426 vs. 1175 kcal , p = 0.001 ) . p<0.05 vs. nonresponder group within pre - forest - walking values table 4table 4.comparison of the subscales of the profile of mood states before ( pre ) and after ( post ) forest walking between the two groupsrespondernonrespondertwo - way anova f and p values ( df=1 , 52)pre post pre postgrouptimeinteractionfpfpfptension - anxiety3.50.60.70.23.40.50.60.30.0140.90860.684<0.0010.0030.960depression1.90.50.40.21.30.40.50.20.6570.42119.265<0.0011.8810.176anger - hostility1.40.40.10.10.70.20.20.11.9580.16812.418<0.0013.1040.084vigor8.30.810.21.09.70.812.60.93.0470.08719.049<0.0010.8570.359fatigue2.70.71.20.42.20.70.60.20.8980.3489.4470.0030.0050.942confusion4.90.53.00.34.90.62.80.30.0770.78230.282<0.0010.1300.720values are shown as the mean sem . p<0.05 vs. nonresponder group within pre - forest - walking values shows changes in the subscales of the poms between the two groups . there were no significant differences between groups in the pre - forest - walking values of the subscales of the poms ( p>0.05 , respectively ) , with the exception of a - h ( p<0.05 ) . forest walking significantly improved the subscales of the poms in both groups ( p<0.05 , respectively ) , with no statistical differences between the two groups ( p>0.05 , respectively ) . only the a - h subscale of the poms in the nr group did not improve after forest walking ( p>0.05 ) . p<0.05 vs. nonresponder group within pre - forest - walking values aee was correlated with map changes in rs ( f [ 1 , 25]=9.858 , r=0.532 , p=0.004 , n=27 ) , but this was not observed in nrs ( f [ 1 , 25]=1.311 , r=0.223 , p=0.263 , n=27 ) . the major findings of the present study were that forest walking may have a health benefit that reduces map and improves the subscales of the poms in middle - aged and elderly people . in addition , enhanced aee during forest walking was associated with changes in map , but enhanced aee did not affect the poms . collectively , these results indicated that forest walking in itself may improve health ; however , greater - walking - related aee might be required to accentuate physiological benefits . forest walking - induced reductions in bp can be explained by changes in sympathetic nerve activity . although the precise mechanisms and interactions between changes in bp and hrv are still unclear in this study , there is a possibility that forest environments may increase parasympathetic nerve activity , resulting in bp decreases . horiuchi et al.7 recently found that both sbp and dbp were reduced in elderly people , but not in younger people , after forest walking . however , their study could not discriminate between the effects of walking and forest environments per se7 . in addition , one interesting study revealed that peh was similar following a short period of high- intensity exercise and a longer period of moderate - intensity exercise that were matched for total work rate , suggesting that total energy expenditure may affect peh20 . another potential explanation may be related to different initial values in bp between groups . in this study , map was calculated as [ ( sbpdbp)/3+dbp ] , indicating that dbp values may be related to map values . indeed , the initial dbp values before forest walking in the r group were significantly higher than those in the nr group . moreover , the initial map values in the r group were also higher in the nrs with no statistical differences between groups . it has been reported that samy levels in individuals exposed to a forest environment were lower than those in individuals in urban environments when both sets of individuals walked in and observed the surrounding landscape5 . our results failed to reveal a marked improvement of these stress markers in both groups . therefore , they did not investigate whether the forest environment per se reduced salivary stress markers . these findings indicate that our results , which showed no difference between before and after forest - walking , may not be unnatural . these results appear to be consistent with our findings , in that the forest environment , including walking in the forest environments , improved the participants poms subscale scores , and it is unlikely that differential ee and/or map changes are related to individuals poms scores . however , one different result was the significant improvement observed in the a - h subscale of the poms in the r group , with it remaining unchanged in the nr group . as the pre- forest - walking values of a - h were significantly lower in the nr group , which showed no a - h feelings , it might be reasonable to expect no further improvement in this subscale . another possibility is the relationship between these negative feelings and risk of cardiovascular disease . moreover , psychosocial stress has been implicated in the pathogenesis of essential hypertension24 , and elevated stress has also been associated with an increased risk of atherosclerosis25 and acute cardiovascular events26 . the mechanisms underlying these links are complex , because it should be considered that effect of exercise per se on improvements of the poms in both groups . however , it was reported that a health promotion program consisting of easy and simple exercise improved psychological attitudes including a subscale of the poms , indicating that consecutive exercise per se may be useful for improvement of psychological stress27 . thus , although future studies are warranted , our results may indicate that forest walking can improve the poms , irrespective of map changes , suggesting that forest walking may be a potential strategy to reduce psychological stress in middle - aged and elderly people . indeed , previous studies have examined effects of forest walking and bathing on bp compared with city walking , and their results showed a greater reduction in bp with forest walking than with city walking2,3,4 . , the forest and city , because numerous factors , such as walking speed , gradients , and environmental conditions , can change the activity energy expenditure . accordingly , we did not want to compel subjects to control their walking speed , etc , because it is likely that walking at a comfortable pace is a more important issue in evaluating effects of forest walking . more importantly , our main aim was to examine effects of energy expenditure on bp during forest walking ; furthermore , our study attempted to suggest a way to walk , even in forest environments for human health . in this regard , our results clearly demonstrated that greater aee may have more positive effects , but mental health assessed by the poms was not affected by differences in aee . although future studies are required to elucidate the underlying mechanisms of forest walking for human heath , the current results can be applied to people who would like to try forest walking and can be used to propose an appropriate walking method under forest conditions . second , our participants were both males and females , some participants were taking medications , and the menstrual cycle in middle - aged women was different between groups . it could not be completely ruled out that these variables has possible effects ; however , our statistical analysis clearly demonstrated no differences between the two groups according to gender , medication , and menstrual cycle status . moreover , the numbers of middle - aged women who had a normal menstrual cycle were relatively small in each group in the present study ( n=2 in the r group and n=3 in the nr group ) . in summary , greater ee during forest walking was associated with a greater reduction in map , but greater ee did not strongly affect changes in the poms . these results suggest that greater - walking - related ee might be required to accentuate physiological beneficial effects on health in middle - aged and aged people , in particular , those who are prehypertensive and hypertensive . furthermore , the forest environment per se can attenuate psychological stress irrespective of aee during walking . with regard to the strategy of forest walking , our results may , therefore , provide useful information for prehypertensive and hypertensive people who can perform a kind of brisk walking and/or people with mental stress who can not .

according to a nationwide survey conducted in 20022004 by the pakistan national eye survey study group , the results of which are reported in jadoon et al . , the prevalence of blindness in pakistan among adults aged 30 years and above is 2.7% . using the 2011 population estimate of the corresponding age groups fifty - five percent of the total blind population in pakistan is estimated to be cataract blind [ tables 13 ] . the prevalence of adult blindness ( 30 years and above ) in pakistan by cause age specific blind population by all causes in pakistan age specific blind population by cause the prevalence of blindness is observed to be higher among older age groups compared with the younger age cohorts . with respect to sex , the prevalence of blindness is lower among females than males in the younger age group of 3039 years . in older age groups of 4059 years overall there are around 624,000 people blind in pakistan in the productive age group of 3059 years . in order to estimate the potential economic gains of rehabilitating the blind in pakistan , we use the average earnings to calculate the productivity gains that can be realized if the blind population in the productive age group ( 3059 years ) is rehabilitated and facilitated to become productive members of society . these productivity gains are analyzed for a short run scenario comprising of a year to a long run horizon spanning over 10 years through appropriate growth and discounting techniques . long - term benefits are computed because health care and rehabilitation programs have an immediate sizeable effect on resources , but their effect on health and other indicators of social wellbeing extend well beyond a year to a lifetime benefit . the estimation of productivity gains was done in several steps . in the first step , data on average monthly wage earnings were obtained for 2003 ( which corresponds to the year of blindness data ) . a disaggregated data of wage rates by sex were used in order to take into account the substantial difference between the average wages of the two sexes . these monthly wage earnings were converted into per annum average wage earnings of an individual . the per annum individual wage earnings were then extrapolated for the next 10 years by applying the official growth rate in nominal wages for the corresponding year and by deflating the nominal wage earnings by the corresponding inflation rate . for this purpose , gdp deflator , which is commonly used to deflate output , was used as a measure of inflation . since we do not have the inflation figures for 20112013 , we used the moving average of inflation in the past 5 years preceding 2011 . in the second step , the extrapolated wage earnings over the 10-year period were aggregated and discounted to estimate the net present value of the future stream of income over the 10-year period . the standard discounting formula was applied to compute the present value of future earnings : where pv denotes present value of future income . yi represents per annum individual wage earning in year i. the discount rate r=0.04 and n=10 is used . the discount rate is based upon the weighted average rate of return on long - term deposits ( 5 years and over ) in 2004 as published by the state bank of pakistan . in the third step , the present value of individual wage earnings over the 10 year period was multiplied by the total number of blind adults in the productive age group of 3059 years to compute the aggregate level of income achieved by rehabilitating the blind population and enabling them to participate in the labor market . as a consequence of this , the rehabilitation of blind would also release the carers productive time , which they can use for wage earnings . in order to estimate that , we first estimated the average hours that carers spend on the care of the disabled . this was done by making use of the recently available data on time use survey conducted by the finance division of pakistan . the survey contains detailed data on unpaid care work that includes housework by women as well as time spent on caring for children , old people , the sick , and disabled , as well as time spent in assisting the community . the survey covered a randomly selected sample of 19,600 household from all four provinces of the country . according to the survey , the mean length of time spent by the carers on physical care and supervision of sick or disabled adults is 119 minutes or 2 hours . this is equal to one - fourth of the total productive time per day ( assuming that the total productive time is 8 hours per day ) . this includes the following activities : physical care of sick or disabled adult ( activity code 541 ) ; accompany sick and disabled ( activity code 551 ) ; supervising sick and disabled adult ( activity code 562 ) ; and travel related to care of sick and disabled adult ( activity code 582 ) . the average number of hours spent on care for disabled is multiplied by the hourly wage rate , which was then converted into monthly and yearly income . the hourly wage rate is derived from average monthly earnings of both sexes and the key assumption made in the computation is that the average hours worked per week are 48 hours as reported in the labor force survey of pakistan . the hourly wage computed in this way turns out to be 21 ( about us $ 0.25 ) . again , these per annum individual wage earnings were extrapolated for the next 10 years by applying the official growth rate in nominal wages for the corresponding year and the discount rate . the total individual wage earnings thus computed over the 10-year period were multiplied by the total number of carers , which is assumed to be equal to the total number of blind in both the productive as well as nonproductive age group . since we do not have data on the division of carers by sex , we applied the wage rate of both sexes as reported in the labor force survey and in table 4 . in the final step , the total potential earnings of the blind as well as the carers were added and expressed as a percentage of the current gdp of pakistan . estimates of average earnings and growth rates in 20032004 used in the study ( pak ) by taking average labor earnings as a proxy for average productivity , the present paper uses a much more rigorous and accurate methodology compared with earlier studies . that take per capita gnp as a proxy for average earnings and hence productivity , in estimating the burden of blindness . it is important to note the gnp per capita includes income from many sources including labor and nonlabor sources . income from nonlabor sources may include rental income or returns on financial investments . while income from nonlabor sources may not be directly affected by blindness , it is the wage income that is mostly foregone by the blind population . in short , average the following key assumptions were made to estimate the gains in productivity of rehabilitating the blind in pakistan : the productivity gains were estimated for individuals in the economically productive age group for which we have data on the prevalence of blindness : 3059 years of age . although the productive age starts from 18 years , however , we do not have data on the prevalence of blindness for age groups below 30 years . the inclusion of younger age groups in the computation is likely to increase further the estimates of economic gains . blindness were considered for this paper , there is likely to be an underestimation as those with moderate or severe visual impairment were not included in the calculations.the foregone earnings of the blind population were computed assuming full utilization of their productive time in case of rehabilitation . this may not appear to be a realistic assumption as all blind persons may not be able to fully utilize their productive time . the possible upward bias in our estimates from this assumption is , however , likely to cancel out with the downward bias of our estimates due to noninclusion of less than 30 years of population in our productive age group.the estimates of average monthly earnings for males , females , and both sexes are taken from the labor force survey ( 20032004 ) of pakistan . the growth rate of wages is also taken from the same survey.the foregone earnings of the carers were computed based upon the evidence from time use survey ( conducted by the ministry of finance , pakistan ) according to which they devote 2 hours of their productive time per day for the care of disabled persons.since we are not aware of the precise division of carers by sex , we used the average hourly wage rate of both sexes as reported in the labor force survey of pakistan ( 20032004).the total number of carers is assumed to be equal to the total number of blind population that includes the blind people in both the productive and unproductive age group.the discount rate used for computing the present value of future stream of income is 4% , which is based upon the weighted average rate of return on long - term deposits ( 5 years and over ) in 2004 as published by the state bank of pakistan . the productivity gains were estimated for individuals in the economically productive age group for which we have data on the prevalence of blindness : 3059 years of age . although the productive age starts from 18 years , however , we do not have data on the prevalence of blindness for age groups below 30 years . the inclusion of younger age groups in the computation is likely to increase further the estimates of economic gains . blindness were considered for this paper , there is likely to be an underestimation as those with moderate or severe visual impairment were not included in the calculations . the foregone earnings of the blind population were computed assuming full utilization of their productive time in case of rehabilitation . this may not appear to be a realistic assumption as all blind persons may not be able to fully utilize their productive time . the possible upward bias in our estimates from this assumption is , however , likely to cancel out with the downward bias of our estimates due to noninclusion of less than 30 years of population in our productive age group . the estimates of average monthly earnings for males , females , and both sexes are taken from the labor force survey ( 20032004 ) of pakistan . the foregone earnings of the carers were computed based upon the evidence from time use survey ( conducted by the ministry of finance , pakistan ) according to which they devote 2 hours of their productive time per day for the care of disabled persons . since we are not aware of the precise division of carers by sex , we used the average hourly wage rate of both sexes as reported in the labor force survey of pakistan ( 20032004 ) . the total number of carers is assumed to be equal to the total number of blind population that includes the blind people in both the productive and unproductive age group . the discount rate used for computing the present value of future stream of income is 4% , which is based upon the weighted average rate of return on long - term deposits ( 5 years and over ) in 2004 as published by the state bank of pakistan . data on the prevalence of blindness by age group and by sex was obtained from the pakistan national eye survey study group that conducted a nationally representative survey of blindness in pakistan , the results of which are reported in jadoon et al . the latest demographic data on pakistan , disaggregated by age and sex , was obtained from the international database of us census of bureau . finally data on current gdp of pakistan and inflation rate was obtained from the economic survey of pakistan . table 5 illustrates our computation results regarding the potential productivity gains of rehabilitating the blind in pakistan , disaggregated by sex and by causes . economic benefits of rehabilitating the blind in pakistan ( all causes ) the results show that if the entire blind population in pakistan ( by all causes ) is rehabilitated , the total economic benefit / productivity gain of 422 billion ( us$ 4.9 billion ) is realizable over a period of 10 years . this translates into 42.2 billion ( us$ 490 million ) per annum . if only the avoidable blindness is treated , the aggregate economic benefit of 361 billion ( us$ 4.25 billion ) over a period of 10 years and an average of 36 billion ( us$ 425 million ) per annum is realizable . the rehabilitation of incurably blind results in the aggregate benefits of 61 billion ( us$ 718 million ) over a period of 10 years and an average of 6.1 billion ( us$ 71.8 million ) per year . similarly , the treatment of cataract blindness results in 217 billion ( us$ 2.55 billion ) over 10 years and an average of 21.7 billion ( us$ 255 million ) per annum . in table 6 , these economic benefits are expressed in terms of the total current gdp of pakistan . table 6 shows that if the entire blind population in pakistan is rehabilitated , economic benefits per annum amount to 0.7% of gdp . this is a significant economic gain that is roughly equal to the total government spending on health in pakistan . if only those people are treated who are blind due to avoidable causes , the economic benefit achieved per annum is equal to 0.6% of gdp . economic benefits of rehabilitating the blind in pakistan the results in table 6 show that even if the rehabilitation of the blind in pakistan costs 0.7% of the gdp per annum , it is still justified on economic grounds . in future , it would be useful to estimate the total cost of rehabilitating the blind in pakistan which can then be compared with the total economic benefits computed in terms of productivity gain above so as to estimate the net economic gain of rehabilitating the blind in pakistan . the world health organization states that there are 285 million people visually impaired and 39 million blind globally . these conditions by themselves may not be life threatening but they have a significant impact on quality of life , and are associated with poverty and social exclusion . of those who are visually impaired , 153 million are in need of a simple pair of spectacles to correct their refractive error . twelve million school children aged between 5 and 15 years are visually impaired for want of a pair of spectacles . at least half of all the people who are blind have cataracts which can be treated easily by surgery . trachoma , a blinding disease spread by flies affects 84 million people in 56 countries and 8 million are blind or visually impaired . river blindness affects 37 million people of whom 750,000 are severely visually impaired or blind . children who are born blind or who become blind and survive have a lifetime of blindness ( 70 million blind years ) ahead of them , with all the associated emotional , social and economic costs to the child , the family and society . it affects their access to education and severely limits their livelihood options in developing countries . indeed , many of the conditions associated with blindness in children are also causes of child mortality , for example , premature birth , measles , vitamin a deficiency , meningitis , and congenital rubella syndrome . women have been found to have a 30% higher risk of blindness than men . in this paper , we have tried to demonstrate , through a country case - study , the opportunity cost of not addressing the needs of the blind in poverty reduction strategies . we have used the average earnings approach to calculate the productivity gains that can be obtained if the blind population in the productive age group ( 3059 years ) is rehabilitated and facilitated to become productive members of society . this approach is based upon the assumption that each person represents a productive resource to society and illness or disability diminishes that person 's productive capacity , which in turn is valued by his or her loss of earning . it is important to note that labor earnings is a much more accurate proxy for average productivity compared with gnp per capita used by earlier studies . in this sense , the present study evaluates the economic benefits in a much more rigorous and accurate manner . the results of our analysis indicate that the rehabilitation of the blind can potentially result in significant productivity gains in the form of increase in earnings by both the blind and their carers . our estimates suggest that the aggregate economic benefits spread over a 10-year period represent 0.7% of current gdp per annum , which is roughly equal to the total public spending on health . our results clearly show that the rehabilitation of the blind in pakistan is not only justified on ethical and humanitarian grounds but also on economic grounds . however , in order to reap these benefits , an effective rehabilitation is not adequate ; subsequent absorption into mainstream economic activity must also be facilitated . although our results are sensitive to the assumptions we made , it turns out that the negative and positive biases that are most likely to be created by two of our most critical assumptions tend to outweigh each other and there is no significant deviation from the results otherwise obtained . more specifically , let us examine the impact of two of our most critical assumptions : the first assumption is the consequence of our data limitation : the estimates of blind population in pakistan are available only for ages 30 years and above , however , the productive age group starts from 18 years and above.we assumed full utilization of the productive time of the blind in case of rehabilitation . the first assumption is the consequence of our data limitation : the estimates of blind population in pakistan are available only for ages 30 years and above , however , the productive age group starts from 18 years and above . we assumed full utilization of the productive time of the blind in case of rehabilitation . our sensitivity analysis indicates that if we include the population from 2029 years and above and apply the same incidence of blindness as that prevailing in the age group of 30 - 34 year , our estimates of economic gains for the 10-year period increase from 421 billion to 688 billion ; a difference of about 267 billion . if on the other hand , we apply the assumption that only 50% of the blind are able to participate in the labor market after being rehabilitated , our estimates of aggregate economic earning decrease from 421 billion to 210 billion : a reduction of 210 billion . the positive bias turns out to be slightly higher that the negative bias thereby bringing in a net increase of about 57 billion in our estimates of economic gain over the 10-year period ( 421 - 210 + 267=478 ) . secondly , we have used the average wage rate and it is possible that many of the people who are blind , if equipped with education and skills , are able to earn more than the average wage rate . frick and foster used economic and epidemiologic modeling to estimate the number of blind individuals globally and the associated economic productivity loss from the year 2000 to 2020 . they concluded that if significant efforts were made to reduce avoidable blindness , this could result in a reduction of 429 million blind person - years and a minimum saving of $ 102 billion for unaccommodated blindness alone over the period from 2003 to 2020 . similarly , taylor et al . in australia attempted to quantify the economic costs of vision loss in australia and assess the impact of a costed intervention package to prevent avoidable vision loss . they found that the intervention package , if implemented , could bring about a 4.8-fold return on investment . our study shows that if the cataract blind are rehabilitated , this alone will result in an economic benefit aggregated over 1 year of 0.38% of the current gdp . eye care has a proven range of low - risk , high - success , and highly cost - effective interventions as measured by cost utility and cost - effective analyses . cataract surgery is one of the most cost - effective interventions in developing countries , costing as little as us $ 15 to us $ 23 per disability - adjusted life - year saved . despite dismal health indicators , pakistan spends a meager amount of its gdp on the health sector . over the past many decades , total public health spending in pakistan has remained below 0.8% of its gdp . within the health sector itself , there are multiple increasingly expensive areas , each clamoring for adequate funding . not all of these areas are expected to reap economic gains nor are all justified on ethical and equity grounds . the present global financial crisis has impacted developing and developed countries alike but some countries are affected more than others . some developing countries may be at a higher risk of not spending enough on social sectors due to the fact that they have approached the international monetary fund ( imf ) for emergency assistance . the imf may impose additional restrictions on these countries to spend less on social sectors and rather focus on cost recovery , which may not be possible under present circumstances . populations in countries affected by or emerging from conflict , with few financial reserves , weak institutions and damaged infrastructure , are especially vulnerable . there is a need to protect health spending in countries where per capita health expenditures are below certain threshold levels , which may be necessary to achieve reasonable health outcomes . this requires policy coordination from both the government and donors not only in priority health sectors but also in other allied areas as well . preventing and treating blindness and improving eye health is one of the most cost - effective health interventions that not only improves the quality of life and promotes social inclusion , but also increases productivity . according to vision 2020the right to sight globally , about 85% of all visual impairment and 75% of blindness although our results are sensitive to the assumptions we made , it turns out that the negative and positive biases that are most likely to be created by two of our most critical assumptions tend to outweigh each other and there is no significant deviation from the results otherwise obtained . more specifically , let us examine the impact of two of our most critical assumptions : the first assumption is the consequence of our data limitation : the estimates of blind population in pakistan are available only for ages 30 years and above , however , the productive age group starts from 18 years and above.we assumed full utilization of the productive time of the blind in case of rehabilitation . the first assumption is the consequence of our data limitation : the estimates of blind population in pakistan are available only for ages 30 years and above , however , the productive age group starts from 18 years and above . we assumed full utilization of the productive time of the blind in case of rehabilitation . our sensitivity analysis indicates that if we include the population from 2029 years and above and apply the same incidence of blindness as that prevailing in the age group of 30 - 34 year , our estimates of economic gains for the 10-year period increase from 421 billion to 688 billion ; a difference of about 267 billion . if on the other hand , we apply the assumption that only 50% of the blind are able to participate in the labor market after being rehabilitated , our estimates of aggregate economic earning decrease from 421 billion to 210 billion : a reduction of 210 billion . the positive bias turns out to be slightly higher that the negative bias thereby bringing in a net increase of about 57 billion in our estimates of economic gain over the 10-year period ( 421 - 210 + 267=478 ) . secondly , we have used the average wage rate and it is possible that many of the people who are blind , if equipped with education and skills , are able to earn more than the average wage rate . frick and foster used economic and epidemiologic modeling to estimate the number of blind individuals globally and the associated economic productivity loss from the year 2000 to 2020 . they concluded that if significant efforts were made to reduce avoidable blindness , this could result in a reduction of 429 million blind person - years and a minimum saving of $ 102 billion for unaccommodated blindness alone over the period from 2003 to 2020 . similarly , taylor et al . in australia attempted to quantify the economic costs of vision loss in australia and assess the impact of a costed intervention package to prevent avoidable vision loss . they found that the intervention package , if implemented , could bring about a 4.8-fold return on investment . our study shows that if the cataract blind are rehabilitated , this alone will result in an economic benefit aggregated over 1 year of 0.38% of the current gdp . eye care has a proven range of low - risk , high - success , and highly cost - effective interventions as measured by cost utility and cost - effective analyses . cataract surgery is one of the most cost - effective interventions in developing countries , costing as little as us $ 15 to us $ 23 per disability - adjusted life - year saved . despite dismal health indicators , pakistan spends a meager amount of its gdp on the health sector . over the past many decades , total public health spending in pakistan has remained below 0.8% of its gdp . within the health sector itself , there are multiple increasingly expensive areas , each clamoring for adequate funding . not all of these areas are expected to reap economic gains nor are all justified on ethical and equity grounds . the present global financial crisis has impacted developing and developed countries alike but some countries are affected more than others . some developing countries may be at a higher risk of not spending enough on social sectors due to the fact that they have approached the international monetary fund ( imf ) for emergency assistance . the imf may impose additional restrictions on these countries to spend less on social sectors and rather focus on cost recovery , which may not be possible under present circumstances . populations in countries affected by or emerging from conflict , with few financial reserves , weak institutions and damaged infrastructure , are especially vulnerable . there is a need to protect health spending in countries where per capita health expenditures are below certain threshold levels , which may be necessary to achieve reasonable health outcomes . besides , entitlement to better health outcomes is a basic human right . this requires policy coordination from both the government and donors not only in priority health sectors but also in other allied areas as well . preventing and treating blindness and improving eye health is one of the most cost - effective health interventions that not only improves the quality of life and promotes social inclusion , but also increases productivity . according to vision 2020the right to sight globally , about 85% of all visual impairment and 75% of blindness one of the most important insights that the human development paradigm offers is that poverty is not so much a problem of the lowness of income as of the inadequacy of income to achieve the necessary functioning and the capability ( or the freedom ) to acquire those functioning . poverty is obviously seen as a serious deprivation of certain capabilities. the implication of this approach on development policy is that poverty must be accepted as social evil irrespective of prevailing inter - country and interpersonal disparities between income and expenditure patterns ; and that ameliorative action must go well beyond just raising the income of the target group of poor . everywhere , it would take transforming more resources to the poor and the physically disabled in order to redress interpersonal capability deficits . it provides the basis to infer that an effective eye health system to eradicate visual impairment must be seen in the context of capabilities approach for necessary functioning of human beings for their livelihood and survival . there are many direct and indirect gains that can be achieved through targeted expenditure in the health and education sector . it is important for these to be documented and used as part of the planning process . equally important , the social sector expenditure that includes spending on education , vocational training as well as spending targeted toward those with special needs may not realize the true benefit if the mechanisms to absorb the excess capacity are not encouraged and facilitated . generally , this would include areas as diverse as job creation and industrialization on one hand and sustainable and equitable rural development and economic empowerment on the other hand . it is important for policy makers at the national level to realize that economic evaluation should be an integral component of the health planning process . furthermore , rehabilitation of the disabled , including blind , can result in significant economic benefits in terms of increase in overall productivity and thus should be included in poverty reduction strategies . however , it must be recognized that this gain is fully realizable only if those with disabilities , such as the rehabilitated blind , are facilitated in realizing their full employment and livelihood development potential .

state and nonstate health programs in developing countries are often influenced by priorities that are defined in the millennium development goals ( mdgs ) . in the wake of recessionary pressures , policy makers in the health sector are often seen to divert significant budgets to some specific health programs and make only token allocations for other health problems that are important but do not fall under the traditional mdg box of health priorities . this paper illustrates the economic argument for investment in one such program : the eye health program and employs a country case study of pakistan to demonstrate that there are significant economic gains that are being foregone by not addressing the needs of the blind in poverty reduction strategies . by applying appropriate growth and discounting factors and using the average wage rate , the paper estimates the total productivity gains that are realizable over a period of 10 years if the blind population in pakistan is rehabilitated and their carers released to participate in the mainstream economic activity . our findings indicate that significant productivity gains accumulated over 10 years , range from 61 billion ( us$ 709 million ) to 421 billion ( us$ 4.9 billion ) depending upon whether the entire blind population or only those affected by a specific cause are rehabilitated . the per annum productivity gains of rehabilitating the entire blind population represents 0.74% of the current gross domestic product of pakistan , which is higher than the total public spending on health . in order to reap these benefits , the subsequent absorption of the rehabilitated blind and their carers into mainstream economic activity is as important as their effective rehabilitation .

The Prevalence of Blindness in Pakistan Materials and Methods Key Assumptions Sources of Data Results Discussion Sensitivity analysis Conclusion

, the prevalence of blindness in pakistan among adults aged 30 years and above is 2.7% . using the 2011 population estimate of the corresponding age groups fifty - five percent of the total blind population in pakistan is estimated to be cataract blind [ tables 13 ] . the prevalence of adult blindness ( 30 years and above ) in pakistan by cause age specific blind population by all causes in pakistan age specific blind population by cause the prevalence of blindness is observed to be higher among older age groups compared with the younger age cohorts . with respect to sex , the prevalence of blindness is lower among females than males in the younger age group of 3039 years . in older age groups of 4059 years overall there are around 624,000 people blind in pakistan in the productive age group of 3059 years . in order to estimate the potential economic gains of rehabilitating the blind in pakistan , we use the average earnings to calculate the productivity gains that can be realized if the blind population in the productive age group ( 3059 years ) is rehabilitated and facilitated to become productive members of society . these productivity gains are analyzed for a short run scenario comprising of a year to a long run horizon spanning over 10 years through appropriate growth and discounting techniques . a disaggregated data of wage rates by sex were used in order to take into account the substantial difference between the average wages of the two sexes . these monthly wage earnings were converted into per annum average wage earnings of an individual . the per annum individual wage earnings were then extrapolated for the next 10 years by applying the official growth rate in nominal wages for the corresponding year and by deflating the nominal wage earnings by the corresponding inflation rate . for this purpose , gdp deflator , which is commonly used to deflate output , was used as a measure of inflation . since we do not have the inflation figures for 20112013 , we used the moving average of inflation in the past 5 years preceding 2011 . in the second step , the extrapolated wage earnings over the 10-year period were aggregated and discounted to estimate the net present value of the future stream of income over the 10-year period . the discount rate is based upon the weighted average rate of return on long - term deposits ( 5 years and over ) in 2004 as published by the state bank of pakistan . in the third step , the present value of individual wage earnings over the 10 year period was multiplied by the total number of blind adults in the productive age group of 3059 years to compute the aggregate level of income achieved by rehabilitating the blind population and enabling them to participate in the labor market . as a consequence of this , the rehabilitation of blind would also release the carers productive time , which they can use for wage earnings . in order to estimate that , we first estimated the average hours that carers spend on the care of the disabled . this was done by making use of the recently available data on time use survey conducted by the finance division of pakistan . the survey contains detailed data on unpaid care work that includes housework by women as well as time spent on caring for children , old people , the sick , and disabled , as well as time spent in assisting the community . this is equal to one - fourth of the total productive time per day ( assuming that the total productive time is 8 hours per day ) . the average number of hours spent on care for disabled is multiplied by the hourly wage rate , which was then converted into monthly and yearly income . the hourly wage rate is derived from average monthly earnings of both sexes and the key assumption made in the computation is that the average hours worked per week are 48 hours as reported in the labor force survey of pakistan . again , these per annum individual wage earnings were extrapolated for the next 10 years by applying the official growth rate in nominal wages for the corresponding year and the discount rate . the total individual wage earnings thus computed over the 10-year period were multiplied by the total number of carers , which is assumed to be equal to the total number of blind in both the productive as well as nonproductive age group . since we do not have data on the division of carers by sex , we applied the wage rate of both sexes as reported in the labor force survey and in table 4 . in the final step , the total potential earnings of the blind as well as the carers were added and expressed as a percentage of the current gdp of pakistan . estimates of average earnings and growth rates in 20032004 used in the study ( pak ) by taking average labor earnings as a proxy for average productivity , the present paper uses a much more rigorous and accurate methodology compared with earlier studies . while income from nonlabor sources may not be directly affected by blindness , it is the wage income that is mostly foregone by the blind population . in short , average the following key assumptions were made to estimate the gains in productivity of rehabilitating the blind in pakistan : the productivity gains were estimated for individuals in the economically productive age group for which we have data on the prevalence of blindness : 3059 years of age . although the productive age starts from 18 years , however , we do not have data on the prevalence of blindness for age groups below 30 years . the inclusion of younger age groups in the computation is likely to increase further the estimates of economic gains . blindness were considered for this paper , there is likely to be an underestimation as those with moderate or severe visual impairment were not included in the calculations.the foregone earnings of the blind population were computed assuming full utilization of their productive time in case of rehabilitation . the possible upward bias in our estimates from this assumption is , however , likely to cancel out with the downward bias of our estimates due to noninclusion of less than 30 years of population in our productive age group.the estimates of average monthly earnings for males , females , and both sexes are taken from the labor force survey ( 20032004 ) of pakistan . the growth rate of wages is also taken from the same survey.the foregone earnings of the carers were computed based upon the evidence from time use survey ( conducted by the ministry of finance , pakistan ) according to which they devote 2 hours of their productive time per day for the care of disabled persons.since we are not aware of the precise division of carers by sex , we used the average hourly wage rate of both sexes as reported in the labor force survey of pakistan ( 20032004).the total number of carers is assumed to be equal to the total number of blind population that includes the blind people in both the productive and unproductive age group.the discount rate used for computing the present value of future stream of income is 4% , which is based upon the weighted average rate of return on long - term deposits ( 5 years and over ) in 2004 as published by the state bank of pakistan . the productivity gains were estimated for individuals in the economically productive age group for which we have data on the prevalence of blindness : 3059 years of age . although the productive age starts from 18 years , however , we do not have data on the prevalence of blindness for age groups below 30 years . the inclusion of younger age groups in the computation is likely to increase further the estimates of economic gains . blindness were considered for this paper , there is likely to be an underestimation as those with moderate or severe visual impairment were not included in the calculations . the foregone earnings of the blind population were computed assuming full utilization of their productive time in case of rehabilitation . the estimates of average monthly earnings for males , females , and both sexes are taken from the labor force survey ( 20032004 ) of pakistan . since we are not aware of the precise division of carers by sex , we used the average hourly wage rate of both sexes as reported in the labor force survey of pakistan ( 20032004 ) . the total number of carers is assumed to be equal to the total number of blind population that includes the blind people in both the productive and unproductive age group . the discount rate used for computing the present value of future stream of income is 4% , which is based upon the weighted average rate of return on long - term deposits ( 5 years and over ) in 2004 as published by the state bank of pakistan . data on the prevalence of blindness by age group and by sex was obtained from the pakistan national eye survey study group that conducted a nationally representative survey of blindness in pakistan , the results of which are reported in jadoon et al . the latest demographic data on pakistan , disaggregated by age and sex , was obtained from the international database of us census of bureau . finally data on current gdp of pakistan and inflation rate was obtained from the economic survey of pakistan . table 5 illustrates our computation results regarding the potential productivity gains of rehabilitating the blind in pakistan , disaggregated by sex and by causes . economic benefits of rehabilitating the blind in pakistan ( all causes ) the results show that if the entire blind population in pakistan ( by all causes ) is rehabilitated , the total economic benefit / productivity gain of 422 billion ( us$ 4.9 billion ) is realizable over a period of 10 years . this translates into 42.2 billion ( us$ 490 million ) per annum . if only the avoidable blindness is treated , the aggregate economic benefit of 361 billion ( us$ 4.25 billion ) over a period of 10 years and an average of 36 billion ( us$ 425 million ) per annum is realizable . the rehabilitation of incurably blind results in the aggregate benefits of 61 billion ( us$ 718 million ) over a period of 10 years and an average of 6.1 billion ( us$ 71.8 million ) per year . similarly , the treatment of cataract blindness results in 217 billion ( us$ 2.55 billion ) over 10 years and an average of 21.7 billion ( us$ 255 million ) per annum . in table 6 , these economic benefits are expressed in terms of the total current gdp of pakistan . table 6 shows that if the entire blind population in pakistan is rehabilitated , economic benefits per annum amount to 0.7% of gdp . this is a significant economic gain that is roughly equal to the total government spending on health in pakistan . if only those people are treated who are blind due to avoidable causes , the economic benefit achieved per annum is equal to 0.6% of gdp . economic benefits of rehabilitating the blind in pakistan the results in table 6 show that even if the rehabilitation of the blind in pakistan costs 0.7% of the gdp per annum , it is still justified on economic grounds . in future , it would be useful to estimate the total cost of rehabilitating the blind in pakistan which can then be compared with the total economic benefits computed in terms of productivity gain above so as to estimate the net economic gain of rehabilitating the blind in pakistan . the world health organization states that there are 285 million people visually impaired and 39 million blind globally . children who are born blind or who become blind and survive have a lifetime of blindness ( 70 million blind years ) ahead of them , with all the associated emotional , social and economic costs to the child , the family and society . it affects their access to education and severely limits their livelihood options in developing countries . indeed , many of the conditions associated with blindness in children are also causes of child mortality , for example , premature birth , measles , vitamin a deficiency , meningitis , and congenital rubella syndrome . in this paper , we have tried to demonstrate , through a country case - study , the opportunity cost of not addressing the needs of the blind in poverty reduction strategies . we have used the average earnings approach to calculate the productivity gains that can be obtained if the blind population in the productive age group ( 3059 years ) is rehabilitated and facilitated to become productive members of society . this approach is based upon the assumption that each person represents a productive resource to society and illness or disability diminishes that person 's productive capacity , which in turn is valued by his or her loss of earning . in this sense , the present study evaluates the economic benefits in a much more rigorous and accurate manner . the results of our analysis indicate that the rehabilitation of the blind can potentially result in significant productivity gains in the form of increase in earnings by both the blind and their carers . our estimates suggest that the aggregate economic benefits spread over a 10-year period represent 0.7% of current gdp per annum , which is roughly equal to the total public spending on health . our results clearly show that the rehabilitation of the blind in pakistan is not only justified on ethical and humanitarian grounds but also on economic grounds . however , in order to reap these benefits , an effective rehabilitation is not adequate ; subsequent absorption into mainstream economic activity must also be facilitated . more specifically , let us examine the impact of two of our most critical assumptions : the first assumption is the consequence of our data limitation : the estimates of blind population in pakistan are available only for ages 30 years and above , however , the productive age group starts from 18 years and above.we assumed full utilization of the productive time of the blind in case of rehabilitation . the first assumption is the consequence of our data limitation : the estimates of blind population in pakistan are available only for ages 30 years and above , however , the productive age group starts from 18 years and above . we assumed full utilization of the productive time of the blind in case of rehabilitation . our sensitivity analysis indicates that if we include the population from 2029 years and above and apply the same incidence of blindness as that prevailing in the age group of 30 - 34 year , our estimates of economic gains for the 10-year period increase from 421 billion to 688 billion ; a difference of about 267 billion . if on the other hand , we apply the assumption that only 50% of the blind are able to participate in the labor market after being rehabilitated , our estimates of aggregate economic earning decrease from 421 billion to 210 billion : a reduction of 210 billion . secondly , we have used the average wage rate and it is possible that many of the people who are blind , if equipped with education and skills , are able to earn more than the average wage rate . in australia attempted to quantify the economic costs of vision loss in australia and assess the impact of a costed intervention package to prevent avoidable vision loss . our study shows that if the cataract blind are rehabilitated , this alone will result in an economic benefit aggregated over 1 year of 0.38% of the current gdp . cataract surgery is one of the most cost - effective interventions in developing countries , costing as little as us $ 15 to us $ 23 per disability - adjusted life - year saved . despite dismal health indicators , pakistan spends a meager amount of its gdp on the health sector . over the past many decades , total public health spending in pakistan has remained below 0.8% of its gdp . within the health sector itself , there are multiple increasingly expensive areas , each clamoring for adequate funding . not all of these areas are expected to reap economic gains nor are all justified on ethical and equity grounds . there is a need to protect health spending in countries where per capita health expenditures are below certain threshold levels , which may be necessary to achieve reasonable health outcomes . preventing and treating blindness and improving eye health is one of the most cost - effective health interventions that not only improves the quality of life and promotes social inclusion , but also increases productivity . more specifically , let us examine the impact of two of our most critical assumptions : the first assumption is the consequence of our data limitation : the estimates of blind population in pakistan are available only for ages 30 years and above , however , the productive age group starts from 18 years and above.we assumed full utilization of the productive time of the blind in case of rehabilitation . the first assumption is the consequence of our data limitation : the estimates of blind population in pakistan are available only for ages 30 years and above , however , the productive age group starts from 18 years and above . we assumed full utilization of the productive time of the blind in case of rehabilitation . our sensitivity analysis indicates that if we include the population from 2029 years and above and apply the same incidence of blindness as that prevailing in the age group of 30 - 34 year , our estimates of economic gains for the 10-year period increase from 421 billion to 688 billion ; a difference of about 267 billion . if on the other hand , we apply the assumption that only 50% of the blind are able to participate in the labor market after being rehabilitated , our estimates of aggregate economic earning decrease from 421 billion to 210 billion : a reduction of 210 billion . secondly , we have used the average wage rate and it is possible that many of the people who are blind , if equipped with education and skills , are able to earn more than the average wage rate . our study shows that if the cataract blind are rehabilitated , this alone will result in an economic benefit aggregated over 1 year of 0.38% of the current gdp . cataract surgery is one of the most cost - effective interventions in developing countries , costing as little as us $ 15 to us $ 23 per disability - adjusted life - year saved . despite dismal health indicators , pakistan spends a meager amount of its gdp on the health sector . over the past many decades , total public health spending in pakistan has remained below 0.8% of its gdp . within the health sector itself , there are multiple increasingly expensive areas , each clamoring for adequate funding . not all of these areas are expected to reap economic gains nor are all justified on ethical and equity grounds . the present global financial crisis has impacted developing and developed countries alike but some countries are affected more than others . preventing and treating blindness and improving eye health is one of the most cost - effective health interventions that not only improves the quality of life and promotes social inclusion , but also increases productivity . according to vision 2020the right to sight globally , about 85% of all visual impairment and 75% of blindness one of the most important insights that the human development paradigm offers is that poverty is not so much a problem of the lowness of income as of the inadequacy of income to achieve the necessary functioning and the capability ( or the freedom ) to acquire those functioning . everywhere , it would take transforming more resources to the poor and the physically disabled in order to redress interpersonal capability deficits . it provides the basis to infer that an effective eye health system to eradicate visual impairment must be seen in the context of capabilities approach for necessary functioning of human beings for their livelihood and survival . there are many direct and indirect gains that can be achieved through targeted expenditure in the health and education sector . it is important for these to be documented and used as part of the planning process . equally important , the social sector expenditure that includes spending on education , vocational training as well as spending targeted toward those with special needs may not realize the true benefit if the mechanisms to absorb the excess capacity are not encouraged and facilitated . it is important for policy makers at the national level to realize that economic evaluation should be an integral component of the health planning process . furthermore , rehabilitation of the disabled , including blind , can result in significant economic benefits in terms of increase in overall productivity and thus should be included in poverty reduction strategies . however , it must be recognized that this gain is fully realizable only if those with disabilities , such as the rehabilitated blind , are facilitated in realizing their full employment and livelihood development potential .

because of increasing concern regarding environmental issues , there is a great demand for the development of efficient sustainable technologies . in the area of organic synthesis , the development of efficient heterogeneous catalytic procedures has become of enormous importance , as shown by the number of reports in this field in recent years . for the industrial syntheses of building blocks and pharmaceutically relevant compounds , the elaboration of environmentally benign and cost - effective methods for carbon carbon or carbon heteroatom bond - formating reactions is of paramount importance . within this family of reactions , the suzuki miyaura cross - coupling reaction is one of the most robust and versatile methods for the formation of the carbon backbone of a target molecule , which is of obvious relevance for further progress in various branches of this field . a number of challenges remain to be overcome to achieve high efficiency under mild reaction conditions ( e.g. , ambient temperature ) in environmentally friendly solvents . in addition , the use of cheaper and less - reactive substrates ( e.g. , aryl chlorides ) also remains a challenge . typical catalysts for these transformations can sometimes become very expensive , owing to the necessity of using transition - metal complexes in combination with different types of ligands . thus , their high cost and environmental impact are major drawbacks for their large - scale application . metal organic frameworks ( mofs ) , also known as coordination polymers , have gained wide interest over the last decade . mofs have shown great potential in gas uptake and separation , as well as in various biomedical applications , such as drug encapsulation and delivery . in addition , mofs can be used as catalyst supports that allow for the recycling of metallic species and also prevent the contamination of the final products . however , for a mof to be a suitable catalytic support it must be stable under the reaction conditions and allow no leaching of the active species , whilst still allowing full accessibility of the starting materials to the reaction site . however , this requirement is not always a simple task . gratifyingly , by a thoughtful design of the linkers or subsequent postsynthetic modifications , it is possible to carefully tailor the inside of the pores so that they can better accommodate the catalyst and prevent it from leaching . one of the materials that meets all of the criteria mentioned above is mil-101cr [ ( cr3f(h2o)2o[(o2c)-c6h4-(co2)]3nh2o ) ] , which was first reported in 2005 by frey and co - workers . mil-101cr has a very high specific surface area ( bet , about 4100 m g ) and contains two types of cages with diameters of 29 and 34 , which have pore apertures of 12 and 16 , respectively . mil-101cr is stable in water , even under very acidic conditions , and can tolerate temperatures as high as 275 c in air . these unique features of mil-101cr have attracted enormous attention from researchers worldwide to explore its potential in various applications , including catalysis . supporting metallic nanoparticles inside a porous matrix is one of the three possible ways of exploiting mofs in catalysis , along with supporting single - site metal complexes or by making use of the structural metal atoms in the framework as catalytically active sites.[13 , 14 ] the main advantage of mofs is that , by controlling the pore size , one can limit the growth of the nanoparticles , thereby obtaining small and well - distributed nanoparticles and avoiding agglomeration . this result is achieved by the infiltration of a metallic precursor into the framework , followed by subsequent reduction , thus leading to the direct formation of active nanoparticles in a controlled manner inside the pores . recent reviews by dhakshinamoorthy and garcia summarized the use of metal nanoparticles that were immobilized inside mofs as heterogeneous catalysts for various reactions , including condensation , hydrogenation , carbon carbon coupling , and alcohol - oxidation reactions , as well as the synthesis of meoh . as such , great efforts have been made to immobilize palladium nanoparticles inside the robust mil-101cr to form pd@mil-101cr as a heterogeneous catalyst , which has been used for suzuki coupling reactions[17 , 18 ] or other palladium - catalyzed reactions , such as the synthesis of indoles through sonogashira cross - coupling followed by a cycloaddition reaction , the aqueous hydrogenation of phenols into cyclohexanones , the synthesis of methyl isobutyl ketone from acetone , the hydrogenation of ketones , and even tandem processes , as cleverly illustrated by corma and co - workers . as a proof of concept , in 2010 , jiang and co - workers published the first report of a suzuki coupling reaction of aryl chlorides in water by using pd@mil-101cr at 80 c with naome as a base ( 1.5 equiv ) and tetrabutylammonium bromide ( 0.6 equiv ) as a co - catalyst under an inert atmosphere . from this starting point , other groups have been working on the elaboration of this method to maintain high conversions whilst trying to perform the reactions under aerobic conditions at lower temperatures . significant progress in the field of functionalized mofs was reported in 2011 by stock and co - workers , who described , for the first time , a rapid and facile method for the postsynthetic functionalization of mil-101cr by covalently grafting amino groups onto the terephthalate linkers . bearing in mind the affinity of palladium for nitrogen atoms , this modification constituted a very important breakthrough , because it minimized the leaching of metallic species . indeed , cao and co - workers followed this idea and presented evidence of an increased stabilization effect of the nanoparticles in amino - functionalized mil-53al - nh2 compared to mil-53al.[18 , 25 ] over the past few years , our groups have undertaken on - going research into the development of new and efficient transition - metal - catalyzed processes for the formation of carbon carbon ( c c ) and carbon heteroatom ( c x ) bonds and the use of mofs in heterogeneous catalysis.[12 , 14 ] during our investigations , we became interested in understanding the parameters that affect the efficiency of suzuki miyaura cross - coupling reactions using pd nanoparticles that are immobilized inside mil-101cr - nh2 . in particular , we were intrigued by the fact that the reported palladium loadings in the mofs were often low , about 13 wt % , [ 16 , 19 , 21 , 23a , 25 ] with only a few exceptions with higher pd loadings ( 56 wt % ) . herein , we report our investigations into the potential and limitations of mil-101cr - nh2 as a catalyst support of pd nanoparticles and its application in suzuki miyaura cross - coupling reactions . we investigated how the loading of pd into mil-101cr - nh2 affected the stability of the material and its performance as a catalyst and we determined the optimal pd loading into the framework . tem and total scattering techniques were employed to study the pd nanoparticles in the mil-101cr - nh2 crystals . the catalyst with the optimized amount of pd ( 8 wt % loaded ) in mil-101cr - nh2 was subjected to a systematic screening of the reaction parameters , thereby leading to efficient suzuki miyaura coupling reactions , with excellent yields for a large substrate scope , including challenging heteroaromatic halides and bulky substrates , under environmentally friendly conditions ( water or water / etoh , ambient temperature , k2co3 ) . thus , herein , we show the importance of tuning the loading of the nanoparticles in the material and of optimization of the reaction conditions for efficient and recyclable catalysis by using mofs , which demonstrates their great potential as supports of metal nanoparticles for practical use and large - scale applications . synthesis and characterization of pd@mil-101cr - nh2 : mil-101cr was synthesized according to a literature procedure . the amino functionality was introduced into mil-101cr in two steps , as reported by stock and co - workers . first , nitration of the phenyl rings was achieved by the treatment of mil-101cr with hno3/h2so4 . in the second step , the quantitative functionalization of mil-101cr was confirmed by both elemental analysis and by h nmr spectroscopy after the digestion of mil-101cr - no2 and mil-101cr - nh2 ( see the supporting information , figure s1 , and the experimental section ) . palladium loadings of 4 , 8 , 12 , and 16 wt % were prepared by the impregnation of mil-101cr - nh2 with the corresponding amount of [ pdcl2(mecn)2 ] in dry ch2cl2 overnight , thus yielding pd@mil-101cr - nh2 , in which pd was coordinated to the amino groups . subsequent reduction into pd by using nabh4 afforded nanosized pd particles inside the mil-101cr - nh2 crystals , pd@mil-101cr - nh2 . the synthesized material was washed with water and etoh to eliminate all of the residues . the purity of the four final materials ( 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 ) was checked by energy - dispersive spectroscopy ( eds ) that was connected to a sem , elemental analysis , and by inductively coupled plasma - optical emission spectrometry ( icp - oes ; supporting information , table s2 ) . the actual pd contents , as determined by icp - oes , for the samples of 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 were 4.38 , 8.06 , 11.29 , and 15.07 wt % , respectively . importantly , no traces of either sn from sncl2 or na from the reducing reagent were found in any of the pd@mil-101cr - nh2 samples . the functionalization of the terephthalate linker first with nitro groups and then with amino groups was also followed by ftir spectroscopy . the ftir spectra and vibration frequencies of our synthesized samples , as shown in the supporting information , figures s2 and s3 and table s3 , corresponded well with literature values . o stretches , were observed in the spectrum of mil-101cr - no2 ( supporting information , figure s3 ) . n stretch at 1258 cm in mil-101cr - nh2 was clearly seen in the spectrum . all of the frequency bands of the mil-101cr framework remained unchanged after the incorporation and reduction of palladium . the pxrd patterns of our synthesized samples of mil-101cr matched with those reported in the literature . the structure of mil-101cr remained intact after all of the postsynthetic modification steps and no obvious changes in the pxrd patterns were detected , which confirmed that mil-101cr - nh2 was chemically and hydrothermally stable and that it could withstand harsh conditions , such as those applied during the nitration step ( with mixtures of concentrated acids ) and during the reduction of pd by using excess nabh4 ( figure 1 ) . the systematic changes in the relative peak intensities in the pxrd patterns with different pd loadings , for example , those within the regions 2=56 and 89 , indicated that pd had been incorporated into the pores of mil-101cr . pxrd patterns of mil-101cr , mil-101cr - no2 , mil-101cr - nh2 , and 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 . the weight losses of the synthesized samples upon heating in air were studied by using tga ( supporting information , figure s4 ) . the thermal stability of mil-101cr seemed to be slightly weakened after amino - functionalization and even more so after immobilization of the pd nanoparticles . the amount of water and solvent molecules in the pores differed between the samples , as shown by the different weight losses during heating . n2-sorption measurements were performed for as - synthesized mil-101cr , amino - functionalized mil-101cr - nh2 , and 4- , 8- , 12- , and 16 wt % pd@mil-101cr the n2-adsorption isotherms , the bet surface areas , and the pore volumes are shown in figure 2 and table 1 . the bet surface areas of mil-101cr and mil-101cr - nh2 are 2966 and 2869 mg , respectively , which are in agreement with the reported values . compared with the parent mil-101cr - nh2 , the bet surface area and pore volume decreased with higher pd loading ( table 1 ) . the adsorption isotherms of all samples show stepwise n2 uptake , at p / p0 values up to 0.25 , thus indicating the existence of different types of cage . nitrogen - adsorption isotherms of mil-101cr , mil-101cr - nh2 , and 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 . the measurements were performed at 77 k. specific surface areas and pore volumes of mil-101cr and the functionalized mil-101cr materials sem analysis showed that the mil-101cr crystals were formed as octahedra , with sizes between 0.3 and 1.0 m ( supporting information , figure s5 ) . tem analysis indicated that the octahedral morphology of mil-101cr was preserved throughout all post - modification steps , which include nitration , reduction of the nitro groups , introduction of pd , and reduction of pd into pd . the sizes of the pd particles ranged from 20 to 30 , which were comparable to the pore diameters of unfunctionalized mil-101cr ( 29 and 34 , figure 3 ) . however , on increasing the pd loading in mil-101cr - nh2 from 11.3 to 15.1 wt % , the size of the pd nanoparticles increased up to about 8 nm ( figure 3 ) . these larger pd nanoparticles were found on the surfaces of the 16 wt % pd@mil-101cr - nh2 crystals , as shown by tem . electron tomography was performed on crystals of 8 wt % pd@mil-101cr - nh2 to study the distribution of the pd nanoparticles . the reconstructed 3d tomographic images show that the nanoparticles are embedded inside the crystals and are homogeneously distributed throughout the crystals ( figure 4 and the supporting information , movies s1 and s2 ) . electron diffraction and high - resolution transmission electron microscopy ( hrtem ) images show that the pd nanoparticles are crystalline ( supporting information , figure s6 ) . tem images of samples of 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 with different pd loadings ( 4.4 , 8.1 , 11.3 , and 15.1 wt % pd , respectively ) . insets show the size distributions of the pd particles as histograms , each of which was formed from about 100 pd particles in several crystals in the tem images . the x axis in the histograms 2d section of the 3d tomographic image of two pd@mil-101cr - nh2 crystals with 8 wt % pd loading , as reconstructed by the electron tomography from a tilt series of 83 tem images with a tilt range of 41 and a tilt step of 0.5. for the tilt series and the full tomogram , see the supporting information , movies s1 and s2 , respectively . a better technique to study the size and distribution of the pd nanoparticles is x - ray total scattering , because tem only gives some local information and does not necessarily represent the nature of the bulk samples . owing to their small particle sizes and the low concentrations of the pd nanoparticles , no bragg peaks for the pd nanoparticles could be clearly seen in the pxrd patterns that were recorded on an in - house powder x - ray diffractometer . therefore , we recorded the total scattering intensities of the 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 samples at the beamline p02.1 , petra iii , desy , by using a high - energy synchrotron source ( 60 kev ) . the pair distribution function ( pdf ) , g(r ) , was obtained by performing a fourier transform of the total scattering intensities , including both bragg peaks and diffuse scattering . this function describes the statistical distribution of all of the interatomic distances in a sample and , thus , is particularly suitable for investigating nanoparticles , which usually give very broadened or even no bragg peaks by using conventional pxrd . the pdfs of the 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 samples were extracted from the total x - ray scattering signal truncated at a maximum q value ( qmax ) of 19 ( figure 5 ; supporting information , figure s7 ) . for comparison , the pdfs that were calculated from the structural model of mil-101cr and from the structure of an ideal pd nanoparticle ( space group fmm ) with a diameter of 25 are also shown in figure 5 ( for the detailed procedure see the supporting information ) . the peaks in the observed pdfs of the 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 samples correspond to the interatomic distances in both mil-101cr and the pd nanoparticle . o distance , which is not present in the calculated pdf of the pure pd nanoparticle ( figure 5 , top ) . the peak at 2.9 corresponds to the shortest distance between two pd atoms in the pd nanoparticle . the ripples on either side of the first peak in the calculated pdf of the pd nanoparticle originate from termination effects that are due to the finite qmax of 19 that was used for the calculations and can be neglected . whereas the peaks in the calculated pdf of the pd nanoparticle are relatively sharp , those above 2.5 in the calculated pdf of mil-101cr are rather broad , owing to multiple overlapping of different atom atom correlations of similar distances . the peak at 1.3 results from overlapping of the nearest c c and c o distances in mil-101cr . the integrated intensity of a well - defined pdf peak yields the coordination number of that specific atomic pair . for example , the integrated intensity of the first peak at 2.9 in the calculated pdf of the pure pd nanoparticle will yield 12 , which corresponds to the coordination number of pd . in case of multielement samples , such as mil-101cr and 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 , the peak intensities in the pdf are multiplied by a weighting factor to take into account the different scattering factors of the chemical species . thus , in the observed pdfs of 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 , the peak intensities can be related to the multiplicity and abundance of the atomic pairs , as well as to atomic scattering factors . by comparing the pdfs of the 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 samples ( figure 5 ) , a change in the relative peak intensities can be observed ; in particular , with higher pd loading , the intensity of the peak at 2.9 , which corresponds to the pd pd distance , increases in comparison with that of the peak at 2.0 , which is assigned to the cr o distance . this result is reasonable because , with higher pd loading , the number of pd atoms and , hence , the number of pd pd distances of 2.9 is expected to increase , whereas the number of cr o distances of 2.0 in the mil-101cr - nh2 should remain constant . when the pdfs of the 12- and 16 wt % pd@mil-101cr - nh2 samples are compared to the pdf of the 25 pd nanoparticles ( calculated ) , one can see that the peaks at r>4.5 mainly originate from the pd nanoparticles . in fact , the pdf of 16 wt % pd@mil-101cr - nh2 can be almost fully described by the structure of the pd nanoparticles with an average size of 25 . the pdf of 12 wt % pd@mil-101cr - nh2 is similar to that of 16 wt % pd@mil-101cr - nh2 , except that the former attenuated at a shorter distance ( r ) . this result indicates that the size of the pd nanoparticles in 16 wt % pd@mil-101cr - nh2 is slightly larger than that in 12 wt % pd@mil-101cr - nh2 . on the other hand , the pdfs of the 4- and 8 wt % pd@mil-101cr - nh2 samples are very similar at r>4.5 and attenuate at much lower r values compared to the pdfs of 12- and 16 wt % pd@mil-101cr - nh2 . this result indicates that the sizes of the pd particles are similar in 4- and 8 wt % pd@mil-101cr - nh2 and are smaller than those in 12- and 16 wt % pd@mil-101cr - nh2 . it is worth mentioning that the pdf can also tell if there is agglomeration of the nanoparticles in the sample . because no peaks are observed at large r values ( > 20 ) , it can be concluded that there is no significant agglomeration of pd nanoparticles in the four pd@mil-101cr - nh2 samples . wt % pd@mil-101cr - nh2 , compared with the calculated pdfs of mil-101cr ( bottom ) and of a pd nanoparticle ( diameter : 25 , top ) . for the sake of clarity , the pdfs are vertically stacked by 10 . miyaura cross - coupling reaction with pd@mil-101cr - nh2 : in an initial series of reactions , we screened the reactivity of p - bromoanisol ( 1 a ) with different organoboron reagents and bases ( table 2 ) . a similar screening of the reactivity of p - bromotoluene is shown in the supporting information ( table s3 ) . unless otherwise specified , water was used as the solvent and the reactions were performed at room temperature . based on the characterization of the different catalysts , screening of the reaction conditions was performed with 8 wt % pd@mil-101cr - nh2 ( 3 mol % pd loading ) . in contrast to 12- and 16 wt % pd@mil-101cr - nh2 , the 8 wt % catalyst did not show agglomeration of palladium on its surface . moreover , 8 wt % pd@mil-101cr - nh2 contained a more favorable wt % pd per gram of material than 4 wt % pd@mil-101cr - nh2 , which is desirable from an atom - economy viewpoint . nevertheless , the catalytic activity of all of the catalysts has been compared ( see below ) . miyaura coupling reactions catalyzed by pd@mil-101cr - nh2 ; optimization of the reaction conditions [ a ] unless otherwise stated , the organoboron reagent ( 0.12 or 0.15 mmol ) , aryl halide ( 0.1 mmol ) , base ( 0.2 mmol ) , and 8 wt % pd@mil-101cr - nh2 ( 4 mg , 0.003 mmol , 3 mol % pd loading ) were suspended in deionized water ( 2 ml ) . the mixture was stirred vigorously at rt for 6 h in a glass vial in air ( see the supporting information ) ; [ b ] yields determined by h nmr spectroscopy with 1,2,4,5-tetrachloro-3-nitrobenzene as an internal standard ; [ c]>95 % conversion into anisole ( i.e. , dehalogenation ) ; [ d ] 4 wt % pd@mil-101cr - nh2 ( 3 mol % pd loading ) ; [ e ] 12 wt % pd@mil-101cr - nh2 ( 3 mol % pd loading ) ; [ f ] 16 wt % pd@mil-101cr - nh2 ( 3 mol % pd loading ) . miyaura coupling reaction greatly affects the efficiency of the reaction . in general , the reactivities of different bases in the coupling reactions of aryl iodides and bromides decreased in the order : oh > co3aco . at high concentrations , bases can lead to the formation of unreactive boronates ( arb(oh)3 ) and , thus , the [ oh]/[arb(oh)2 ] ratio needs to be controlled . moreover , the countercations can have an unexpected deceleration effect on the transmetalation step , by coordinating to the hydroxo ligand of [ arpd(oh)l2 ] intermediates . with these arguments in mind , we screened a wide selection of bases and observed the stability of the 8 wt % pd@mil-101cr - nh2 catalyst , as shown in table 2 . when phb(oh)2 ( 2 a ) and cs2co3 were used , 4-methoxybiphenyl ( 3 a ) was obtained in quantitative yield within only 6 h ( table 2 , entry 1 ) . we were particularly interested in finding a less - expensive base than cs2co3 that could afford comparably high yields without harming the material . thus , a range of other bases was tested ( table 2 , entries 212 ) . sodium hydroxide also afforded excellent results ( table 2 , entry 2 ) ; however , mil-101cr - nh2 was not stable under these strongly basic conditions ( supporting information , figure s8 ) . for this reason , carbonates were preferred because the release of hydroxy anions into the aqueous medium was slower . this property prevented the decomposition of the framework during the catalysis and also allowed for better control over the [ oh]/[arb(oh)2 ] ratio . other carbonates , such as na2co3 , k2co3 , and baco3 afforded compound 3 a in 71 % , 82 % , and 94 % yield , respectively ( table 2 , entries 35 ) . csf gave a comparable yield to those that were obtained with carbonates ( table 2 , entry 6 ) . acetates have been shown to be a poor choice of base , because they lead to a slow transmetalation step . although various amounts of the coupling product ( 3 a ) were detected in several experiments with naoac as the base , most of the starting material underwent dehalogenation to produce anisole , which occurred faster than the coupling reaction ( table 2 , entry 7 ) . we also tested a number of organic bases ( table 2 , entries 811 ) because their liquid nature may facilitate the large - scale application of this reaction . potential problems owing to pore blocking in the case of insufficient solubility of the base would also be avoided . we found that several bases , such as ipr2nh , ipr2etn , and et3n , afforded the cross - coupled product ( 3 a ) in very high yields ( table 2 , entries 810 ) . a comparative leaching study was performed by using one inorganic base ( cs2co3 ) and one organic base ( ipr2nh ) in the coupling reaction of compound 1 a with compound 2 b in water at room temperature . after completion of the reaction ( 6 h ) , the mixture was filtered through a short pad of celite and the pd content in the filtrate was analyzed by icp - oes analysis . for the reaction with cs2co3 , a remarkable stability of the catalyst was observed and only 0.17 ppm pd was detected in the filtrate . higher pd content was detected in the filtrate when ipr2nh was used ( 5.64 ppm ) , which , although still relatively low , was not negligible . this higher leaching was a consequence of the high affinity of palladium for nitrogenated ligands . furthermore , if a chelating base ( ethylenediamine ) was used ( table 2 , entry 11 ) , all of the palladium was extracted from the support and the material lost its catalytic activity . experiments that were performed in the absence of base showed conversions from 3 % to 21 % ( table 2 , entry 12 ) . this result may be attributed to the presence of free amino groups in the framework , which can act as basic functionalities . solvent and substrate polarity : under the conditions shown in table 2 , entry 1 , we compared the reactivity of compound 1 a with those of p - bromotoluene ( 1 b ) and electron - poor bromide 1 c ( table 2 , entries 13 and 14 ) . surprisingly , in repeated experiments , the neutral ( 1 b ) and electron - rich substrates ( 1 a ) reached full conversion within 6 h , whereas the electron - poor compound 1 c was converted into the corresponding biphenyl in only 84 % yield . this unexpected reactivity , which was opposite to that in organic solvents , owing to the electronic nature of these substrates , may be attributed to the different solubilities of compounds 1 a1 c in water . indeed , when water was replaced by a mixture of etoh and water ( 1:1 , in which differences in solubility were minimized ) , bromide 1 c afforded the corresponding coupling product in excellent yield within only 30 min ( table 2 , entry 15 ) . organoboron reagents : next , we evaluated other organoboron derivatives . to clearly observe any differences in their reactivities , the reactions were stopped after only 4 h and only 1.2 equivalents of the organoboron reagents were used ( table 2 , entries 1618 ) . pinacol phenylboronate ( 2 b ) afforded the highest yield , followed by phenyltrifluoroborate potassium salt ( 2 c ) and phenylboronic acid ( 2 a ) . the lower reactivity of compound 2 a can be explained by its preference to form boroxine trimers , which are inactive under these mild reaction conditions . thus , we decided to continue our investigation by using pinacolate ester 2 b. furthermore , the use of compound 2 b compensated for the loss of reactivity that was observed when cs2co3 was replaced by k2co3 and quantitative yields could be obtained with the latter base by using only 1.2 equivalents of phbpin ( table 2 , entry 19 ) . comparison of pd@mil-101cr - nh2 catalysts with different pd loadings : with the optimized conditions in hand , we compared the activities of the 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 catalysts ( table 2 , entries 2023 ) . the reactions were analyzed before full conversion had occurred ( 3 h ) by using a pd loading of 3 mol % . whereas the reactions with 4- and 8 wt % pd@mil-101cr - nh2 afforded very similar and satisfactory yields ( table 2 , entries 20 and 21 ) , the reactions with 12- and 16 wt % pd@mil-101cr - nh2 afforded significantly lower yields ( table 2 , entries 22 and 23 ) . this behavior was in agreement with the tem and pdf analyses , which showed that the nanoparticles in the 12- and 16 wt % pd@mil-101cr - nh2 catalysts were larger with broader size distributions . the poorer performance of the mofs with higher pd loadings may be explained by an increased difficulty in the diffusion of the substrates and the products through these materials , owing to pore blocking . leaching tests : the stability and performance of the immobilized nanoparticles were evaluated according to the approach reported by sheldon et al . a hot - filtration test was performed by stirring the 8 wt % pd@mil-101cr - nh2 catalyst in boiling water for 15 min , followed by filtration through a pad of celite . the filtrate was subsequently used as the solvent for the reaction of compound 1 a with compound 2 a under similar reaction conditions to those in table 2 , entry 1 , but in the absence of the palladium catalyst . the pd leached into solution after catalysis when carbonates were used as the base was only 0.17 ppm ( see above ) . together with the high recyclability of the catalyst ( see below ) , these results clearly demonstrate the stability of the pd nanoparticles in mil-101cr - nh2 . substrate scope : based on this series of observations ( table 2 ) , we screened a variety of substrates with 8 wt % pd@mil-101cr - nh2 as the catalyst ( 3 mol % pd loading ) and k2co3 as the base , because k2co3 is environmentally benign and about 20-times cheaper than cs2co3 . moreover , potassium has been shown to be the countercation that infers the least - intense decelerating effect in the transmetalation step . the reactions were either performed in water or in water / etoh mixtures ( 1:1 v / v ) . aryl bromides 1 a1 c were easily coupled within short reaction times ( 6 h in water , 30 min in water / etoh ) at room temperature ( table 3 , entries 13 ) . 2-bromonaphthalene ( 1 d ) , which showed very low solubility in water at ambient temperatures , afforded compound 3 d in only 22 % yield . however , 97 % yield was obtained after only 2 h in water / etoh , in which the solubility of the substrate was increased ( table 3 , entry 4 ) . interestingly , the reaction of 9-bromoanthracene ( 1 e ) proceeded very slowly , even in water / etoh after a prolonged reaction time ( table 3 , entry 5 ) . this result may be due to the large size of this substrate , which results in limited access to 8 wt % pd@mil-101cr - nh2 , and the catalysis mainly occurred at the pd nanoparticles on the external surfaces of the mof catalyst particles . importantly , heteroaromatic substrates 1 f and 1 g could also be coupled in high yields ( table 3 , entries 67 ) . however , a lower yield was obtained for 2-bromopyridine ( 1 h ; table 3 , entry 8) . slightly higher leaching of palladium was observed for substrates that contained heteroatoms : 3.10 ppm pd was found in the filtrate after the reaction of bromopyridine ( 1 g ) , whereas only 0.17 ppm was found after the coupling of compound 1 a ( see above ) . the coupling reactions of aryl chlorides required more elevated temperatures , which , disappointingly , were more favorable for the formation of byproducts ( that is , dehalogenation ( arh ) or homocoupling ( ar ar ) products ) . activated aryl chlorides 1 i and 1 j successfully reacted in water and they returned good - to - excellent yields with little or no formation of undesired byproducts ( table 3 , entries 9 and 10 ) . however , if etoh was used as a co - solvent , both activated and inactivated substrates 1 j1 m returned very good conversions but only fair yields , with the concomitant formation of significant amounts of homocoupling and dehalogenation products ( table 3 , entries 1013 ) . suzuki coupling reactions of various aryl bromides and chlorides catalyzed by 8wt % pd@mil-101cr - nh2 [ a ] unless otherwise stated , the pinacol phenylboronate ( 0.12 mmol ) , aryl halide ( 0.1 mmol ) , k2co3 ( 0.2 mmol ) , and 8 wt % pd@mil-101cr - nh2 ( 4 mg , 0.003 mmol , 3 mol % pd loading ) were suspended in either deionized water ( 2 ml ) or water / etoh ( 1:1 ) . then , the mixture was stirred vigorously at rt in air for 6 h ( water ) or 30 min ( water / etoh ) in a glass vial ( see the supporting information ) ; [ b ] yield determined by h nmr spectroscopy with 1,2,4,5-tetrachloro-3-nitrobenzene as an internal standard ; yield of the isolated product is given in parentheses ; [ c ] 2 h ; [ d ] 16 h ; [ e ] 80 c ; [ f ] 96 % conversion , 13 % homocoupling ; [ g ] 89 % conversion , 9 % homocoupling , 16 % dehalogenation ; [ h ] 68 % conversion , 13 % homocoupling ; [ i ] 89 % conversion , 12 % homocoupling , 15 % dehalogenation ; [ j ] 98 % conversion ; toluene was the main byproduct ; [ k ] 100 % conversion , 12 % homocoupling , 32 % dehalogenation ; [ l ] 44 % conversion ; anisole was the main byproduct ; [ m ] 79 % conversion , 10 % homocoupling , 23 % dehalogenation . recycling tests : the recyclability of 8 wt % pd@mil-101cr - nh2 was investigated in the reaction of p - bromotoluene . the experiments were performed on a larger scale ( 1 mmol ) than in previous investigations to facilitate separation and minimize handling errors . after each run , the solid catalyst was separated by centrifugation , washed with water and etoh ( twice each ) , dried under vacuum , and reused . as shown in figure 6 , the catalyst remained highly active , even after 10 cycles . interestingly , the pxrd pattern after the tenth cycle was different from that of mil-101cr - nh2 , but similar to that of another phase , presumably mil-88b - nh2 ( supporting information , figure s9 ) . this phase - transformation phenomenon is currently under investigation in our laboratories . tem analysis showed the presence of some limited aggregation of pd nanoparticles on the surfaces of the mof catalysts after the 10th cycle ( supporting information , figure s10 ) . because the size of the nanoparticles remained unchanged elemental analysis of the recycled 8 wt % pd@mil-101cr - nh2 material showed an overall loss of 0.96 wt % pd after 10 runs . yields of between 97 % and > 99 % were obtained in the recycling runs . based on these excellent results , we decreased the pd loading by 20 times to 0.15 mol % and performed the reactions on the same scale and under similar conditions to those in table 2 , entry 19 , and the reaction proceeded to completion within 4 h. this result indicates that pd@mil-101cr - nh2 is a highly efficient catalyst . recycling experiments on a 1 mmol scale ; reaction time of each run : 0.5 h. in summary , we have provided evidence for a strong relationship between the amount of palladium that is loaded into mil-101cr - nh2 and the catalytic properties of the corresponding system . the mil-101cr - nh2 material was found to be an excellent support for palladium nanoparticles . the size , distribution , and reactivity of palladium nanoparticles were shown to be influenced by the amount of palladium metal that was impregnated into the framework . the nanoparticles were investigated by using both conventional and advanced methods , such as electron tomography and pdf analysis . the best performance was observed for mil-101cr - nh2 that contained about 8 wt % palladium nanoparticles . this result indicates that an optimal ratio was achieved between the amount of loaded catalyst and the number of pores that remained accessible for the diffusion of the substrates and the products . aryl and heteroaryl bromides and activated aryl chlorides were successfully coupled within short reaction times under mild conditions ( ambient temperature and 80 c , respectively ) in benign solvents ( water and etoh ) . our investigations have also shown that the solubility and hydrophobicity of the substrates significantly affect their ability to access the catalyst pores in water , but this limitation is minimized by employing etoh as a co - solvent . the excellent recyclability of the 8 wt % pd@mil-101cr - nh2 catalyst makes it a promising candidate for large - scale suzuki this thorough study of the structural and catalytic properties of metallic nanoparticles that are supported on a mof material can stand as a model for detailed investigations of other related heterogeneous systems , in search for a better understanding of the underlying mechanism of heterogeneous catalysis with mofs . brentano geometry on a panalytical xpert pro diffractometer that was equipped with a pixel detector and cuk1 radiation ( =1.5406 ) . sem and energy - dispersive spectroscopy ( eds ) were performed on a jeol-7000f field - emission scanning electron microscope operating at 12 kv . tem observations were performed on a jeol tem ( jem-2100f ) operating at 200 kv . the mil-101cr samples were dispersed in absolute etoh and then treated by ultrasonication for 2 min . a droplet of the suspension was transferred onto a copper grid and dried in air . a tilt series of tem images for electron tomography was recorded with a tilt step of 0.5 and an angular range of 41 ( 83 images in total ) . ten iterations of the art algorithm were performed in tomoj with a relaxation coefficient of 0.1 . thermogravimetric analysis ( tga ) was performed under a flow of air between 20 and 500 c with a heating rate of 2 c min on a thermogravimetric analyzer ( perkin elmer tga 7 ) that was equipped with a platinum pan . the samples were degassed by heating at 120 c overnight prior to the sorption measurements . specific surface areas were calculated from the data in the adsorption branch at p / p0=0.050.30 . elemental analysis was performed on a carlo erba flash 1112 elemental analyzer and the cr and pd contents were determined by inductively coupled plasma - optical emission spectrometry ( icp - oes ) on a varian vista mpx icp - oes at medac ltd , chobham , uk . high - energy x - ray diffraction experiments were carried out at a fixed energy of approximately 60 kev at a wavelength of 0.2073 ( determined by using a ceo2 standard ) at the beamline p02.1 of petra iii , desy , hamburg . the 2d diffraction images were integrated into a linear scattering signal by using the fit2d software . the pair distribution function ( pdf ) , g(r ) , was obtained by sine fourier transform of the normalized scattering intensity to a maximum q value , qmax4=19 , by using the pdfgetx2 software . the pdf of the pd nanoparticles was calculated with the pdfgui software ( for a detailed procedure , see the supporting information ) . h nmr spectra were recorded at 400 mhz ; c nmr spectra were recorded at 100 mhz on a bruker advance spectrometer . h and c nmr chemical shifts ( ) are reported in ppm relative to tetramethylsilane , with the solvent resonance as an internal standard ( cdcl3 : h=7.26 ppm , c=77.16 ppm ; [ d6]dmso : h=2.50 ppm , c=39.5 ppm ) . high - resolution mass spectra ( hrms ) were recorded on a bruker microtof esi - tof mass spectrometer . synthesis of pd@mil-101cr - nh2 : samples of mil-101cr ( cr3f(h2o)2o[(co2)-c6h4-(co2)]3nh2o),[9 , 44 ] mil-101cr - no2 ( cr3(h2o)2o[(co2)-c6h3no2-(co2)]3nh2o ) , and mil-101cr - nh2 ( cr3(h2o)2o[(co2)-c6h3nh2-(co2)]3nh2o ) were prepared according to literature procedures . samples of pd@mil-101cr - nh2 with four different loadings of pd nanoparticles that were intended to be 4 , 8 , 12 , and 16 wt % pd were prepared by using analogous procedures , as given below . synthesis of mil-101cr - nh2-pdcl2 : for mil-101cr - nh2-pdcl2 affording 8 wt % pd@mil-101cr - nh2 , the following procedure was used : a mixture of mil-101cr - nh2 ( 0.240 g , 3.1510 mol ) , [ pdcl2(mecn)2 ] ( 0.055 g , 2.1110 mmol ) , and anhydrous ch2cl2 ( 30 ml ) was stirred overnight at rt ( 18 h ) . the solid was obtained by centrifugation , washed with anhydrous ch2cl2 ( 210 ml ) , and dried at 40 c for 2 h under vacuum . to obtain samples with 4 , 12 , and 16 wt % pd loading , the amount of [ pdcl2(mecn)2 ] that was used to afford 8 wt % pd loading was multiplied by 0.5 , 2 , and 3 , respectively ; the amount of solvent was kept the same . synthesis of 8 wt % pd@mil-101cr - nh2 : nabh4 ( 0.076 g , 2 mmol ) was added portion - wise to a mixture of mil-101cr - nh2-pdcl2 ( 0.140 g ) and etoh ( 30 ml ) at 0 c . the reaction was stirred for an additional 2 h at 0 c to complete the reduction . the solid was obtained by centrifugation , washed twice with water ( 45 ml ) and once with etoh ( 45 ml ) , and the dark - green solid was dried at 85 c overnight ( 18 h ) in air . digestion of mil-101cr : in a typical experiment , the material ( 10 mg ) was suspended in an aqueous solution of naoh ( 2.0 m , 1 ml ) and ultrasonicated for 30 min . the water was evaporated under vacuum and the residue was analyzed by h nmr spectroscopy in deuterated dmso ( supporting information , figure s1 ) . miyaura cross - coupling reactions : unless otherwise stated , pinacol phenylboronate ( 24.5 mg , 0.12 mmol ) , the aryl bromide ( 0.1 mmol ) , k2co3 ( 27.6 mg , 0.2 mmol ) , and 8 wt % pd@mil-101cr - nh2 ( 4 mg , 0.003 mmol ) were suspended in deionized water ( 2 ml ) and the mixture was stirred vigorously at rt for 6 h. then , the mixture was extracted into ch2cl2 ( 4 ml ) , the organic phase was separated , the volatile compounds were evaporated , and the residue was analyzed by h nmr spectroscopy . to isolate the final product , the reaction was repeated on a larger scale ( 4 ) and the crude reaction mixture was purified by column chromatography on silica gel ( pentane or pentane / etoac mixtures ) . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors

palladium nanoparticles have been immobilized into an amino - functionalized metal organic framework ( mof ) , mil-101cr - nh2 , to form pd@mil-101cr - nh2 . four materials with different loadings of palladium have been prepared ( denoted as 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 ) . the effects of catalyst loading and the size and distribution of the pd nanoparticles on the catalytic performance have been studied . the catalysts were characterized by using scanning electron microscopy ( sem ) , transmission electron microscopy ( tem ) , fourier - transform infrared ( ftir ) spectroscopy , powder x - ray diffraction ( pxrd ) , n2-sorption isotherms , elemental analysis , and thermogravimetric analysis ( tga ) . to better characterize the palladium nanoparticles and their distribution in mil-101cr - nh2 , electron tomography was employed to reconstruct the 3d volume of 8 wt % pd@mil-101cr - nh2 particles . the pair distribution functions ( pdfs ) of the samples were extracted from total scattering experiments using high - energy x - rays ( 60 kev ) . the catalytic activity of the four mof materials with different loadings of palladium nanoparticles was studied in the suzuki miyaura cross - coupling reaction . the best catalytic performance was obtained with the mof that contained 8 wt % palladium nanoparticles . the metallic palladium nanoparticles were homogeneously distributed , with an average size of 2.6 nm . excellent yields were obtained for a wide scope of substrates under remarkably mild conditions ( water , aerobic conditions , room temperature , catalyst loading as low as 0.15 mol % ) . the material can be recycled at least 10 times without alteration of its catalytic properties .

Introduction Results and Discussion Conclusion Experimental Section Supporting Information

within this family of reactions , the suzuki miyaura cross - coupling reaction is one of the most robust and versatile methods for the formation of the carbon backbone of a target molecule , which is of obvious relevance for further progress in various branches of this field . as such , great efforts have been made to immobilize palladium nanoparticles inside the robust mil-101cr to form pd@mil-101cr as a heterogeneous catalyst , which has been used for suzuki coupling reactions[17 , 18 ] or other palladium - catalyzed reactions , such as the synthesis of indoles through sonogashira cross - coupling followed by a cycloaddition reaction , the aqueous hydrogenation of phenols into cyclohexanones , the synthesis of methyl isobutyl ketone from acetone , the hydrogenation of ketones , and even tandem processes , as cleverly illustrated by corma and co - workers . [12 , 14 ] during our investigations , we became interested in understanding the parameters that affect the efficiency of suzuki miyaura cross - coupling reactions using pd nanoparticles that are immobilized inside mil-101cr - nh2 . herein , we report our investigations into the potential and limitations of mil-101cr - nh2 as a catalyst support of pd nanoparticles and its application in suzuki miyaura cross - coupling reactions . tem and total scattering techniques were employed to study the pd nanoparticles in the mil-101cr - nh2 crystals . the catalyst with the optimized amount of pd ( 8 wt % loaded ) in mil-101cr - nh2 was subjected to a systematic screening of the reaction parameters , thereby leading to efficient suzuki miyaura coupling reactions , with excellent yields for a large substrate scope , including challenging heteroaromatic halides and bulky substrates , under environmentally friendly conditions ( water or water / etoh , ambient temperature , k2co3 ) . in the second step , the quantitative functionalization of mil-101cr was confirmed by both elemental analysis and by h nmr spectroscopy after the digestion of mil-101cr - no2 and mil-101cr - nh2 ( see the supporting information , figure s1 , and the experimental section ) . palladium loadings of 4 , 8 , 12 , and 16 wt % were prepared by the impregnation of mil-101cr - nh2 with the corresponding amount of [ pdcl2(mecn)2 ] in dry ch2cl2 overnight , thus yielding pd@mil-101cr - nh2 , in which pd was coordinated to the amino groups . the purity of the four final materials ( 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 ) was checked by energy - dispersive spectroscopy ( eds ) that was connected to a sem , elemental analysis , and by inductively coupled plasma - optical emission spectrometry ( icp - oes ; supporting information , table s2 ) . the actual pd contents , as determined by icp - oes , for the samples of 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 were 4.38 , 8.06 , 11.29 , and 15.07 wt % , respectively . the structure of mil-101cr remained intact after all of the postsynthetic modification steps and no obvious changes in the pxrd patterns were detected , which confirmed that mil-101cr - nh2 was chemically and hydrothermally stable and that it could withstand harsh conditions , such as those applied during the nitration step ( with mixtures of concentrated acids ) and during the reduction of pd by using excess nabh4 ( figure 1 ) . pxrd patterns of mil-101cr , mil-101cr - no2 , mil-101cr - nh2 , and 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 . n2-sorption measurements were performed for as - synthesized mil-101cr , amino - functionalized mil-101cr - nh2 , and 4- , 8- , 12- , and 16 wt % pd@mil-101cr the n2-adsorption isotherms , the bet surface areas , and the pore volumes are shown in figure 2 and table 1 . nitrogen - adsorption isotherms of mil-101cr , mil-101cr - nh2 , and 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 . however , on increasing the pd loading in mil-101cr - nh2 from 11.3 to 15.1 wt % , the size of the pd nanoparticles increased up to about 8 nm ( figure 3 ) . these larger pd nanoparticles were found on the surfaces of the 16 wt % pd@mil-101cr - nh2 crystals , as shown by tem . electron tomography was performed on crystals of 8 wt % pd@mil-101cr - nh2 to study the distribution of the pd nanoparticles . electron diffraction and high - resolution transmission electron microscopy ( hrtem ) images show that the pd nanoparticles are crystalline ( supporting information , figure s6 ) . tem images of samples of 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 with different pd loadings ( 4.4 , 8.1 , 11.3 , and 15.1 wt % pd , respectively ) . the x axis in the histograms 2d section of the 3d tomographic image of two pd@mil-101cr - nh2 crystals with 8 wt % pd loading , as reconstructed by the electron tomography from a tilt series of 83 tem images with a tilt range of 41 and a tilt step of 0.5. for the tilt series and the full tomogram , see the supporting information , movies s1 and s2 , respectively . a better technique to study the size and distribution of the pd nanoparticles is x - ray total scattering , because tem only gives some local information and does not necessarily represent the nature of the bulk samples . owing to their small particle sizes and the low concentrations of the pd nanoparticles , no bragg peaks for the pd nanoparticles could be clearly seen in the pxrd patterns that were recorded on an in - house powder x - ray diffractometer . therefore , we recorded the total scattering intensities of the 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 samples at the beamline p02.1 , petra iii , desy , by using a high - energy synchrotron source ( 60 kev ) . the pair distribution function ( pdf ) , g(r ) , was obtained by performing a fourier transform of the total scattering intensities , including both bragg peaks and diffuse scattering . the pdfs of the 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 samples were extracted from the total x - ray scattering signal truncated at a maximum q value ( qmax ) of 19 ( figure 5 ; supporting information , figure s7 ) . the peaks in the observed pdfs of the 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 samples correspond to the interatomic distances in both mil-101cr and the pd nanoparticle . in case of multielement samples , such as mil-101cr and 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 , the peak intensities in the pdf are multiplied by a weighting factor to take into account the different scattering factors of the chemical species . thus , in the observed pdfs of 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 , the peak intensities can be related to the multiplicity and abundance of the atomic pairs , as well as to atomic scattering factors . by comparing the pdfs of the 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 samples ( figure 5 ) , a change in the relative peak intensities can be observed ; in particular , with higher pd loading , the intensity of the peak at 2.9 , which corresponds to the pd pd distance , increases in comparison with that of the peak at 2.0 , which is assigned to the cr o distance . when the pdfs of the 12- and 16 wt % pd@mil-101cr - nh2 samples are compared to the pdf of the 25 pd nanoparticles ( calculated ) , one can see that the peaks at r>4.5 mainly originate from the pd nanoparticles . in fact , the pdf of 16 wt % pd@mil-101cr - nh2 can be almost fully described by the structure of the pd nanoparticles with an average size of 25 . this result indicates that the size of the pd nanoparticles in 16 wt % pd@mil-101cr - nh2 is slightly larger than that in 12 wt % pd@mil-101cr - nh2 . on the other hand , the pdfs of the 4- and 8 wt % pd@mil-101cr - nh2 samples are very similar at r>4.5 and attenuate at much lower r values compared to the pdfs of 12- and 16 wt % pd@mil-101cr - nh2 . this result indicates that the sizes of the pd particles are similar in 4- and 8 wt % pd@mil-101cr - nh2 and are smaller than those in 12- and 16 wt % pd@mil-101cr - nh2 . because no peaks are observed at large r values ( > 20 ) , it can be concluded that there is no significant agglomeration of pd nanoparticles in the four pd@mil-101cr - nh2 samples . wt % pd@mil-101cr - nh2 , compared with the calculated pdfs of mil-101cr ( bottom ) and of a pd nanoparticle ( diameter : 25 , top ) . miyaura cross - coupling reaction with pd@mil-101cr - nh2 : in an initial series of reactions , we screened the reactivity of p - bromoanisol ( 1 a ) with different organoboron reagents and bases ( table 2 ) . based on the characterization of the different catalysts , screening of the reaction conditions was performed with 8 wt % pd@mil-101cr - nh2 ( 3 mol % pd loading ) . in contrast to 12- and 16 wt % pd@mil-101cr - nh2 , the 8 wt % catalyst did not show agglomeration of palladium on its surface . moreover , 8 wt % pd@mil-101cr - nh2 contained a more favorable wt % pd per gram of material than 4 wt % pd@mil-101cr - nh2 , which is desirable from an atom - economy viewpoint . miyaura coupling reactions catalyzed by pd@mil-101cr - nh2 ; optimization of the reaction conditions [ a ] unless otherwise stated , the organoboron reagent ( 0.12 or 0.15 mmol ) , aryl halide ( 0.1 mmol ) , base ( 0.2 mmol ) , and 8 wt % pd@mil-101cr - nh2 ( 4 mg , 0.003 mmol , 3 mol % pd loading ) were suspended in deionized water ( 2 ml ) . , dehalogenation ) ; [ d ] 4 wt % pd@mil-101cr - nh2 ( 3 mol % pd loading ) ; [ e ] 12 wt % pd@mil-101cr - nh2 ( 3 mol % pd loading ) ; [ f ] 16 wt % pd@mil-101cr - nh2 ( 3 mol % pd loading ) . with these arguments in mind , we screened a wide selection of bases and observed the stability of the 8 wt % pd@mil-101cr - nh2 catalyst , as shown in table 2 . furthermore , if a chelating base ( ethylenediamine ) was used ( table 2 , entry 11 ) , all of the palladium was extracted from the support and the material lost its catalytic activity . comparison of pd@mil-101cr - nh2 catalysts with different pd loadings : with the optimized conditions in hand , we compared the activities of the 4- , 8- , 12- , and 16 wt % pd@mil-101cr - nh2 catalysts ( table 2 , entries 2023 ) . whereas the reactions with 4- and 8 wt % pd@mil-101cr - nh2 afforded very similar and satisfactory yields ( table 2 , entries 20 and 21 ) , the reactions with 12- and 16 wt % pd@mil-101cr - nh2 afforded significantly lower yields ( table 2 , entries 22 and 23 ) . this behavior was in agreement with the tem and pdf analyses , which showed that the nanoparticles in the 12- and 16 wt % pd@mil-101cr - nh2 catalysts were larger with broader size distributions . together with the high recyclability of the catalyst ( see below ) , these results clearly demonstrate the stability of the pd nanoparticles in mil-101cr - nh2 . substrate scope : based on this series of observations ( table 2 ) , we screened a variety of substrates with 8 wt % pd@mil-101cr - nh2 as the catalyst ( 3 mol % pd loading ) and k2co3 as the base , because k2co3 is environmentally benign and about 20-times cheaper than cs2co3 . this result may be due to the large size of this substrate , which results in limited access to 8 wt % pd@mil-101cr - nh2 , and the catalysis mainly occurred at the pd nanoparticles on the external surfaces of the mof catalyst particles . suzuki coupling reactions of various aryl bromides and chlorides catalyzed by 8wt % pd@mil-101cr - nh2 [ a ] unless otherwise stated , the pinacol phenylboronate ( 0.12 mmol ) , aryl halide ( 0.1 mmol ) , k2co3 ( 0.2 mmol ) , and 8 wt % pd@mil-101cr - nh2 ( 4 mg , 0.003 mmol , 3 mol % pd loading ) were suspended in either deionized water ( 2 ml ) or water / etoh ( 1:1 ) . recycling tests : the recyclability of 8 wt % pd@mil-101cr - nh2 was investigated in the reaction of p - bromotoluene . because the size of the nanoparticles remained unchanged elemental analysis of the recycled 8 wt % pd@mil-101cr - nh2 material showed an overall loss of 0.96 wt % pd after 10 runs . based on these excellent results , we decreased the pd loading by 20 times to 0.15 mol % and performed the reactions on the same scale and under similar conditions to those in table 2 , entry 19 , and the reaction proceeded to completion within 4 h. this result indicates that pd@mil-101cr - nh2 is a highly efficient catalyst . recycling experiments on a 1 mmol scale ; reaction time of each run : 0.5 h. in summary , we have provided evidence for a strong relationship between the amount of palladium that is loaded into mil-101cr - nh2 and the catalytic properties of the corresponding system . the best performance was observed for mil-101cr - nh2 that contained about 8 wt % palladium nanoparticles . the excellent recyclability of the 8 wt % pd@mil-101cr - nh2 catalyst makes it a promising candidate for large - scale suzuki this thorough study of the structural and catalytic properties of metallic nanoparticles that are supported on a mof material can stand as a model for detailed investigations of other related heterogeneous systems , in search for a better understanding of the underlying mechanism of heterogeneous catalysis with mofs . high - energy x - ray diffraction experiments were carried out at a fixed energy of approximately 60 kev at a wavelength of 0.2073 ( determined by using a ceo2 standard ) at the beamline p02.1 of petra iii , desy , hamburg . the pair distribution function ( pdf ) , g(r ) , was obtained by sine fourier transform of the normalized scattering intensity to a maximum q value , qmax4=19 , by using the pdfgetx2 software . the pdf of the pd nanoparticles was calculated with the pdfgui software ( for a detailed procedure , see the supporting information ) . samples of pd@mil-101cr - nh2 with four different loadings of pd nanoparticles that were intended to be 4 , 8 , 12 , and 16 wt % pd were prepared by using analogous procedures , as given below . synthesis of mil-101cr - nh2-pdcl2 : for mil-101cr - nh2-pdcl2 affording 8 wt % pd@mil-101cr - nh2 , the following procedure was used : a mixture of mil-101cr - nh2 ( 0.240 g , 3.1510 mol ) , [ pdcl2(mecn)2 ] ( 0.055 g , 2.1110 mmol ) , and anhydrous ch2cl2 ( 30 ml ) was stirred overnight at rt ( 18 h ) . synthesis of 8 wt % pd@mil-101cr - nh2 : nabh4 ( 0.076 g , 2 mmol ) was added portion - wise to a mixture of mil-101cr - nh2-pdcl2 ( 0.140 g ) and etoh ( 30 ml ) at 0 c . miyaura cross - coupling reactions : unless otherwise stated , pinacol phenylboronate ( 24.5 mg , 0.12 mmol ) , the aryl bromide ( 0.1 mmol ) , k2co3 ( 27.6 mg , 0.2 mmol ) , and 8 wt % pd@mil-101cr - nh2 ( 4 mg , 0.003 mmol ) were suspended in deionized water ( 2 ml ) and the mixture was stirred vigorously at rt for 6 h. then , the mixture was extracted into ch2cl2 ( 4 ml ) , the organic phase was separated , the volatile compounds were evaporated , and the residue was analyzed by h nmr spectroscopy .

hepatitis c virus ( hcv ) infection affects over 185 million individuals worldwide and approximately 5.2 million in the us . hcv is a major cause of severe disease in the us , with mortality rates of 4.7 per 100,000 reported in 2010 [ 14 ] . approximately 70% of people infected with hcv develop chronic infection and 30% are known to spontaneously clear the infection ; however , these rates vary by ancestry , gender , and other factors [ 57 ] . chronic infection with hcv results in hepatic inflammation , microfibrosis and may ultimately cause cirrhosis , hepatocellular carcinoma , and death due to liver failure . prior to december 2013 , the standard of care for hcv consisted of 2448 weeks of pegylated interferon ( peg - ifn ) and ribavirin ( rbv ) with a hcv protease inhibitor added for genotype 1 infection [ 8 , 9 ] . after december 2013 , interferon - free regimens have been approved with directly acting antivirals ( daas ) . treatment outcomes have been traditionally defined as either nonresponse ( nr ) ( which includes null response and early relapse ) or sustained virologic response ( svr ) defined as a persistent undetectable serum hcv rna or viral load 24 weeks after the conclusion of treatment [ 8 , 9 ] . nr is considered a treatment failure , whereas svr has been considered a functional cure and has been associated with improved clinical outcomes . however , an estimated 3% of patients will relapse after achieving svr with reemergence of the same virus after peg - ifn / rbv treatment . peripheral blood mononuclear cells ( pbmcs ) have been cited as a possible viral reservoir with previous studies suggesting that 926% of svr patients may have residual virus in pbmcs [ 1319 ] . however , hcv infection of pbmcs is controversial , with some studies demonstrating that there may not be true replicative infection within pbmcs and , rather , that the presence of viral rna may be due circulating virions in the serum passively diffusing into cells [ 20 , 21 ] . it has been demonstrated that genes involved in the inflammatory response are dysregulated in the setting of chronic hcv infection and liver disease with resulting differences in circulating cytokine profiles [ 22 , 23 ] . however , it is an open question how durable these changes are after interferon treatment . a prior study by our group demonstrated that patients with svr had significantly different serum cytokine profiles compared to patients exposed to hcv but with no evidence of ever having clinical infection ( spontaneous clearance , sc ) . prior studies have examined gene expression of a limited number of genes in pbmcs using pcr - based assays in svr patients [ 24 , 25 ] . newer technologies employ multiplex oligonucleotide based arrays to directly quantify and count copy numbers in a sample without the variation inherent in techniques that require nucleic acid amplification [ 26 , 27 ] . we sought to determine if there was a persistent inflammatory response in patients who attained svr after interferon treatment . to this end , we used a multiplex array to investigate pbmc gene expression in patients previously treated for hcv to determine if differences in mrna transcription persisted despite successful treatment . the study was approved by stanford university institutional review board and was conducted under guidelines established by the declaration of helsinki . written informed consent was obtained from all patients . we performed a cross - sectional study of patients with a history of hcv infection from the veterans affairs palo alto health care system ( vapahcs ) . potential study participants were identified from a clinical case registry of previously hcv antibody tested and/or treated patients at the vapahcs and were recruited into the study from june 2010 to september 2014 . the first arm included patients with hcv infection who had completed interferon - containing hcv treatment between 6 months and 5 years prior to study enrollment . these patients were categorized as either svr or rebound / relapse / nonresponse ( nr ) . svr patients were defined as having an undetectable hcv viral load ( abbott realtime hcv , abbott molecular , des plaines , il ) at least 24 weeks after completing therapy . nr patients were defined as any patient who received hcv therapy and either never had an undetectable hcv viral load or had a recurrence of viremia either during or subsequent to treatment . also included were vapahcs patients who were exposed to hcv and spontaneously cleared the virus ( sc ) . sc patients were defined as antibody positive by commercial assay ( architect anti - hcv assay , abbott molecular , des plaines , il ) but who had 2 undetectable hcv rna viral load results at least 1 month apart without any treatment . patients who had hiv coinfection , were on any systemic immunomodulatory medications ( including glucocorticoid , anti - tnf- antibodies , and antineoplastic agents ) , and could not attend study visits , or from whom sufficient human nucleic acid could not be isolated for analysis , were excluded . both medical records and patient interview were used to obtain additional information on age , medication use , and concurrent medical conditions . to approximate the stage of liver disease , the fibrosis-4 ( fib-4 ) score as described by vallet - pichard et al . was calculated using each patient 's age , aspartate aminotransferase ( ast ) and alanine transaminase ( alt ) serum levels , and platelet count . any patient with a fib-4 score greater than 3.25 was considered to have significant fibrosis comparable to a fibrotest score of f3-f4 . an age adjusted charlson comorbidity index score was also calculated for each person . for the purposes of the charlson score , all patients with prior hcv , except for those with a fib-4 score greater than 3.25 , were identified as having mild liver disease based on the fact that they were all chronically infected with hcv . those with a fib-4 score greater than 3.25 were identified as having moderate to severe liver disease . other conditions identified in one or more patients included diabetes , chronic obstructive pulmonary disease , connective tissue disease , peripheral vascular disease , lymphoma , any tumor , myocardial infarction , and congestive heart failure . data on statin and systemic prescription nonsteroidal anti - inflammatory drugs ( nsaid ) use at the time of the blood draw was collected and tabulated . over - the - counter nsaid use could not accurately be accounted for in all cases , so it was not included . all patients included in the analysis had at least one blood draw at the time of enrollment . a smaller subset of patients in the svr and nr groups underwent a second blood draw 36 weeks later as an internal control to ensure that study measurements were stable over time . pbmcs were isolated from blood collected in cell preparation tubes ( cpt ) ( becton , dickinson and company , franklin lakes , nj ) per manufacturer instructions and stored at 80c . total rna was extracted from 2.0 105 10 cells using allprep dna / rna mini kits ( qiagen , valencia , ca ) as per manufacturer instructions and placed into 50 l of rnase - free water . concentration of total rna was measured by spectrophotometry and then concentrated or diluted to a target concentration of between 20 and 60 ng/l . gene expression was directly measured via counts of corresponding messenger rna ( mrna ) in each sample using an ncounter ( nanostring , seattle , wa ) gx human inflammation kit , which is a multiplex assay for 184 genes involved in the human inflammatory response ( listed in supplemental table 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/958231 ) . the ncounter system allows for direct detection and counting of nucleic acid via reporter probes appended with multiple fluorophore barcodes and biotinylated capture - probes that attach to microscopic beads , which are then affixed to lanes in a translucent cartridge and read in an optical scanner . batches of 12 separate samples at one time were prepared as per manufacturer instructions , with 100300 ng of total rna hybridized with probes at 65c for 1618 hours before being placed into the automated ncounter prep station ( nanostring ) in which samples were affixed to cartridges . cartridges were then immediately placed into the ncounter digital analyzer ( nanostring ) optical scanner and read at a goal resolution of 550 fields of view ( fov ) , which is the maximum resolution for this instrument . the raw nanostring gene expression data were normalized using negative controls , positive controls , and housekeeping genes via nsolver version 2.0 software ( nanostring ) . the arithmetic mean plus 2 standard deviations ( sds ) of the internal negative controls in each sample was subtracted from the gene expression count to ensure that any nonspecific mrna detection was excluded . the geometric mean of the 6 internal positive controls was used to normalize the data so that comparisons could be made across samples and to minimize distortion from batch effects . gene expressions were then normalized using the geometric mean of 5 housekeeping genes ( gapdh , cltc , hprt1 , pgk1 , and tubb ) chosen due to their consistent expression in pbmc . batch effects were assessed with a confirmatory mixed batch of samples selected from each prior run with adjustments made as appropriate . the in - group means of gene expression count were compared via two - tailed pairwise analysis with nonparametric distribution assumed . given the number of simultaneous tests and an expected increase in type 1 error , a bonferroni corrected threshold p value of < 0.001 was used to indicate significance . age , fib-4 , age adjusted charlson index scores , and time since treatment were compared between groups with kruskal - wallis test for nonparametric data . chi - squared tests were performed for comparison of categorical data such as sex , race , and hcv genotype . in the final analysis , 55 patients ( 18 nr , 22 svr , and 15 sc patients ) met both inclusion and exclusion criteria and were included in the study . after an initial database search and contact by mail , 79 patients were initially screened by phone for inclusion in the study . of those , 3 patients were excluded due to refusal to participate , 5 patients were excluded due to receipt of interferon prior to the study period ( before 12/2008 ) , 8 were unable to participate in their initial visit , 1 patient was consented but was unable to complete blood draw , 1 patient was consented but rna was not recovered from his samples , 3 patients were excluded due to the presence of immunomodulatory medications , and 3 patients were excluded due to the presence of hiv . demographic and clinically relevant data including hcv genotype , fib-4 and age adjusted charlson comorbidity scores , and statin or nsaid use are reported in table 1 . the sc population was significantly younger than the other two groups , with a median age of 58 years compared to 62 and 63 years ( p = 0.047 ) . due to vapahcs patient demographics , whites and males were overrepresented in this study when compared to the overall us hcv population , although there were no significant differences between groups . as expected , fib-4 scores were significantly higher in the nr group compared to sc or svr ( p = 0.001 ) . age adjusted charlson comorbidity scores were significantly different between the three groups ( p < 0.001 ) ; however , the difference appeared to be driven primarily by the amount of nonsevere liver disease ( 1 point ) in the svr group and cirrhotic liver disease in the nr group ( 3 points ) . rates of prescription statin and nsaid use were similar between groups ( p = 0.761 and 0.948 , resp . ) . between the svr and nr group , there was a significantly higher percentage of genotype 1 patients in the svr group ( p = 0.027 ) , which was unexpected and likely due to local treatment patterns in the last 5 years at vapahcs after the introduction of hcv protease inhibitors in 2011 . time from last interferon treatment ( median 2.0 years in svr group , 2.6 years in nr , p = 0.348 ) and type of treatment ( p = 0.116 ) were not significantly different between groups . as many inflammatory mediators are not primarily transcribed in pbmc , not all genes assessed had significant transcription as detected by our primary assay . of 184 inflammatory mediator genes assessed , 127 had significant expression levels ( mean copy numbers > 10 in any group ) for reliable detection after normalization with negative controls , positive controls , and housekeeping genes and were included in further analysis . this null expression group included important inflammatory mediators primarily expressed in neutrophils , endothelial cells , tissue based macrophages , or other tissues , including il-1a , il-4 , il-6 , and interferons and . a full list of assessed genes and expression is available in supplemental table 1 . when analyzed as individual samples , it was observed that overall pattern of gene expression between svr and nr patients was highly similar , while sc patients differed from the other two groups . a heat map of gene expression with clustering using a spearman correlation is shown in figure 1 and is notable for the uniform clustering of sc patients together . it is of note that the best - fit line for mean expression of svr genes maps precisely onto that of nr genes with an r of 0.98 , while the correlation between svr and sc gene expression was reduced with an r of 0.88 . using a significance threshold of < 0.001 ( corrected for multiple testing ) , mean mrna counts were significantly different for 42 genes when comparing svr to sc patients , 29 genes comparing nr to sc , and no genes comparing svr to nr . differential expression of 24 genes was significantly different in both svr and nr groups when compared to sc and is shown in tables 2 and 3 . expression of 12 genes was upregulated and expression of 12 genes was downregulated . a full data set is available in supplementary table 2 . specific expression patterns were suggestive of increased transcription factor activation for promoters of cellular proliferation in pbmcs and resulting innate immune activity in nr and svr patients versus sc patients . although all 24 genes that were differentially expressed in the same direction in nr and svr patients versus sc patients are potentially important inflammatory cascade regulators and effectors , a few of these are worth special mention as they may be of particular importance in interferon mediated immune response . it is of note that ap-1 components fos ( approximately 3-fold higher in both svr and nr versus sc , p < 0.001 for both ) and jun ( 2.2-fold and 2.4-fold in both svr and nr versus sc , p = 0.054 and 0.028 , resp . ) were both elevated as was cebpb ( ccaat / enhancer - binding protein beta ) ( 1.9-fold and 1.8-fold in both svr and nr versus sc , resp . ap-1 is a known transcription factor for toll - like receptor 4 ( tlr4 ) , which had a mild but nonsignificant elevation of transcription in the svr and nr groups compared to sc ( fold changes of 1.2 and 1.2 , p = 0.04 and 0.02 , resp . ) . significant elevations in transcription of tlr4 downstream effector myeloid differentiation primary response gene 88 ( myd88 ) were also seen ( fold changes of 1.2 and 1.6 , resp . , p < 0.001 for both ) [ 30 , 31 ] . the anti - inflammatory cytokine gene il-10 was not highly expressed in any group ( counts < 25 in all groups , no transcription in sc ) . however , there was a significant elevation in both svr and nr versus sc , perhaps consistent with feedback regulation from stimulation of the myd88 pathway . it is of note that some transcription factors that favor an adaptive response including histone deacetylase 4 ( hdac4 ) were downregulated in svr and nr compared to the sc group ( hdac4 fold changes of 1.4 and 1.3 , resp . , p < 0.001 for both ) we found that the pattern of pbmc gene expression in svr patients , who received peg - ifn treatment a median of 2 years prior , more closely resembles nr patients with active hcv infection than sc patients who have been exposed to hcv but are uninfected and who have no history of chronic viral infection or prolonged peg - ifn exposure . given that there is ongoing viremia in nr patients and svr patients have achieved clinical cure , it may be expected that nr patients would have a different pattern of expression in pbmcs from svr patients ; however , we found no difference . this indicates that there is a durable inflammatory transcriptional response in pbmcs that persists long after treatment . the specific pattern of gene expression demonstrates that genes related to innate immune activation such as fos , cebpb , and myd88 are more transcriptionally active in both svr and chronically viremic patients , while adaptive immune mediators such as hdac4 are downregulated [ 3033 ] . the pattern of activation found in this data can be understood in the light of the findings of dill et al . who demonstrated in liver tissue that although the jak / stat pathway is transiently activated in response to pegylated ifn- , other secondary pathways soon become the main mediators of effector response after sustained therapy and maybe responsible for the benefit of the pegylated form of the molecule . a potential explanation for this pattern of activation may be liver disease itself and prior injury from chronic hcv infection . lack of relative improvement in fibrosis after treatment may be an important predictor of outcomes in svr patients . gene expression of macrophages from liver tissue has demonstrated upregulation of chemotactic factors and proinflammatory cytokines such as il-8 in prior studies . however , in this study population , the fibrosis scores of svr patients were similar to those of sc patients and significantly different from chronically hcv infected nr patients , indicating that continued hepatic injury is most likely not occurring . this is also consistent with what is known about liver disease outcomes in svr patients as 5-year mortality and progression to cirrhosis drop significantly . pairing peripheral pbmc gene expression data with concurrent gene expression from liver macrophages along with histology from liver biopsy samples may be a useful line of further investigation . another possible explanation is a durable effect from long - term exogenous peg - ifn exposure . however , hcv infected patients who achieve svr receive much longer exposure to exogenous peg - ifn therapy than any other patient group , and this pattern of activation may mimic chronic viral infection , particularly when compared to nr group that also had an extended duration ( although significantly less than the svr group ) of exogenous peg - ifn . the addition of a treatment naive chronically hcv infected comparison group may be useful in future studies to explore this possibility . a similar study in patients who receive interferon - free treatment regimens and obtain svr may also resolve this hypothesis . the explanation for this data with the most salient clinical consequences is either intra- or extrahepatic reservoirs of residual hcv infection . as demonstrated by hara et al . , late relapse in many cases is likely due to reemergence of prior infection rather than reinfection with a different strain . as noted above , several studies have investigated the possibility of persistent viral reservoirs , including pbmcs themselves , although true infection of pbmc is controversial [ 1321 ] . two previous studies have used rt - pcr to assay pbmc gene expression in svr patients and postulated residual infection as the etiology . pham et al . assessed transcripts of 9 cytokines in 22 chronically hcv infected patients and 29 patients with resolved disease compared with 15 healthy controls and found significant elevations in ifn- and tnf- in resolved patients versus chronically infected patients . however , the resolved group included both svr and sc patients grouped together , and the chronic infection group included both treatment experienced and treatment naive patients . our study found negligible expression of ifn- transcripts and no significant difference between groups in tnf-. radkowski et al . analyzed 25 cytokine transcripts in 49 patients with svr divided into a group with residual infection as defined by very low levels of hcv in serum or pbmc detected with a home - brew ultrasensitive rt - pcr assay or by presence of ns3 protein via immunofluorescence in pbmc versus those without . there was a high rate ( 46.9% ) of residual viremia found in this study , and in those patients there were significant elevations in il-6 , il-8 , il-12,tnf- , and mip-1 ( ccl4 ) when compared to svr patients without evidence of residual virus . our study did not find significant differences between groups in the transcription of il-12 , tnf- , or ccl4 and did not detect significant transcription of il-6 in any group . we did find , however , that il-8 appeared upregulated in both svr and nr patients compared to sc with fold changes of 7.49 and 5.73 ( p = 0.005 and 0.08 , resp . ) , although these changes did not reach the threshold for significance in the final analysis . our study differs from prior work in several ways , using an entirely different methodology to assay gene expression of many more genes simultaneously as well as a fundamentally different way of defining comparison groups , which may explain differences in results . ours is the only study to assay expression of upstream genes whose products regulate overall transcription patterns rather than only transcripts for effector cytokines , which are not always reliably expressed in pbmcs as opposed to other tissue compartments . the demographics of our patient population are not representative of the us hcv infected population at large in terms of gender or race ; therefore , generalizability may be limited . the small numbers in each comparison group may not have allowed sufficient power to show a difference and increased the likelihood of type ii error . as with any multiplex detection system reliant on binding to probes , uniform annealing conditions across 184 probe sets with different sequences may lead to falsely higher counts in probe / target sets with higher affinity and falsely lower or null counts in probe / target sets with lower affinity . another important limitation is that the time since infection for the sc patients is unknown . this data is not obtainable due to the fact that acute infection with hcv is most often asymptomatic and a positive hcv antibody with negative serum hcv rna pcr was identified in our sc sample on routine screening for at risk populations after the acute phase of infection . there is also a large distribution ( 6 months to 5 years ) in the time since treatment for both the nr and svr patients ; however , the median time since treatment is not significantly different between groups ; therefore , we believe these groups are comparable . in addition , our study is limited by assaying the total blood pbmc population as opposed to specific mononuclear cell subpopulations or liver tissue where important changes may be taking place . however , we believe changes in pbmc mrna expression are important for our understanding of the systemic immune response after hcv treatment . further investigation is warranted to explain why interferon treated patients who obtain svr and a functional cure have a similar pattern of macrophage and lymphocyte gene expression compared to those with chronic hcv infection who did not respond to treatment and also had prior exogenous interferon exposure . this is an important question in the daa era of hcv therapy , as retreatment is now possible for patients with late relapse , and further monitoring of peg - ifn treated patients with svr may be useful to identify late relapse . this may also be clinically important for monitoring peg - ifn treated patients for long - term consequences of prior infection such as progression to cirrhosis and hcc as well as immunogenic consequences of hcv infection such as cryoglobulinemia and porphyria cutanea tarda . further studies investigating the same phenomenon in patients treated with interferon - free regimens are warranted . patients who achieved clinical cure of hcv infection with peg - ifn based regimens appear to have pbmc gene expression patterns that closely resemble chronically hcv infected patients who have failed treatment and are markedly different from patients who spontaneously clear their hcv infection . it is therefore reasonable to conclude that an inflammatory response persists in pbmcs long after successful peg - ifn treatment for hcv infection .

background . inflammatory gene expression in peripheral blood mononuclear cells ( pbmcs ) is altered in chronic hepatitis c virus ( hcv ) infection . duration of changes after pegylated interferon- ( peg - ifn- ) based hcv treatment is unclear . methods . pbmc mrna expression of 184 inflammatory response genes was analyzed ( ncounter gx human inflammation kit , nanostring ) from peg - ifn treatment nonresponders ( nr , n = 18 ) , sustained virologic responders ( svr , n = 22 ) , and spontaneous clearers ( sc , n = 15 ) . logistic regression was used for comparison . results . median time from last treatment was 2 and 2.7 years in svr and nr , respectively ( p = ns ) . mean mrna counts were significantly different for 42 and 29 genes comparing svr to sc patients and nr to sc , respectively , and no genes comparing svr to nr . differential expression of 24 genes was significantly different in both svr and nr groups compared to sc . among these 24 acute and chronic inflammatory cascade genes , significant upregulation was noted for proinflammatory transcription regulators fos , cebpb , and myd88 in svr and nr compared to sc . hdac4 was significantly downregulated in svr and nr compared to the sc group . conclusions . pbmc inflammatory gene expression patterns in svr resemble nr more than sc patients . a generalized inflammatory response persists in pbmcs long after successful peg - ifn treatment for hcv infection .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

hepatitis c virus ( hcv ) infection affects over 185 million individuals worldwide and approximately 5.2 million in the us . approximately 70% of people infected with hcv develop chronic infection and 30% are known to spontaneously clear the infection ; however , these rates vary by ancestry , gender , and other factors [ 57 ] . chronic infection with hcv results in hepatic inflammation , microfibrosis and may ultimately cause cirrhosis , hepatocellular carcinoma , and death due to liver failure . prior to december 2013 , the standard of care for hcv consisted of 2448 weeks of pegylated interferon ( peg - ifn ) and ribavirin ( rbv ) with a hcv protease inhibitor added for genotype 1 infection [ 8 , 9 ] . after december 2013 , interferon - free regimens have been approved with directly acting antivirals ( daas ) . treatment outcomes have been traditionally defined as either nonresponse ( nr ) ( which includes null response and early relapse ) or sustained virologic response ( svr ) defined as a persistent undetectable serum hcv rna or viral load 24 weeks after the conclusion of treatment [ 8 , 9 ] . however , an estimated 3% of patients will relapse after achieving svr with reemergence of the same virus after peg - ifn / rbv treatment . peripheral blood mononuclear cells ( pbmcs ) have been cited as a possible viral reservoir with previous studies suggesting that 926% of svr patients may have residual virus in pbmcs [ 1319 ] . however , hcv infection of pbmcs is controversial , with some studies demonstrating that there may not be true replicative infection within pbmcs and , rather , that the presence of viral rna may be due circulating virions in the serum passively diffusing into cells [ 20 , 21 ] . it has been demonstrated that genes involved in the inflammatory response are dysregulated in the setting of chronic hcv infection and liver disease with resulting differences in circulating cytokine profiles [ 22 , 23 ] . a prior study by our group demonstrated that patients with svr had significantly different serum cytokine profiles compared to patients exposed to hcv but with no evidence of ever having clinical infection ( spontaneous clearance , sc ) . prior studies have examined gene expression of a limited number of genes in pbmcs using pcr - based assays in svr patients [ 24 , 25 ] . we sought to determine if there was a persistent inflammatory response in patients who attained svr after interferon treatment . to this end , we used a multiplex array to investigate pbmc gene expression in patients previously treated for hcv to determine if differences in mrna transcription persisted despite successful treatment . we performed a cross - sectional study of patients with a history of hcv infection from the veterans affairs palo alto health care system ( vapahcs ) . the first arm included patients with hcv infection who had completed interferon - containing hcv treatment between 6 months and 5 years prior to study enrollment . these patients were categorized as either svr or rebound / relapse / nonresponse ( nr ) . nr patients were defined as any patient who received hcv therapy and either never had an undetectable hcv viral load or had a recurrence of viremia either during or subsequent to treatment . also included were vapahcs patients who were exposed to hcv and spontaneously cleared the virus ( sc ) . sc patients were defined as antibody positive by commercial assay ( architect anti - hcv assay , abbott molecular , des plaines , il ) but who had 2 undetectable hcv rna viral load results at least 1 month apart without any treatment . patients who had hiv coinfection , were on any systemic immunomodulatory medications ( including glucocorticoid , anti - tnf- antibodies , and antineoplastic agents ) , and could not attend study visits , or from whom sufficient human nucleic acid could not be isolated for analysis , were excluded . both medical records and patient interview were used to obtain additional information on age , medication use , and concurrent medical conditions . was calculated using each patient 's age , aspartate aminotransferase ( ast ) and alanine transaminase ( alt ) serum levels , and platelet count . for the purposes of the charlson score , all patients with prior hcv , except for those with a fib-4 score greater than 3.25 , were identified as having mild liver disease based on the fact that they were all chronically infected with hcv . other conditions identified in one or more patients included diabetes , chronic obstructive pulmonary disease , connective tissue disease , peripheral vascular disease , lymphoma , any tumor , myocardial infarction , and congestive heart failure . all patients included in the analysis had at least one blood draw at the time of enrollment . a smaller subset of patients in the svr and nr groups underwent a second blood draw 36 weeks later as an internal control to ensure that study measurements were stable over time . gene expression was directly measured via counts of corresponding messenger rna ( mrna ) in each sample using an ncounter ( nanostring , seattle , wa ) gx human inflammation kit , which is a multiplex assay for 184 genes involved in the human inflammatory response ( listed in supplemental table 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/958231 ) . batches of 12 separate samples at one time were prepared as per manufacturer instructions , with 100300 ng of total rna hybridized with probes at 65c for 1618 hours before being placed into the automated ncounter prep station ( nanostring ) in which samples were affixed to cartridges . cartridges were then immediately placed into the ncounter digital analyzer ( nanostring ) optical scanner and read at a goal resolution of 550 fields of view ( fov ) , which is the maximum resolution for this instrument . the raw nanostring gene expression data were normalized using negative controls , positive controls , and housekeeping genes via nsolver version 2.0 software ( nanostring ) . the arithmetic mean plus 2 standard deviations ( sds ) of the internal negative controls in each sample was subtracted from the gene expression count to ensure that any nonspecific mrna detection was excluded . the geometric mean of the 6 internal positive controls was used to normalize the data so that comparisons could be made across samples and to minimize distortion from batch effects . gene expressions were then normalized using the geometric mean of 5 housekeeping genes ( gapdh , cltc , hprt1 , pgk1 , and tubb ) chosen due to their consistent expression in pbmc . the in - group means of gene expression count were compared via two - tailed pairwise analysis with nonparametric distribution assumed . given the number of simultaneous tests and an expected increase in type 1 error , a bonferroni corrected threshold p value of < 0.001 was used to indicate significance . age , fib-4 , age adjusted charlson index scores , and time since treatment were compared between groups with kruskal - wallis test for nonparametric data . chi - squared tests were performed for comparison of categorical data such as sex , race , and hcv genotype . in the final analysis , 55 patients ( 18 nr , 22 svr , and 15 sc patients ) met both inclusion and exclusion criteria and were included in the study . of those , 3 patients were excluded due to refusal to participate , 5 patients were excluded due to receipt of interferon prior to the study period ( before 12/2008 ) , 8 were unable to participate in their initial visit , 1 patient was consented but was unable to complete blood draw , 1 patient was consented but rna was not recovered from his samples , 3 patients were excluded due to the presence of immunomodulatory medications , and 3 patients were excluded due to the presence of hiv . demographic and clinically relevant data including hcv genotype , fib-4 and age adjusted charlson comorbidity scores , and statin or nsaid use are reported in table 1 . the sc population was significantly younger than the other two groups , with a median age of 58 years compared to 62 and 63 years ( p = 0.047 ) . due to vapahcs patient demographics , whites and males were overrepresented in this study when compared to the overall us hcv population , although there were no significant differences between groups . as expected , fib-4 scores were significantly higher in the nr group compared to sc or svr ( p = 0.001 ) . age adjusted charlson comorbidity scores were significantly different between the three groups ( p < 0.001 ) ; however , the difference appeared to be driven primarily by the amount of nonsevere liver disease ( 1 point ) in the svr group and cirrhotic liver disease in the nr group ( 3 points ) . rates of prescription statin and nsaid use were similar between groups ( p = 0.761 and 0.948 , resp . ) between the svr and nr group , there was a significantly higher percentage of genotype 1 patients in the svr group ( p = 0.027 ) , which was unexpected and likely due to local treatment patterns in the last 5 years at vapahcs after the introduction of hcv protease inhibitors in 2011 . time from last interferon treatment ( median 2.0 years in svr group , 2.6 years in nr , p = 0.348 ) and type of treatment ( p = 0.116 ) were not significantly different between groups . of 184 inflammatory mediator genes assessed , 127 had significant expression levels ( mean copy numbers > 10 in any group ) for reliable detection after normalization with negative controls , positive controls , and housekeeping genes and were included in further analysis . this null expression group included important inflammatory mediators primarily expressed in neutrophils , endothelial cells , tissue based macrophages , or other tissues , including il-1a , il-4 , il-6 , and interferons and . when analyzed as individual samples , it was observed that overall pattern of gene expression between svr and nr patients was highly similar , while sc patients differed from the other two groups . a heat map of gene expression with clustering using a spearman correlation is shown in figure 1 and is notable for the uniform clustering of sc patients together . it is of note that the best - fit line for mean expression of svr genes maps precisely onto that of nr genes with an r of 0.98 , while the correlation between svr and sc gene expression was reduced with an r of 0.88 . using a significance threshold of < 0.001 ( corrected for multiple testing ) , mean mrna counts were significantly different for 42 genes when comparing svr to sc patients , 29 genes comparing nr to sc , and no genes comparing svr to nr . differential expression of 24 genes was significantly different in both svr and nr groups when compared to sc and is shown in tables 2 and 3 . expression of 12 genes was upregulated and expression of 12 genes was downregulated . specific expression patterns were suggestive of increased transcription factor activation for promoters of cellular proliferation in pbmcs and resulting innate immune activity in nr and svr patients versus sc patients . although all 24 genes that were differentially expressed in the same direction in nr and svr patients versus sc patients are potentially important inflammatory cascade regulators and effectors , a few of these are worth special mention as they may be of particular importance in interferon mediated immune response . it is of note that ap-1 components fos ( approximately 3-fold higher in both svr and nr versus sc , p < 0.001 for both ) and jun ( 2.2-fold and 2.4-fold in both svr and nr versus sc , p = 0.054 and 0.028 , resp . ) were both elevated as was cebpb ( ccaat / enhancer - binding protein beta ) ( 1.9-fold and 1.8-fold in both svr and nr versus sc , resp . ap-1 is a known transcription factor for toll - like receptor 4 ( tlr4 ) , which had a mild but nonsignificant elevation of transcription in the svr and nr groups compared to sc ( fold changes of 1.2 and 1.2 , p = 0.04 and 0.02 , resp . ) significant elevations in transcription of tlr4 downstream effector myeloid differentiation primary response gene 88 ( myd88 ) were also seen ( fold changes of 1.2 and 1.6 , resp . however , there was a significant elevation in both svr and nr versus sc , perhaps consistent with feedback regulation from stimulation of the myd88 pathway . it is of note that some transcription factors that favor an adaptive response including histone deacetylase 4 ( hdac4 ) were downregulated in svr and nr compared to the sc group ( hdac4 fold changes of 1.4 and 1.3 , resp . , p < 0.001 for both ) we found that the pattern of pbmc gene expression in svr patients , who received peg - ifn treatment a median of 2 years prior , more closely resembles nr patients with active hcv infection than sc patients who have been exposed to hcv but are uninfected and who have no history of chronic viral infection or prolonged peg - ifn exposure . given that there is ongoing viremia in nr patients and svr patients have achieved clinical cure , it may be expected that nr patients would have a different pattern of expression in pbmcs from svr patients ; however , we found no difference . this indicates that there is a durable inflammatory transcriptional response in pbmcs that persists long after treatment . the specific pattern of gene expression demonstrates that genes related to innate immune activation such as fos , cebpb , and myd88 are more transcriptionally active in both svr and chronically viremic patients , while adaptive immune mediators such as hdac4 are downregulated [ 3033 ] . a potential explanation for this pattern of activation may be liver disease itself and prior injury from chronic hcv infection . lack of relative improvement in fibrosis after treatment may be an important predictor of outcomes in svr patients . gene expression of macrophages from liver tissue has demonstrated upregulation of chemotactic factors and proinflammatory cytokines such as il-8 in prior studies . however , in this study population , the fibrosis scores of svr patients were similar to those of sc patients and significantly different from chronically hcv infected nr patients , indicating that continued hepatic injury is most likely not occurring . this is also consistent with what is known about liver disease outcomes in svr patients as 5-year mortality and progression to cirrhosis drop significantly . pairing peripheral pbmc gene expression data with concurrent gene expression from liver macrophages along with histology from liver biopsy samples may be a useful line of further investigation . another possible explanation is a durable effect from long - term exogenous peg - ifn exposure . however , hcv infected patients who achieve svr receive much longer exposure to exogenous peg - ifn therapy than any other patient group , and this pattern of activation may mimic chronic viral infection , particularly when compared to nr group that also had an extended duration ( although significantly less than the svr group ) of exogenous peg - ifn . the explanation for this data with the most salient clinical consequences is either intra- or extrahepatic reservoirs of residual hcv infection . , late relapse in many cases is likely due to reemergence of prior infection rather than reinfection with a different strain . two previous studies have used rt - pcr to assay pbmc gene expression in svr patients and postulated residual infection as the etiology . assessed transcripts of 9 cytokines in 22 chronically hcv infected patients and 29 patients with resolved disease compared with 15 healthy controls and found significant elevations in ifn- and tnf- in resolved patients versus chronically infected patients . however , the resolved group included both svr and sc patients grouped together , and the chronic infection group included both treatment experienced and treatment naive patients . our study found negligible expression of ifn- transcripts and no significant difference between groups in tnf-. analyzed 25 cytokine transcripts in 49 patients with svr divided into a group with residual infection as defined by very low levels of hcv in serum or pbmc detected with a home - brew ultrasensitive rt - pcr assay or by presence of ns3 protein via immunofluorescence in pbmc versus those without . there was a high rate ( 46.9% ) of residual viremia found in this study , and in those patients there were significant elevations in il-6 , il-8 , il-12,tnf- , and mip-1 ( ccl4 ) when compared to svr patients without evidence of residual virus . we did find , however , that il-8 appeared upregulated in both svr and nr patients compared to sc with fold changes of 7.49 and 5.73 ( p = 0.005 and 0.08 , resp . ) our study differs from prior work in several ways , using an entirely different methodology to assay gene expression of many more genes simultaneously as well as a fundamentally different way of defining comparison groups , which may explain differences in results . ours is the only study to assay expression of upstream genes whose products regulate overall transcription patterns rather than only transcripts for effector cytokines , which are not always reliably expressed in pbmcs as opposed to other tissue compartments . another important limitation is that the time since infection for the sc patients is unknown . this data is not obtainable due to the fact that acute infection with hcv is most often asymptomatic and a positive hcv antibody with negative serum hcv rna pcr was identified in our sc sample on routine screening for at risk populations after the acute phase of infection . there is also a large distribution ( 6 months to 5 years ) in the time since treatment for both the nr and svr patients ; however , the median time since treatment is not significantly different between groups ; therefore , we believe these groups are comparable . however , we believe changes in pbmc mrna expression are important for our understanding of the systemic immune response after hcv treatment . further investigation is warranted to explain why interferon treated patients who obtain svr and a functional cure have a similar pattern of macrophage and lymphocyte gene expression compared to those with chronic hcv infection who did not respond to treatment and also had prior exogenous interferon exposure . this is an important question in the daa era of hcv therapy , as retreatment is now possible for patients with late relapse , and further monitoring of peg - ifn treated patients with svr may be useful to identify late relapse . this may also be clinically important for monitoring peg - ifn treated patients for long - term consequences of prior infection such as progression to cirrhosis and hcc as well as immunogenic consequences of hcv infection such as cryoglobulinemia and porphyria cutanea tarda . patients who achieved clinical cure of hcv infection with peg - ifn based regimens appear to have pbmc gene expression patterns that closely resemble chronically hcv infected patients who have failed treatment and are markedly different from patients who spontaneously clear their hcv infection . it is therefore reasonable to conclude that an inflammatory response persists in pbmcs long after successful peg - ifn treatment for hcv infection .

cardiovascular gene delivery has been previously investigated for mitigation of in - stent restenosis.14 the use of the stent itself as a platform for gene delivery is attractive because it is site specific , potentially helping to avoid distal spread of therapeutic dna and viral vectors . for example , a wide range of therapeutic dna and viruses were delivered from the stent surface to reduce the extent of restenosis.57 the above studies have all used polymer - coated stents to elute the genes . this polymer component , however , has been shown to cause local inflammatory responses and late thrombosis.8,9 the use of a technology that does not need a polymer layer may simplify the complex device coating formulation . for example , fishbein et al has investigated alternatives to polymer coatings.10,11 they reported that pretreatment of metal alloy surfaces with an aqueous bisphosphonate solution , polyallylamine bisphosphonate ( paa - bp ) , enables adenoviral expressing inducible nitric oxide synthase vectors binding to bare metal , which showed effective gene vector delivery and resulted in significant inhibition of restenosis in vivo . biocompatibility evaluation in rats also showed that there was no difference between paa - bp - modified stents and control bare metal stents in the inflammatory response . since the late 1970s , it has been demonstrated that phosphonic acid , rpo3h2 ( r is an organic component ) , readily reacts with a wide range of metal salts and oxides , leading to a rich variety of one- to three - dimensional metal organic frameworks , also called metal phosphonates . the cohesion of these networks results from the formation of metal phosphonate ionocovalent bonds , which usually provide highly stable architectures.12,13 moreover , phosphonic acids show a high compatibility with other organic functional groups and both their ester and acid forms can generally be used for surface modifications . in the study by levy et al , paa - bp that can both interact with metal oxide surfaces and provide reactive sites for chemical conjugation was synthesized by a direct michael addition of paa to the activated double bond of vinylidenebisphosphonic acid . the metallic surface could be modified through aminobisphosphonates exposure , thereby attaching to the metallic surface a derivatizable polybisphosphonate molecule that could , in turn , be covalently conjugated with vector - binding agents . gene delivery vectors such as naked plasmids , viral vectors , and nonviral nanodelivery have been successfully used in experimental gene therapy.1416 however , there are several recurring issues that have led to reconsideration of their use in human clinical trials . these include inefficient in vivo gene transfer , high cytotoxicity , strong inflammatory reactions , and potential risk of viral dna integration to host genome . previous studies by the authors group have reported a novel plasmid dna vector composed of plasmid dna / anti - dna antibody / cationic lipid tri - complex ( dac micelles ) , which exhibits high transfection efficiency , high nuclear entry , and low toxicity both in vitro and in vivo.17,18 this complex formed stable nanoscaled micelles with a mean particle diameter around 360 nm and a zeta potential of 15 mv by self - assembling process . the authors studies also showed that dac micelles are structurally stable under cell culture conditions ; furthermore , the entire complex can remain intact throughout cellular processing including nuclear entry . the authors group has focused on covalent linking of dac micelles on the collagen - coated stents in order to deliver plasmid dna by a vector tethering mechanism.19,20 this approach has been shown to be feasible , with high levels of regional expression in vitro and in vivo . in the current study , the intention was to explore new applications of dac micelles in gene delivery and further investigate the binding stability and the controlled delivery behavior of dac micelles bound on a paa - bp - modified stent surface . first , the stent surface was modified with paa - bp , thereby enabling the retention of a paa - bp molecular monolayer that permits the attachment ( via vector - binding molecules ) of dac gene vectors . after modification , the paa - bp - modified stent surface was activated by n - succinimidyl-3-(2-pyridyldithiol)-propionate ( spdp ) . these thiol - reactive sites were then further reacted in each instance to attach dac micelles for vector tethering . the novel plasmid dna delivery system based on paa - bp - modified stents was characterized by assessments of gene transfection efficiency . 316 l stainless steel foils were obtained from goodfellow corporation ( coraopolis , pa , usa ) . mustang 316 l stainless steel coronary stents ( 2.5 13 mm ) were a gift from microport medical co , ltd ( shanghai , people s republic of china ) . 316 l stainless steel foils were used for x - ray photoelectron spectroscopy ( xps ) and atomic force microscopy ( afm ) of the modified steel surface , while 316 l stainless steel stents were applied to in vitro gene expression . paa - bp was a gift that ivan s alferiev synthesized . the plasmid encoding a green fluorescent protein ( gfp ) with enhanced fluorescence ( pegfp - n3 ) was purchased from clontech laboratories ( mountain view , ca , usa ) . monoclonal mouse anti - bovine dna antibody ( immunoglobulin m ) recognizing double - strand and single - strand dna ( 1 mg / ml ) was obtained from us biological ( swampscott , ma , usa ) and nonspecific mouse immunoglobulin m antibody was from rockland immunochemicals inc ( gilbertsville , pa , usa ) . rat arterial smooth muscle cell ( a10 ) was purchased from american tissue type collection ( manassas , va , usa ) . lipofectamine 2000 was from life technologies ( carls - bad , ca , usa ) . sodium iodide ( nai ) was from ge healthcare ( little chalfont , uk ) . gfp expression was assessed using fluorescent microscopy with a filter calibrated to detect fluorescein isothiocyanate ( eclipse e800 ; nikon corporation , tokyo , japan ) . to clean the stainless steel surface of any impurities , the 316 l stainless steel foils were rinsed twice in 1.5 n nitric acid ( about 30 seconds each time ) , washed copiously with double distilled water , and incubated twice in isopropanol at 55c with shaking ( 15 minutes each time ) . for stent cleaning / surface activation , the additional steps of chloroform incubation ( 15 minutes , 55c , shaking ) and heating ( in a glass vial ) at 250c for 2 hours were taken . for paa - bp modification , the cleaned metal samples were exposed to 3% ( w / v ) aqueous paa - bp ( ph 5.5 ) at 60c for 4 hours . upon completion of incubation with paa - bp the surface elemental composition was analyzed by xps escalabmk ii ( vg scienta , uppsala , sweden ) equipped with an aluminum k x - ray source . surface profiles were visualized by using a dimension 3100 afm ( veeco instruments , santa barbara , ca , usa ) . imaging was performed in the intermittent noncontact ( tapping ) mode by using oscillating linear silicon tips with a resonance frequency range of 300350 hz . each data scan was collected over a 25 m area at a scanning frequency of 0.50 hz . the paa - bp - modified stents were reacted with spdp ( 20 mg / ml ) at room temperature for 2 hours followed with dithiothreitol to introduce sulfhydryl groups on the stents . separately , the mouse monoclonal anti - dna antibody was reacted with an excess of spdp to introduce dithiol groups on the antibody molecules . the dithiol - activated anti - dna antibody was then chemically linked to the stents through the thiol exchange reaction . the antibody - bound stent was then incubated in a pegfp - n3 dna solution ( 20 g pure plasmid dna in 200 l dulbecco s modified eagle s medium [ dmem ] ) at 37c for 1 hour followed by an extensive rinse with copious phosphate buffered saline ( pbs ) solution . the stents were further reacted with lipofectamine 2000 reagent ( 5 l/200 l ) and incubated at room temperature for 30 minutes before incubation with cells . a nonspecific antibody was attached to the modified stents in the same manner as the contrast . in some cases , physical absorption of the antibody onto paa - bp - modified stents was performed and used as the control . rhodamine - labeled dna was used to assess the anchoring of dac micelles on paa - bp - modified stents under a fluorescence microscope ( nikon eclipse e800 ) . anti - dna antibody was iodinated by a modified chloramine - t procedure.21 a 250 g aliquot of anti - dna antibody ( 2.2 mg / ml ) was mixed with 3.5 l nai ( 37 mbq ) and 60 l chloramine - t ( 5 mg / ml , ph 7.5 ) at room temperature and incubated for 3 minutes . the reaction was stopped by adding 80 l of a sodium metabisulfite solution ( 10 mg / ml , ph7.5 ) . i - labeled antibody was purified by gel filtration on a sephadex g-50 column ( sigma - aldrich , st louis , mo , usa ) to remove unreacted iodine . the i - labeled antibody was activated with spdp and chemically linked on the stents as described above ( n = 5 ) . a control group was made by directly soaking the paa - bp - modified stents in the i - labeled antibody solution ( n = 5 ) . both types of antibody - loaded stents were subjected to the same pbs rinse procedure . to remove the unbound antibody , the eluting solution was monitored using a gamma counter until the level of radioactivity was close to the background . the binding capacity of the antibody on the stents was determined by measuring the radioactivity remaining on the stents . to evaluate the binding stability of the i - labeled antibody , stents were incubated in pbs at 37c with shaking ( 140 rpm ) . at predetermined time intervals , the incubation solution was replaced with the same volume of fresh pbs and the radioactivity remaining on the stents was measured with a gamma counter . the stents were incubated in a 1 10 a10 cell ( rat arterial smooth muscle cells ) suspension at 37c for 1 hour before being placed into 35 mm cell culture plates . a suspension of 1 10 a10 cells in dmem was added . the cells were incubated in serum - free medium for 5 hours followed by the addition of fetal bovine serum to a final concentration of 5% . the culture medium was changed to growth medium ( dmem + 10% fetal bovine serum + 1% penicillin / streptomycin ) after 24 hours . the pegfp - n3 gene expression , as demonstrated by fluorescein isothiocyanate - positive cells , was observed on a nikon eclipse e800 fluorescent microscope ( nikon eclipse e800 ) equipped with spot version 3.02 software ( spot imaging solutions , sterling heights , mi , usa ) and photographs were taken after 3 days of cell culture . data for all experiments were expressed as the mean standard error of the mean . the significance of differences was assessed using student s t - test or analysis of variance . 316 l stainless steel foils were obtained from goodfellow corporation ( coraopolis , pa , usa ) . mustang 316 l stainless steel coronary stents ( 2.5 13 mm ) were a gift from microport medical co , ltd ( shanghai , people s republic of china ) . 316 l stainless steel foils were used for x - ray photoelectron spectroscopy ( xps ) and atomic force microscopy ( afm ) of the modified steel surface , while 316 l stainless steel stents were applied to in vitro gene expression . paa - bp was a gift that ivan s alferiev synthesized . the plasmid encoding a green fluorescent protein ( gfp ) with enhanced fluorescence ( pegfp - n3 ) was purchased from clontech laboratories ( mountain view , ca , usa ) . monoclonal mouse anti - bovine dna antibody ( immunoglobulin m ) recognizing double - strand and single - strand dna ( 1 mg / ml ) was obtained from us biological ( swampscott , ma , usa ) and nonspecific mouse immunoglobulin m antibody was from rockland immunochemicals inc ( gilbertsville , pa , usa ) . rat arterial smooth muscle cell ( a10 ) was purchased from american tissue type collection ( manassas , va , usa ) . lipofectamine 2000 was from life technologies ( carls - bad , ca , usa ) . sodium iodide ( nai ) was from ge healthcare ( little chalfont , uk ) . gfp expression was assessed using fluorescent microscopy with a filter calibrated to detect fluorescein isothiocyanate ( eclipse e800 ; nikon corporation , tokyo , japan ) . to clean the stainless steel surface of any impurities , the 316 l stainless steel foils were rinsed twice in 1.5 n nitric acid ( about 30 seconds each time ) , washed copiously with double distilled water , and incubated twice in isopropanol at 55c with shaking ( 15 minutes each time ) . for stent cleaning / surface activation , the additional steps of chloroform incubation ( 15 minutes , 55c , shaking ) and heating ( in a glass vial ) at 250c for 2 hours were taken . for paa - bp modification , the cleaned metal samples were exposed to 3% ( w / v ) aqueous paa - bp ( ph 5.5 ) at 60c for 4 hours . upon completion of incubation with paa - bp the surface elemental composition was analyzed by xps escalabmk ii ( vg scienta , uppsala , sweden ) equipped with an aluminum k x - ray source . surface profiles were visualized by using a dimension 3100 afm ( veeco instruments , santa barbara , ca , usa ) . imaging was performed in the intermittent noncontact ( tapping ) mode by using oscillating linear silicon tips with a resonance frequency range of 300350 hz . each data scan was collected over a 25 m area at a scanning frequency of 0.50 hz . the paa - bp - modified stents were reacted with spdp ( 20 mg / ml ) at room temperature for 2 hours followed with dithiothreitol to introduce sulfhydryl groups on the stents . separately , the mouse monoclonal anti - dna antibody was reacted with an excess of spdp to introduce dithiol groups on the antibody molecules . the dithiol - activated anti - dna antibody was then chemically linked to the stents through the thiol exchange reaction . the antibody - bound stent was then incubated in a pegfp - n3 dna solution ( 20 g pure plasmid dna in 200 l dulbecco s modified eagle s medium [ dmem ] ) at 37c for 1 hour followed by an extensive rinse with copious phosphate buffered saline ( pbs ) solution . the stents were further reacted with lipofectamine 2000 reagent ( 5 l/200 l ) and incubated at room temperature for 30 minutes before incubation with cells . a nonspecific antibody was attached to the modified stents in the same manner as the contrast . in some cases , physical absorption of the antibody onto paa - bp - modified stents rhodamine - labeled dna was used to assess the anchoring of dac micelles on paa - bp - modified stents under a fluorescence microscope ( nikon eclipse e800 ) . anti - dna antibody was iodinated by a modified chloramine - t procedure.21 a 250 g aliquot of anti - dna antibody ( 2.2 mg / ml ) was mixed with 3.5 l nai ( 37 mbq ) and 60 l chloramine - t ( 5 mg / ml , ph 7.5 ) at room temperature and incubated for 3 minutes . the reaction was stopped by adding 80 l of a sodium metabisulfite solution ( 10 mg / ml , ph7.5 ) . i - labeled antibody was purified by gel filtration on a sephadex g-50 column ( sigma - aldrich , st louis , mo , usa ) to remove unreacted iodine . the i - labeled antibody was activated with spdp and chemically linked on the stents as described above ( n = 5 ) . a control group was made by directly soaking the paa - bp - modified stents in the i - labeled antibody solution ( n = 5 ) . both types of antibody - loaded stents were subjected to the same pbs rinse procedure . to remove the unbound antibody , the eluting solution was monitored using a gamma counter until the level of radioactivity was close to the background . the binding capacity of the antibody on the stents was determined by measuring the radioactivity remaining on the stents . to evaluate the binding stability of the i - labeled antibody , stents were incubated in pbs at 37c with shaking ( 140 rpm ) . at predetermined time intervals , the incubation solution was replaced with the same volume of fresh pbs and the radioactivity remaining on the stents was measured with a gamma counter . the stents were incubated in a 1 10 a10 cell ( rat arterial smooth muscle cells ) suspension at 37c for 1 hour before being placed into 35 mm cell culture plates . a suspension of 1 10 a10 cells in dmem was added . the cells were incubated in serum - free medium for 5 hours followed by the addition of fetal bovine serum to a final concentration of 5% . the culture medium was changed to growth medium ( dmem + 10% fetal bovine serum + 1% penicillin / streptomycin ) after 24 hours . the pegfp - n3 gene expression , as demonstrated by fluorescein isothiocyanate - positive cells , was observed on a nikon eclipse e800 fluorescent microscope ( nikon eclipse e800 ) equipped with spot version 3.02 software ( spot imaging solutions , sterling heights , mi , usa ) and photographs were taken after 3 days of cell culture . data for all experiments were expressed as the mean standard error of the mean . the significance of differences was assessed using student s t - test or analysis of variance . in this study , the 316 l stainless steel substrates mainly composed of iron , chromium , and nickel were modified through paa - bp exposure , thereby attaching to the metallic surface a derivatizable polybisphosphonate molecule that could , in turn , be covalently conjugated with dac micelles for local gene delivery . a heterobifunctional crosslinker , spdp , was used to chemically link the dac micelles on the metal stent via a paa - bp monolayer that provided the reactive amine groups . the basic chemistry for dac micelles for binding on paa - bp - modified stent surface is : ( 1 ) to introduce sulfhydryl functional groups on the paa - bp monolayer ; ( 2 ) to derivatize the anti - dna antibody with pyridyl disulfides ; ( 3 ) to couple the dithiol antibody to the sulfhydryl - activated paa - bp monolayer ; and ( 4 ) to form stable dac micelles with incubation with plasmid dna and cationic lipid . thus , dac micelles were covalently linked on the paa - bp - modified stents . xps confirmed the presence of a paa - bp molecular monolayer on the steel surface , demonstrating the emergence of a characteristic phosphorus 2p signal in the xps of the treated sample ; a phosphorus signal is not present in the control 316 l steel ( figure 1a ) . furthermore , the characteristic iron 2p peaks of the steel substrate were still present in the xps from the paa - bp - modified sample ( figure 1b ) , indicating that the thickness of the paa - bp coordination layer is less than the effective xps sampling depth ( 5 nm ) . afm images of the surface of the bare and paa - bp - modified metal surface are shown in figure 2 . afm images show that the paa - bp - modified metal surface displayed substantially increased roughness ( root mean square : 25 5 nm , measured on 2 m 2 m region ) , compared with that of bare surface ( 11 3 nm ) . the increase of local surface roughness following paa - bp modification illustrates that topographical as well as chemical changes to the metal surface can occur as a result of surface modification . it is likely that a thin , rough , but stable paa - bp monolayer was formed on the metal surface . the anti - dna antibody was successfully labeled with radioisotope i. unreacted i was completely separated from the i - labeled antibodies ( i - antibody ) by gel filtration on a sephadex g-50 column . the radiochemical purity reached a level of 98% , as determined by paper chromatography . the final concentration of the dithiol derivative i - antibody ( after spdp activation ) was approximately 60 g / ml , as determined by coomassie brilliant blue g-250 . the results show that the amount of i - antibody chemically linked on the paa - bp - modified stents was 17-fold higher than on the physically adsorbed control stents ( figure 3a ; p < 0.01 ) . the chemically linked i - antibody showed a sustained release over a course of 21 days and approximately 50% of the total bound antibody was retained on the stents at the end of 21 days . however , the physically adsorbed antibody on the control stents was almost completely eluted out within 3 days ( figure 3b ) . there was no significant difference in the binding capacity of i - antibody chemically linked on the paa - bpp - modified stents ( about 0.68 g ) and the collagen - coated stents ( about 0.76 g ) previously studied by the authors group , in which anti - dna antibody was covalently immobilized on the collagen - coated stents in the same way as the paa - bp - modified stents.19 the antibody released from collagen - coated stents was relatively faster than the paa - bp - modified stents , which shows that only 25% of the total bound antibody was retained on the collagen - coated stents at the end of 16 days . the results indicate that the method of paa - bp - modified stents was superior to the collagen - coated stents in the binding stability of the antibody . dac micelles were successfully immobilized onto paa - bp - modified stents as demonstrated by immunofluorescence studies using rhodamine - labeled dna ; control stents that did not contain sulfhydryl - linked anti - dna antibody demonstrated faint autofluorescence ( figure 4 ) . the result confirmed specific anchoring of micelles on spdp - activated paa - bp - modified stents . further inspection of figure 4a revealed that the anchoring of dac micelles was generally uniform and devoid of large - scale patches of defects . it was previously reported that plasmid dna / polyethyleneimine polyplexes nonspecifically adsorbed on hydrophobic surfaces were highly aggregated , resulting in reduced gene transfection efficiency.22,23 in the current study , the chemically linked dac micelles were less aggregated and better distributed on the surface of paa - bp - modified stent , which would increase the chance of being transported within adhered cells through substrate - mediated gene transfection . the paa - bp - modified stents deposited with dac micelles demonstrated efficient gfp gene transfection in a10 cells . the stents retrieved from cell culture after 72 hours of incubation contained numerous gfp - transfected cells that infiltrated the paa - bp coating on the stents , indicating a highly localized and efficient gene delivery pattern ( figure 5a ) . however , on the control stents that had been immobilized with nonspecific antibody and incubated with the same amount pegfp - n3 , only a few transfected gfp - positive cells were found ( figure 5b ) . the authors group has focused on covalent linking of dac micelles on the collagen - coated stents in order to deliver the plasmid dna by a vector tethering mechanism.19,20 this approach has been shown to be feasible , with high levels of regional expression and no detectable spreading of the vector beyond the implanted artery . however , polymer - coated gene delivery stents is problematic because of harmful properties of the polymer coatings.8,9 therefore , in the current study , dac micelles were directly immobilized onto the surface of paa - bp - modified stents without the use of a polymer coating . in vitro transfection data showed that dac micelles linked on paa - bp - modified stents by chemical and immunoreaction ensured highly localized and efficient gene transfection . this is simply because surface - immobilized dac micelles have more chance of being in close proximity or in contact with cells , which can lead to high and long - lasting gene expression by immediate internalization and sustained release effects.24,25 in addition , the formation of nanoscale micelles can be crucial for cellular internalization of dna and subsequent trafficking within cells.22,26 in this study , the 316 l stainless steel substrates mainly composed of iron , chromium , and nickel were modified through paa - bp exposure , thereby attaching to the metallic surface a derivatizable polybisphosphonate molecule that could , in turn , be covalently conjugated with dac micelles for local gene delivery . a heterobifunctional crosslinker , spdp , was used to chemically link the dac micelles on the metal stent via a paa - bp monolayer that provided the reactive amine groups . the basic chemistry for dac micelles for binding on paa - bp - modified stent surface is : ( 1 ) to introduce sulfhydryl functional groups on the paa - bp monolayer ; ( 2 ) to derivatize the anti - dna antibody with pyridyl disulfides ; ( 3 ) to couple the dithiol antibody to the sulfhydryl - activated paa - bp monolayer ; and ( 4 ) to form stable dac micelles with incubation with plasmid dna and cationic lipid . thus , xps confirmed the presence of a paa - bp molecular monolayer on the steel surface , demonstrating the emergence of a characteristic phosphorus 2p signal in the xps of the treated sample ; a phosphorus signal is not present in the control 316 l steel ( figure 1a ) . furthermore , the characteristic iron 2p peaks of the steel substrate were still present in the xps from the paa - bp - modified sample ( figure 1b ) , indicating that the thickness of the paa - bp coordination layer is less than the effective xps sampling depth ( 5 nm ) . afm images of the surface of the bare and paa - bp - modified metal surface are shown in figure 2 . afm images show that the paa - bp - modified metal surface displayed substantially increased roughness ( root mean square : 25 5 nm , measured on 2 m 2 m region ) , compared with that of bare surface ( 11 3 nm ) . the increase of local surface roughness following paa - bp modification illustrates that topographical as well as chemical changes to the metal surface can occur as a result of surface modification . it is likely that a thin , rough , but stable paa - bp monolayer was formed on the metal surface . the anti - dna antibody was successfully labeled with radioisotope i. unreacted i was completely separated from the i - labeled antibodies ( i - antibody ) by gel filtration on a sephadex g-50 column . the radiochemical purity reached a level of 98% , as determined by paper chromatography . the final concentration of the dithiol derivative i - antibody ( after spdp activation ) was approximately 60 g / ml , as determined by coomassie brilliant blue g-250 . the results show that the amount of i - antibody chemically linked on the paa - bp - modified stents was 17-fold higher than on the physically adsorbed control stents ( figure 3a ; p < 0.01 ) . the chemically linked i - antibody showed a sustained release over a course of 21 days and approximately 50% of the total bound antibody was retained on the stents at the end of 21 days . however , the physically adsorbed antibody on the control stents was almost completely eluted out within 3 days ( figure 3b ) . there was no significant difference in the binding capacity of i - antibody chemically linked on the paa - bpp - modified stents ( about 0.68 g ) and the collagen - coated stents ( about 0.76 g ) previously studied by the authors group , in which anti - dna antibody was covalently immobilized on the collagen - coated stents in the same way as the paa - bp - modified stents.19 the antibody released from collagen - coated stents was relatively faster than the paa - bp - modified stents , which shows that only 25% of the total bound antibody was retained on the collagen - coated stents at the end of 16 days . the results indicate that the method of paa - bp - modified stents was superior to the collagen - coated stents in the binding stability of the antibody . dac micelles were successfully immobilized onto paa - bp - modified stents as demonstrated by immunofluorescence studies using rhodamine - labeled dna ; control stents that did not contain sulfhydryl - linked anti - dna antibody demonstrated faint autofluorescence ( figure 4 ) . the result confirmed specific anchoring of micelles on spdp - activated paa - bp - modified stents . further inspection of figure 4a revealed that the anchoring of dac micelles was generally uniform and devoid of large - scale patches of defects . it was previously reported that plasmid dna / polyethyleneimine polyplexes nonspecifically adsorbed on hydrophobic surfaces were highly aggregated , resulting in reduced gene transfection efficiency.22,23 in the current study , the chemically linked dac micelles were less aggregated and better distributed on the surface of paa - bp - modified stent , which would increase the chance of being transported within adhered cells through substrate - mediated gene transfection . the paa - bp - modified stents deposited with dac micelles demonstrated efficient gfp gene transfection in a10 cells . the stents retrieved from cell culture after 72 hours of incubation contained numerous gfp - transfected cells that infiltrated the paa - bp coating on the stents , indicating a highly localized and efficient gene delivery pattern ( figure 5a ) . however , on the control stents that had been immobilized with nonspecific antibody and incubated with the same amount pegfp - n3 , only a few transfected gfp - positive cells were found ( figure 5b ) . the authors group has focused on covalent linking of dac micelles on the collagen - coated stents in order to deliver the plasmid dna by a vector tethering mechanism.19,20 this approach has been shown to be feasible , with high levels of regional expression and no detectable spreading of the vector beyond the implanted artery . however , polymer - coated gene delivery stents is problematic because of harmful properties of the polymer coatings.8,9 therefore , in the current study , dac micelles were directly immobilized onto the surface of paa - bp - modified stents without the use of a polymer coating . in vitro transfection data showed that dac micelles linked on paa - bp - modified stents by chemical and immunoreaction ensured highly localized and efficient gene transfection . this is simply because surface - immobilized dac micelles have more chance of being in close proximity or in contact with cells , which can lead to high and long - lasting gene expression by immediate internalization and sustained release effects.24,25 in addition , the formation of nanoscale micelles can be crucial for cellular internalization of dna and subsequent trafficking within cells.22,26 through surface modification with paa - bp and subsequent anchoring of dac micelles , functionalized stents with paa - bp and dna were created for cardiovascular gene delivery . the dac micelle - immobilized paa - bp - modified stents were successful as a gene delivery system , giving rise to efficient and localized gene delivery to arterial smooth muscle cells . gene delivery using dac micelle - tethered stent - based paa - bp functionalization should be suitable for a wide array of single or multiple therapeutic gene strategies , and could be used on cardiovascular metallic implants for achieving efficient gene therapy .

purpose : to investigate the anchoring of plasmid dna / anti - dna antibody / cationic lipid tri - complex ( dac micelles ) onto bisphosphonate - modified 316 l coronary stents for cardiovascular site - specific gene delivery.methods:stents were first modified with polyallylamine bisphosphonate ( paa - bp ) , thereby enabling the retention of a paa - bp molecular monolayer that permits the anchoring ( via vector - binding molecules ) of dac micelles . dac micelles were then chemically linked onto the paa - bp - modified stents by using n - succinimidyl-3-(2-pyridyldithiol)-propionate ( spdp ) as a crosslinker . rhodamine - labeled dna was used to assess the anchoring of dac micelles , and radioactive - labeled antibody was used to evaluate binding capacity and stability . dac micelles ( encoding green fluorescent protein ) were tethered onto the paa - bp - modified stents , which were assessed in cell culture . the presence of a paa - bp molecular monolayer on the steel surface was confirmed by x - ray photoelectron spectroscopy and atomic force microscope analysis.results:the anchoring of dac micelles was generally uniform and devoid of large - scale patches of defects . isotopic quantification confirmed that the amount of antibody chemically linked on the stents was 17-fold higher than that of the physical adsorbed control stents and its retention time was also significantly longer . in cell culture , numerous green fluorescent protein - positive cells were found on the paa - bp modified stents , which demonstrated high localization and efficiency of gene delivery.conclusion:the dac micelle - immobilized paa - bp - modified stents were successful as a gene delivery system . gene delivery using dac micelle - tethered stent - based paa - bp functionalization should be suitable for a wide array of single or multiple therapeutic gene strategies , and could be used on cardiovascular metallic implants for achieving efficient gene therapy .

Introduction Materials and methods Materials Metal surface modification with functional PAA-BP Surface characterization Protocol for preparing DAC micelle-tethered PAA-BP-modified stents Binding capacity and stability of Cell transfection by DAC micelle-tethered stents in vitro Statistical analysis Results and discussion Chemical reasoning of tethering DAC micelles onto the PAA-BP-modified metal surface Surface characterization of a functional PAA-BP-modified metal surface Binding capacity and stability of Anchoring of rhodamine-labeled DAC micelles on PAA-BP-modified stents In vitro gene transfection mediated by DAC micelle-tethered stents Conclusion

for example , fishbein et al has investigated alternatives to polymer coatings.10,11 they reported that pretreatment of metal alloy surfaces with an aqueous bisphosphonate solution , polyallylamine bisphosphonate ( paa - bp ) , enables adenoviral expressing inducible nitric oxide synthase vectors binding to bare metal , which showed effective gene vector delivery and resulted in significant inhibition of restenosis in vivo . biocompatibility evaluation in rats also showed that there was no difference between paa - bp - modified stents and control bare metal stents in the inflammatory response . previous studies by the authors group have reported a novel plasmid dna vector composed of plasmid dna / anti - dna antibody / cationic lipid tri - complex ( dac micelles ) , which exhibits high transfection efficiency , high nuclear entry , and low toxicity both in vitro and in vivo.17,18 this complex formed stable nanoscaled micelles with a mean particle diameter around 360 nm and a zeta potential of 15 mv by self - assembling process . in the current study , the intention was to explore new applications of dac micelles in gene delivery and further investigate the binding stability and the controlled delivery behavior of dac micelles bound on a paa - bp - modified stent surface . first , the stent surface was modified with paa - bp , thereby enabling the retention of a paa - bp molecular monolayer that permits the attachment ( via vector - binding molecules ) of dac gene vectors . after modification , the paa - bp - modified stent surface was activated by n - succinimidyl-3-(2-pyridyldithiol)-propionate ( spdp ) . the novel plasmid dna delivery system based on paa - bp - modified stents was characterized by assessments of gene transfection efficiency . 316 l stainless steel foils were used for x - ray photoelectron spectroscopy ( xps ) and atomic force microscopy ( afm ) of the modified steel surface , while 316 l stainless steel stents were applied to in vitro gene expression . the paa - bp - modified stents were reacted with spdp ( 20 mg / ml ) at room temperature for 2 hours followed with dithiothreitol to introduce sulfhydryl groups on the stents . the dithiol - activated anti - dna antibody was then chemically linked to the stents through the thiol exchange reaction . in some cases , physical absorption of the antibody onto paa - bp - modified stents was performed and used as the control . rhodamine - labeled dna was used to assess the anchoring of dac micelles on paa - bp - modified stents under a fluorescence microscope ( nikon eclipse e800 ) . the i - labeled antibody was activated with spdp and chemically linked on the stents as described above ( n = 5 ) . a control group was made by directly soaking the paa - bp - modified stents in the i - labeled antibody solution ( n = 5 ) . the binding capacity of the antibody on the stents was determined by measuring the radioactivity remaining on the stents . 316 l stainless steel foils were used for x - ray photoelectron spectroscopy ( xps ) and atomic force microscopy ( afm ) of the modified steel surface , while 316 l stainless steel stents were applied to in vitro gene expression . the paa - bp - modified stents were reacted with spdp ( 20 mg / ml ) at room temperature for 2 hours followed with dithiothreitol to introduce sulfhydryl groups on the stents . separately , the mouse monoclonal anti - dna antibody was reacted with an excess of spdp to introduce dithiol groups on the antibody molecules . the dithiol - activated anti - dna antibody was then chemically linked to the stents through the thiol exchange reaction . in some cases , physical absorption of the antibody onto paa - bp - modified stents rhodamine - labeled dna was used to assess the anchoring of dac micelles on paa - bp - modified stents under a fluorescence microscope ( nikon eclipse e800 ) . the i - labeled antibody was activated with spdp and chemically linked on the stents as described above ( n = 5 ) . a control group was made by directly soaking the paa - bp - modified stents in the i - labeled antibody solution ( n = 5 ) . in this study , the 316 l stainless steel substrates mainly composed of iron , chromium , and nickel were modified through paa - bp exposure , thereby attaching to the metallic surface a derivatizable polybisphosphonate molecule that could , in turn , be covalently conjugated with dac micelles for local gene delivery . a heterobifunctional crosslinker , spdp , was used to chemically link the dac micelles on the metal stent via a paa - bp monolayer that provided the reactive amine groups . the basic chemistry for dac micelles for binding on paa - bp - modified stent surface is : ( 1 ) to introduce sulfhydryl functional groups on the paa - bp monolayer ; ( 2 ) to derivatize the anti - dna antibody with pyridyl disulfides ; ( 3 ) to couple the dithiol antibody to the sulfhydryl - activated paa - bp monolayer ; and ( 4 ) to form stable dac micelles with incubation with plasmid dna and cationic lipid . thus , dac micelles were covalently linked on the paa - bp - modified stents . xps confirmed the presence of a paa - bp molecular monolayer on the steel surface , demonstrating the emergence of a characteristic phosphorus 2p signal in the xps of the treated sample ; a phosphorus signal is not present in the control 316 l steel ( figure 1a ) . furthermore , the characteristic iron 2p peaks of the steel substrate were still present in the xps from the paa - bp - modified sample ( figure 1b ) , indicating that the thickness of the paa - bp coordination layer is less than the effective xps sampling depth ( 5 nm ) . afm images of the surface of the bare and paa - bp - modified metal surface are shown in figure 2 . afm images show that the paa - bp - modified metal surface displayed substantially increased roughness ( root mean square : 25 5 nm , measured on 2 m 2 m region ) , compared with that of bare surface ( 11 3 nm ) . the anti - dna antibody was successfully labeled with radioisotope i. unreacted i was completely separated from the i - labeled antibodies ( i - antibody ) by gel filtration on a sephadex g-50 column . the results show that the amount of i - antibody chemically linked on the paa - bp - modified stents was 17-fold higher than on the physically adsorbed control stents ( figure 3a ; p < 0.01 ) . the chemically linked i - antibody showed a sustained release over a course of 21 days and approximately 50% of the total bound antibody was retained on the stents at the end of 21 days . there was no significant difference in the binding capacity of i - antibody chemically linked on the paa - bpp - modified stents ( about 0.68 g ) and the collagen - coated stents ( about 0.76 g ) previously studied by the authors group , in which anti - dna antibody was covalently immobilized on the collagen - coated stents in the same way as the paa - bp - modified stents.19 the antibody released from collagen - coated stents was relatively faster than the paa - bp - modified stents , which shows that only 25% of the total bound antibody was retained on the collagen - coated stents at the end of 16 days . the results indicate that the method of paa - bp - modified stents was superior to the collagen - coated stents in the binding stability of the antibody . dac micelles were successfully immobilized onto paa - bp - modified stents as demonstrated by immunofluorescence studies using rhodamine - labeled dna ; control stents that did not contain sulfhydryl - linked anti - dna antibody demonstrated faint autofluorescence ( figure 4 ) . the result confirmed specific anchoring of micelles on spdp - activated paa - bp - modified stents . further inspection of figure 4a revealed that the anchoring of dac micelles was generally uniform and devoid of large - scale patches of defects . it was previously reported that plasmid dna / polyethyleneimine polyplexes nonspecifically adsorbed on hydrophobic surfaces were highly aggregated , resulting in reduced gene transfection efficiency.22,23 in the current study , the chemically linked dac micelles were less aggregated and better distributed on the surface of paa - bp - modified stent , which would increase the chance of being transported within adhered cells through substrate - mediated gene transfection . the paa - bp - modified stents deposited with dac micelles demonstrated efficient gfp gene transfection in a10 cells . the stents retrieved from cell culture after 72 hours of incubation contained numerous gfp - transfected cells that infiltrated the paa - bp coating on the stents , indicating a highly localized and efficient gene delivery pattern ( figure 5a ) . however , on the control stents that had been immobilized with nonspecific antibody and incubated with the same amount pegfp - n3 , only a few transfected gfp - positive cells were found ( figure 5b ) . the authors group has focused on covalent linking of dac micelles on the collagen - coated stents in order to deliver the plasmid dna by a vector tethering mechanism.19,20 this approach has been shown to be feasible , with high levels of regional expression and no detectable spreading of the vector beyond the implanted artery . however , polymer - coated gene delivery stents is problematic because of harmful properties of the polymer coatings.8,9 therefore , in the current study , dac micelles were directly immobilized onto the surface of paa - bp - modified stents without the use of a polymer coating . in vitro transfection data showed that dac micelles linked on paa - bp - modified stents by chemical and immunoreaction ensured highly localized and efficient gene transfection . this is simply because surface - immobilized dac micelles have more chance of being in close proximity or in contact with cells , which can lead to high and long - lasting gene expression by immediate internalization and sustained release effects.24,25 in addition , the formation of nanoscale micelles can be crucial for cellular internalization of dna and subsequent trafficking within cells.22,26 in this study , the 316 l stainless steel substrates mainly composed of iron , chromium , and nickel were modified through paa - bp exposure , thereby attaching to the metallic surface a derivatizable polybisphosphonate molecule that could , in turn , be covalently conjugated with dac micelles for local gene delivery . a heterobifunctional crosslinker , spdp , was used to chemically link the dac micelles on the metal stent via a paa - bp monolayer that provided the reactive amine groups . the basic chemistry for dac micelles for binding on paa - bp - modified stent surface is : ( 1 ) to introduce sulfhydryl functional groups on the paa - bp monolayer ; ( 2 ) to derivatize the anti - dna antibody with pyridyl disulfides ; ( 3 ) to couple the dithiol antibody to the sulfhydryl - activated paa - bp monolayer ; and ( 4 ) to form stable dac micelles with incubation with plasmid dna and cationic lipid . thus , xps confirmed the presence of a paa - bp molecular monolayer on the steel surface , demonstrating the emergence of a characteristic phosphorus 2p signal in the xps of the treated sample ; a phosphorus signal is not present in the control 316 l steel ( figure 1a ) . furthermore , the characteristic iron 2p peaks of the steel substrate were still present in the xps from the paa - bp - modified sample ( figure 1b ) , indicating that the thickness of the paa - bp coordination layer is less than the effective xps sampling depth ( 5 nm ) . afm images of the surface of the bare and paa - bp - modified metal surface are shown in figure 2 . afm images show that the paa - bp - modified metal surface displayed substantially increased roughness ( root mean square : 25 5 nm , measured on 2 m 2 m region ) , compared with that of bare surface ( 11 3 nm ) . the results show that the amount of i - antibody chemically linked on the paa - bp - modified stents was 17-fold higher than on the physically adsorbed control stents ( figure 3a ; p < 0.01 ) . the chemically linked i - antibody showed a sustained release over a course of 21 days and approximately 50% of the total bound antibody was retained on the stents at the end of 21 days . there was no significant difference in the binding capacity of i - antibody chemically linked on the paa - bpp - modified stents ( about 0.68 g ) and the collagen - coated stents ( about 0.76 g ) previously studied by the authors group , in which anti - dna antibody was covalently immobilized on the collagen - coated stents in the same way as the paa - bp - modified stents.19 the antibody released from collagen - coated stents was relatively faster than the paa - bp - modified stents , which shows that only 25% of the total bound antibody was retained on the collagen - coated stents at the end of 16 days . the results indicate that the method of paa - bp - modified stents was superior to the collagen - coated stents in the binding stability of the antibody . dac micelles were successfully immobilized onto paa - bp - modified stents as demonstrated by immunofluorescence studies using rhodamine - labeled dna ; control stents that did not contain sulfhydryl - linked anti - dna antibody demonstrated faint autofluorescence ( figure 4 ) . the result confirmed specific anchoring of micelles on spdp - activated paa - bp - modified stents . further inspection of figure 4a revealed that the anchoring of dac micelles was generally uniform and devoid of large - scale patches of defects . it was previously reported that plasmid dna / polyethyleneimine polyplexes nonspecifically adsorbed on hydrophobic surfaces were highly aggregated , resulting in reduced gene transfection efficiency.22,23 in the current study , the chemically linked dac micelles were less aggregated and better distributed on the surface of paa - bp - modified stent , which would increase the chance of being transported within adhered cells through substrate - mediated gene transfection . the paa - bp - modified stents deposited with dac micelles demonstrated efficient gfp gene transfection in a10 cells . the stents retrieved from cell culture after 72 hours of incubation contained numerous gfp - transfected cells that infiltrated the paa - bp coating on the stents , indicating a highly localized and efficient gene delivery pattern ( figure 5a ) . however , on the control stents that had been immobilized with nonspecific antibody and incubated with the same amount pegfp - n3 , only a few transfected gfp - positive cells were found ( figure 5b ) . the authors group has focused on covalent linking of dac micelles on the collagen - coated stents in order to deliver the plasmid dna by a vector tethering mechanism.19,20 this approach has been shown to be feasible , with high levels of regional expression and no detectable spreading of the vector beyond the implanted artery . however , polymer - coated gene delivery stents is problematic because of harmful properties of the polymer coatings.8,9 therefore , in the current study , dac micelles were directly immobilized onto the surface of paa - bp - modified stents without the use of a polymer coating . in vitro transfection data showed that dac micelles linked on paa - bp - modified stents by chemical and immunoreaction ensured highly localized and efficient gene transfection . this is simply because surface - immobilized dac micelles have more chance of being in close proximity or in contact with cells , which can lead to high and long - lasting gene expression by immediate internalization and sustained release effects.24,25 in addition , the formation of nanoscale micelles can be crucial for cellular internalization of dna and subsequent trafficking within cells.22,26 through surface modification with paa - bp and subsequent anchoring of dac micelles , functionalized stents with paa - bp and dna were created for cardiovascular gene delivery . the dac micelle - immobilized paa - bp - modified stents were successful as a gene delivery system , giving rise to efficient and localized gene delivery to arterial smooth muscle cells . gene delivery using dac micelle - tethered stent - based paa - bp functionalization should be suitable for a wide array of single or multiple therapeutic gene strategies , and could be used on cardiovascular metallic implants for achieving efficient gene therapy .

natural uranium ( nu ) is an alpha particle emitter radionuclide of the actinide series and a ubiquitous environmental trace metal found in almost all types of rocks , soils , plants , and water . surface water and especially ground water play a significant role in the migration and redistribution of this nuclide in the environment . increased nu levels in groundwater are associated with uranium - rich ores and its high solubility under oxidising conditions in soft and bicarbonate - rich waters . consequently , populations may be exposed to nu in some countries and regions with high nu levels in drinking water [ 24 ] . the harmful effects on human health of high levels of nu in drinking water are naturally of great interest to the scientific community and the general public . nu comprises three isotopes : u , 99.28% ; u , 0.715% ; and u , 5.5 10% . techniques have been developed in which uranium ore is chemically enriched , thereby increasing the concentration of u to 24% . the different stages of nuclear fuel cycle lead to the production of enriched uranium ( eu ) and a by - product with a lower proportion of u , called depleted uranium ( du ) . the radiological hazard is more important into the following order : eu > nu > du , but all these uranium have the same chemical toxicity . although the central nervous system is a target organ for many toxic heavy metals and is a radiosensitive organ [ 68 ] few studies have looked for in vivo neurological and neurobehavioural effects following internal contamination with uranium . a few studies on nuclear workers or gulf war veterans have looked at its brain effects [ 911 ] . experimental studies show that after exposure , uranium can reach the brain and lead to neurobehavioural effects on locomotor activity , the sleep - wake cycle , memory , and anxiety . almost all of these data have been recorded with du and supraenvironmental levels ( i.e. , 40 mgl ) . the toxicity of du is expected to be mainly chemical rather than radioactive , so the radiological hazards of uranium have been little investigated . several in vivo studies have shown that uranium can affect the brain , but a still more sensitive approach is necessary to overcome the specific limitations due to low doses . metabolomics , the comprehensive analysis of a wide range of metabolites , provides a novel tool in the search for new biomarkers of exposure or diagnostic and is an alternative and complementary approach to establish more -omics techniques such as genomics , transcriptomics , or proteomics . metabolomics provides the ultimate response of a biological system through the analysis of small molecules ( < 1000 da ) and the characterisation of metabolic phenotypes . metabolomics has recently been found efficient for identifying a discriminant metabolic signature of chronic low - dose cesium 137 or uranium contamination in urine of rats [ 13 , 14 ] . . the metabolites in cerebrospinal fluid ( csf ) reflect central nervous system metabolism and the balance between blood and csf the purpose of the present work was to establish for the first time whether chronic exposure to nu can induce behavioural effects and whether the csf metabolome is modified . to mimic environmental contamination of drinking water , especially among children , who are known to be a sensitive subgroup in toxicology and radiobiology [ 17 , 18 ] , male rats were exposed from birth to adulthood , that is , over a continuous 9-month period , through lactation and next drinking water containing concentrations of nu known not to be toxic to the kidneys . we used nu concentrations of 1.5 , 10 , and 40 mgl , since the highest concentration of naturally occurring uranium in spring water is 12 mgl . in order to determine the influence of sex , female rats were exposed to the highest nu concentration ( 40 mgl ) female sprague - dawley rats ( n = 48 ) were purchased at gestational day 18 ( charles river , france ) and were individually housed under standard conditions ( 21 1c ) with a 12:00 h/12:00 h light / dark cycle ( lights on from 08:00 a.m. to 08:00 p.m. ) . the study was conducted in accordance with french legislation concerning the protection of animals used for experimental purposes . all procedures were performed by scientists certified by the french ministry of agriculture ( license of first author number 92 - 254 ) . at the birth of their pups , mothers were subdivided into four groups ( n = 12 mothers for each group ) . one group was contaminated using mineral drinking water supplemented with nu ( in its uranyl nitrate form ; from areva ) at a concentration of 1.5 mgl ( dose about 0.04 mgday per female rat ) . a second group was contaminated using mineral drinking water supplemented with nu ( in its uranyl nitrate form ; from areva ) at a concentration of 10 mgl ( dose about 0.25 mgday per female rat ) . a third group was contaminated with nu in drinking water at a concentration of 40 mgl ( dose about 1 mgday per female rat ) . control mothers drank noncontaminated water ( fourth group ) . after weaning on postnatal day 21 , male pups were still exposed to nu via drinking water ( 1.5 , 10 , or 40 mgl ) until they reached 9 months of age . female pups were also still exposed to nu via drinking water ( 40 mgl ) until they reached 9 months of age . one male or female offspring per litter was assigned to behavioural tests ( n = 12 for each group ) . health parameters , that is , body weight , water consumption , and food intake , were measured at the end of nu exposure ( at 9 months of age ) . male or female pups ( n = 12 for each experimental group ) were submitted to behavioural evaluation tests at 9 months of age . six days are necessary to perform all behavioural tests following this order . on the first and second days , each animal was individually placed in an open field ( 45 45 cm ) and was monitored by an automated activity monitoring system ( bioseb , chaville , france ) . lateral and horizontal movements were recorded over a 15 min session , only on the first day . on the third day when the rats were acclimated to the open field , they were tested in a two - object recognition task . the animal was placed in the open field with two identical objects for 3 min ( first session ) . after a 1-hour delay , the rat was returned to the open field and allowed to explore two objects , one identical to those presented at the first session ( familiar object ) and the other different ( novel object ) , for an additional 3 min period ( second session ) . spatial working memory was assessed on the fourth day in a y - maze with three arms ( 70 cm long , 50 cm high , 10 cm wide at the bottom , and 20 cm wide at the top ) which converged at an equal angle . each rat was placed at the centre of the maze and was allowed to move freely through the maze for a 10 min test session . anxiety was assessed on day five in an elevated plus maze comprising a wooden cross at a height of 70 cm with two open ( 10 cm70 cm ) and two closed arms with walls ( 10 cm55 cm70 cm ) , arranged such that the arms of the same type were opposite to each other and connected by a common open central platform ( 5 cm5 cm ) . at the beginning of the session , the rat was placed at the centre of the maze always facing the same open arm . standard spatiotemporal measures were recorded , including the number of entries in the open and closed arms and the cumulative time spent in the different parts of the maze ( open and closed arms ) . an arm entry was recorded if all four of the animal 's paws were in the arm . between the testing of each animal , the maze was cleaned with a 10% ethanol solution . the forced swimming test , which was the most stressful test , was performed last , on day 6 . the rats were individually placed in a glass cylinder ( height of 60 cm and diameter of 40 cm ) , containing enough water such that the hind legs could not reach the bottom of the cylinder but the tail could . the water was maintained at 2325c and the rats were left for 10 min . immobility was measured during the last 5 min of the test ( the animal was judged to be immobile when it floated in an upright position and made only minimal movements to keep its head above water ) . all the tests were recorded by a video camera and were read by an observer blind to the exposure conditions . at the end of behavioural tests , each animal was euthanised with isoflurane , placed prone on the stereotaxic instrument and the head of the rat was fixed in a holder . a terminal csf sample was obtained by direct insertion of an insulin syringe needle ( myjector , 29 g 9 1/200 ) via the arachnoid membrane into the cisterna magna . for this purpose a skin incision was made followed by a horizontal incision in the descending part of the trapezius muscle to reveal the arachnoid membrane . each sample was transferred into a polypropylene tube , immediately snap frozen in liquid nitrogen , and stored at 80c for further analysis . previous experiments have shown that collecting up to 100 l using this technique and these conditions provides haemoglobin - free csf samples . metabolomics analyses have been performed by criblage biologique marseille ( cribiom ) platform . for the analysis of csf , protein of 50 l was removed by methanol precipitation using 200 l of cold methanol ( 20c ) followed by 5 min centrifugation at 14000 rpm . the supernatant was recovered and filtered through 10 kda filters to remove all proteins . the extracts were evaporated to dryness under a stream of nitrogen at room temperature and redissolved with 25 l of water / acetonitrile = 90/10 ( v / v ) . to check for data quality , a blank sample ( deionised water ) and a pool sample ( a mixture of all csf samples ) the samples were analysed on a dionex ultimate 3000 ( thermo fisher scientific , france ) coupled to a q - exactive plus mass spectrometer ( thermo fisher scientific , france ) . the lc conditions were autosampler temperature , 4c ; column temperature , 40c ; solvent flow , 0.4 ml / min ( solvent a : water , 10 mm ammonium formate , 0.1% formic acid , and solvent b : acetonitrile , 10 mm ammonium formate , 0.1% formic acid ) ; and gradient , 5% b for 1 min , 550% b for 2 min , 5097% b for 6 min , 97% b for 2 min , 975% b for 1 min , and 5% b for 4 min ( running time , 16 min ) . the ms conditions were as follows : acquisition mode , positive electrospray ionisation , and full scan 801000 m / z ; capillary voltage , 4.5 kv ; capillary temperature , 320c ; cone voltage , 55 v ; drying gas flow rate , 8 lmin . multivariate statistical analyses were performed using simca - p+ ( version 12 , umetrics ) . statistical models were validated by anova in the cross - validation mode , where p values less than 0.05 were considered significant . the robustness of the models was assessed by calculating the explained variance values ( r2y ) and predicted variances ( q2y ) and by the decrease to negative values of the predicted variance after multiple permutations . principal component analysis and partial least squares discriminant analysis ( pls - da ) were performed on the processed data in log 10[1 + 10e ] and scaled in pareto mode . data quality and filtering was performed using appropriately tuned xcms and less stable features removal [ 14 , 25 ] . to select the most discriminant variables , we found most appropriate to examine the clustering of features with the variable score values as calculated by hierarchical cluster analysis applied to wc loadings of the pls - da model . the most discriminant mass features were tentatively annotated using mzeddb from the chemical formulas generated from the accurately measured masses ( accuracy < 5 ppm ) generated by the thermo xcalibur qual browser molecular formula engine . the kegg compound i d of any hits was recorded , and all recorded ids were inserted into the kegg mapper ( http://www.genome.jp/kegg/tool/map_pathway2.html ) for tentative pathway identification . samples ( cerebral cortex and csf ) were prepared by adding 8 ml of 70% ultrapure nitric acid and 2 ml of hydrogen peroxide . samples were then mineralised using a 1000 w microwave ( ethos touch ; milestone microwave laboratory systems ; begamo , italy ) with a 20 min ramp to 180c , followed by 10 min at 180c . the uranium content of samples was determined using an inductively coupled plasma mass spectrometer ( icpms - vgpq , excell , thermo electron corporation ) with bismuth ( 1 gl ) as internal standard . for uranium , values were expressed as nanograms per gram of fresh tissue or nanograms per l of csf and presented as mean sem . in all the experiments , data are expressed as mean sem and were analysed by two - way anova with the main factors of group and dose . body weight was not significantly modified in rats exposed to 1.5 , 10 , or 40 mgl nu compared with the control group ( table 1 ) . food intake was also not significantly changed in nu - exposed rats in comparison with control rats ( table 1 ) . daily water consumption did not significantly change in rats exposed to 1.5 , 10 , or 40 mgl nu , when compared with control rats ( table 1 ) . the locomotor and exploratory behaviours of rats were assessed by the total number of lines crossed and total number of rearings in the open field over 15 minutes . the results for both parameters are depicted in figure 1 . in rats exposed to 1.5 , 10 , or 40 mgl nu , no significant effect on lines crossed or rearing the medium - term memory of rats was assessed by the time spent exploring novel and familiar objects . , all groups of animals spent the same overall time exploring the left and right objects ( figure 2(a ) ) . but during the second session , control group rats spent significantly more time exploring the novel object than the familiar object ( 5.1 1.4 versus 2.3 0.4 s ) ( figure 2(b ) ) . groups exposed to 1.5 , 10 , or 40 mgl nu did not prefer the novel object to the familiar object ( figure 2(b ) ) . this indicated a loss of medium - term memory in rats exposed to nu without dose effect . the spatial working memory capacities of rats were assessed by spontaneous alternation and number of arm visits , in the y - maze . the percentage alternation was significantly higher than 50% , indicating that spatial memory was present for the four groups . however , in rats exposed to 40 mgl nu , a significant decrease in the percentage of spontaneous alternation was observed in comparison with the control group ( 16% , p < 0.05 ) ( figure 3(b ) ) . this decrease in alternation behaviour was not associated with changes of general locomotor activity , measured as the number of arm visits ( figure 3(a ) ) . for rats exposed to 1.5 or 10 mgl nu , no significant effect was found on the percentage alternation or on the number of visits to each arm , when compared with controls ( figure 3 ) . the anxiety - like behaviour of rats was assessed by the time spent in the closed arms and the number of closed arm entries in the elevated plus maze . rats exposed to 40 mgl nu spent significantly more time in the closed arms than did the controls ( 31% , p < 0.01 ) , the rats exposed to 1.5 mgl nu ( + 18% , p < 0.05 ) , and the rats exposed to 10 mgl nu ( + 18% , p < 0.05 ) ( figure 4(b ) ) . their number of visits to the closed arms did not differ significantly from that of the other groups ( figure 4(a ) ) . for rats exposed to 1.5 or 10 mgl nu , no significant difference was observed in the time spent in or the number of visits to the closed arms compared with controls ( figure 4 ) . the depressive - like behaviour of rats was assessed by the time they spent immobile during the 5 last minutes of the test . the immobility time did not differ in rats exposed to 1.5 or 10 mgl nu in comparison with control rats ( figure 5 ) , but it increased significantly ( + 163% , p < 0.05 ) when rats were exposed to 40 mgl for 9 months ( figure 5 ) . as this parameter is usually used to evaluate depressive - like behaviour , this result indicates that the depressive - like behaviour was not affected by exposure to 1.5 or 10 mgl nu but increased after 9 months of exposure to 40 mgl nu . the number of lines crossed during the open - field test and the number of closed arm entries in the elevated plus maze increased significantly ( + 26% , p < 0.05 and + 27% , p < 0.05 , resp . ) in females exposed to nu compared with controls ( figures 6(a1 ) and 6(c1 ) ) . the number of rearings during the open - field test , spontaneous alternation and the number of arm visits in the y - maze , the time spent in the closed arms of the elevated plus maze , and the immobility time during the forced swimming test were not significantly modified in female rats exposed to 40 mgl nu compared with control female rats ( figure 6 ) . during the object recognition test , exposed and controls groups spent significantly more time exploring the novel object than the familiar object ( figure 6(d ) ) . the variables responsible for the discrimination between control rats and rats exposed to 40 mgl nu are shown in figure 7 . using principal component analysis , pls - da and hierarchical ascendant classification , a model was created with the 86 most discriminating variables from the initial 1244 detected csf analytical features . this model was built on a single pls - da component and it was validated by permutation tests and cv - anova ( p = 3.91721e ) . from this model , we were able to observe and select the best discriminating variables of the 86 significant ones that discriminate control from exposed rats , for male , female , and both animals . when comparing males to females , from the top 18 control to nu discriminating variables , 7 were found exclusively related to female rats from control rats and 7 others to male rats . four variables were common for female rats and male rats , corresponding to variables discriminating exposed rats versus control rats , regardless of gender ( figure 7 ) . these 4 variables have been putatively identified as n2-succinyl - l - arginine , n4-acetylaminobutanoate , and n - methylsalsolinol , which decreased in nu - exposed rats compared to control rats , and butyric acid , which increased in nu - exposed rats ( table 2 ) . this metabolomics analysis thus showed that some metabolites differed in the csf of male versus female and the nu - exposed versus control groups , whereas others were only specific of nu exposure , irrespective of gender . uranium concentrations in the cortex of male rats exposed to 10 or 40 mgl nu were significantly increased ( resp . , + 110% and + 218% ) in comparison with control rats ( table 1 ) . no significant difference was observed between rats exposed to 1.5 mgl nu and control rats ( table 1 ) . the concentration of uranium in csf increased significantly in male rats exposed to 40 mgl nu compared with control rats ( 19.7 6.0 ngl versus 8.1 2.5 ngl , p < 0.05 ) . although there is an undeniable risk of radiological toxicity from orally ingested nu , the hazards of nu have been little investigated , especially after chronic exposure . the primary objective of this experimental study was to obtain new data to shed light on the long - term central effects of nu chronically ingested through drinking water at environmental doses . more specifically , we sought to obtain ( i ) a phenotypic brain signature associated with nu exposure and ( ii ) a comparison of the gender related response to nu to reveal any sexual dimorphism associated with exposure . the strength of the present study lies in its combination of a wide range of uranium levels ( 1.5 to 40 mgl ) , a large panel of behavioural tests ( locomotor activity , memory , anxiety , and depression ) , and the use of a highly relevant , innovative , and sensitive metabolomics approach , which yields a metabolic fingerprint relevant to nu exposure . body weight , food intake , and water consumption were evaluated as endpoints of the general toxicity of nu . they were not significantly affected by nu exposure in our experimental conditions , which is in accordance with previous studies in adult rats chronically exposed to du [ 27 , 28 ] . we observed no substantial brain weight loss or macroscopic brain tissue damage suggestive of deterioration in health status . these results are not surprising since low doses of nu were used in the present study . to study the potential adverse effects of uranium on the neurobehaviour of rats chronically exposed to nu , we first investigated total activity . the open - field procedure involves primary motor activity , evaluated by calculating the number of line crossings , and exploratory activity , evaluated by calculating the number of rearings on the hind limbs . in our experimental conditions , motor activity and exploratory activity were not significantly modified in male rats exposed to nu , regardless of the dose . these results are in accordance with a report that there was no significant effect on motor / exploratory activity after 9-month exposure to 40 mgl du or 4%-eu in adult rats . however , the opposite results , that is , hyperactivity or hypoactivity , in terms of line crossing and rearing or the distance travelled , have also been observed in rats exposed to du [ 2931 ] . locomotor activity is closely related to the cholinergic system and exploratory rearing behaviour is evidently related to glutamatergic mechanisms . it is also known that dopamine modulates glutamatergic inputs in the brain and that dopamine - excitatory amino acid interaction is involved in the locomotor behaviour . verification is therefore needed where uranium - induced impairment of activity is mediated by changes in glutamatergic and dopaminergic neurotransmitters . we also analysed the effects of nu on emotional behaviour using the forced swimming test , which is generally considered as an animal model of depression . forced swimming data revealed that male rats that received the highest dose of nu showed increased susceptibility to depressive - like behaviour . the results of the present study suggest also that exposure to 40 mgl nu significantly affects anxiety - like behaviour in the elevated plus maze test , one of the tests most frequently used in behavioural psychopharmacology to assess the potential anxiolytic properties of drugs . this result is consistent with previous data demonstrating a significant effect on anxiety - like behaviour in rats exposed to 4%-eu or du since birth [ 27 , 37 ] . monoamine transport systems might play a physiological role in the response to mood disorders induced by uranium . among them , oct2 , a member of the polyspecific organic cation transporter family , is expressed notably in the limbic system , is implicated in anxiety and depression - related behaviour , and could be a target of uranium . data obtained with the y - maze test indicated that exposure of male rats to 40 mgl nu resulted in impairment of spatial working memory , as previously observed in adult rats exposed to 4%-eu for 9 months , but not in adult rats exposed to du for 9 months , suggesting that the percent of u plays a role . whatever the dose used in the present experimental study these results suggest that sensitivity to nu may differ according to the kind of memory studied . the hippocampus for working spatial memory and the hippocampus / entorhinal cortex for medium - term memory may be responsible for this differential sensitivity . our behavioural tests showed a strong gender effect , with changes more evidently in males than in females . the single study to date of the involvement of sexual dimorphism in the effects of uranium on behaviour showed increased locomotor activity in male rats , but not females , after ingestion of du . brain differences between males and females are a common phenomenon , since sexual differentiation in the brain takes place during a perinatal sensitive window as a result of gonadal steroid hormone - induced developmental organisation . there is considerable evidence for the involvement of sex steroid hormones , such as oestrogen and androgen , in neurotransmitter systems and consequently in possible interactions with cognitive impairment . a noael ( no - observed - adverse - effect level ) threshold , less than 1.5 mgl , may be suggested on the basis of these observed behavioural effects . recently demonstrated that the brain is the organ most sensitive to chronic exposure by du ingestion . it is becoming evident that uptake transporters are essential in mediating the entry of large numbers of xenobiotics into cells . some transporters present at the blood - brain barrier , such as organic anion transporting polypeptide 1c1 ( oatp1c1 ) and monocarboxylate transporter 8 ( mct8 ) , may alter brain development , locomotion , or cognition . this raises the question of their role after nu exposure and thus opens up new perspectives in studying the mechanisms of its toxicity . the exact mechanisms underlying these neurotoxic effects of uranium have not yet been specifically addressed and are probably complex . legrand et al . have demonstrated that some steps of neurogenesis , that is , cell proliferation and cell death , are disturbed during prenatal and postnatal brain development after du exposure . these effects on neurogenesis could impair synaptic plasticity and might cause cognitive dysfunction in adulthood . we used a ms - based metabolomics approach to reveal any metabolic disruption associated to nu exposure that we found highly suitable to reveal low - dose radionuclide contamination [ 13 , 14 ] . the objective of this preliminary study was to use csf metabolomics to discriminate between groups of rats exposed or not to nu and to determine whether there is a difference between males and females . we have shown here that some variables specifically discriminated either male and/or female rats exposed to nu from controls at the onset of behavioural deficits , whereas others were only nu specific , irrespective of gender . among these latter variables that discriminated exposed and nonexposed rats to nu , 4 metabolites were putatively identified as n2-succinyl - l - arginine , n4-acetylaminobutanoate , n - methylsalsolinol , and butyrate . this result demonstrates for the first time that nu has an effect on metabolism of csf . for instance , the two first belong to the metabolic pathway of arginine and proline and n - methylsalsolinol is implicated on the balance impairment between dopamine and acetylcholine . these results could be paralleled to studies using metabolomics as a diagnostic marker in neurodegenerative diseases and/or cognitive impairment . butyric acid which can be found in csf ( database accession number hmdb00039 ) is also an end - product of ketone bodies metabolism ( kegg pathway map00650 ) , which are compounds used by brain for alternative energy production to glucose . when injected in csf it has been associated with memory function as well as mood stabilization in rats . in conclusion , our study demonstrates that the behavioural approach and the application of metabolomics are relevant in the field of low - doses radiation toxicology . our finding is the first evaluation of the nu - induced health risk in the case of chronic environmental exposure . it suggests that exposure to low - dose nu during development and adulthood can have an impact on behaviour and on the csf metabolome , highlighting an impact on the brain function and activity in our rat model . the next question is to find out whether and how the changes in the csf metabolome are related to behavioural changes . the goal now is to continue the identification of these metabolites in order to understand signalling pathways that could explain the behavioural observed effects .

natural uranium ( nu ) , a component of the earth 's crust , is not only a heavy metal but also an alpha particle emitter , with chemical and radiological toxicity . populations may therefore be chronically exposed to nu through drinking water and food . since the central nervous system is known to be sensitive to pollutants during its development , we assessed the effects on the behaviour and the cerebrospinal fluid ( csf ) metabolome of rats exposed for 9 months from birth to nu via lactation and drinking water ( 1.5 , 10 , or 40 mgl1 for male rats and 40 mgl1 for female rats ) . medium - term memory decreased in comparison to controls in male rats exposed to 1.5 , 10 , or 40 mgl1 nu . in male rats , spatial working memory and anxiety- and depressive - like behaviour were only altered by exposure to 40 mgl1 nu and any significant effect was observed on locomotor activity . in female rats exposed to nu , only locomotor activity was significantly increased in comparison with controls . lc - ms metabolomics of csf discriminated the fingerprints of the male and/or female nu - exposed and control groups . this study suggests that exposure to environmental doses of nu from development to adulthood can have an impact on rat brain function .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

natural uranium ( nu ) is an alpha particle emitter radionuclide of the actinide series and a ubiquitous environmental trace metal found in almost all types of rocks , soils , plants , and water . surface water and especially ground water play a significant role in the migration and redistribution of this nuclide in the environment . consequently , populations may be exposed to nu in some countries and regions with high nu levels in drinking water [ 24 ] . the harmful effects on human health of high levels of nu in drinking water are naturally of great interest to the scientific community and the general public . although the central nervous system is a target organ for many toxic heavy metals and is a radiosensitive organ [ 68 ] few studies have looked for in vivo neurological and neurobehavioural effects following internal contamination with uranium . experimental studies show that after exposure , uranium can reach the brain and lead to neurobehavioural effects on locomotor activity , the sleep - wake cycle , memory , and anxiety . the toxicity of du is expected to be mainly chemical rather than radioactive , so the radiological hazards of uranium have been little investigated . metabolomics , the comprehensive analysis of a wide range of metabolites , provides a novel tool in the search for new biomarkers of exposure or diagnostic and is an alternative and complementary approach to establish more -omics techniques such as genomics , transcriptomics , or proteomics . metabolomics has recently been found efficient for identifying a discriminant metabolic signature of chronic low - dose cesium 137 or uranium contamination in urine of rats [ 13 , 14 ] . the metabolites in cerebrospinal fluid ( csf ) reflect central nervous system metabolism and the balance between blood and csf the purpose of the present work was to establish for the first time whether chronic exposure to nu can induce behavioural effects and whether the csf metabolome is modified . to mimic environmental contamination of drinking water , especially among children , who are known to be a sensitive subgroup in toxicology and radiobiology [ 17 , 18 ] , male rats were exposed from birth to adulthood , that is , over a continuous 9-month period , through lactation and next drinking water containing concentrations of nu known not to be toxic to the kidneys . we used nu concentrations of 1.5 , 10 , and 40 mgl , since the highest concentration of naturally occurring uranium in spring water is 12 mgl . in order to determine the influence of sex , female rats were exposed to the highest nu concentration ( 40 mgl ) female sprague - dawley rats ( n = 48 ) were purchased at gestational day 18 ( charles river , france ) and were individually housed under standard conditions ( 21 1c ) with a 12:00 h/12:00 h light / dark cycle ( lights on from 08:00 a.m. to 08:00 p.m. ) . one group was contaminated using mineral drinking water supplemented with nu ( in its uranyl nitrate form ; from areva ) at a concentration of 1.5 mgl ( dose about 0.04 mgday per female rat ) . a second group was contaminated using mineral drinking water supplemented with nu ( in its uranyl nitrate form ; from areva ) at a concentration of 10 mgl ( dose about 0.25 mgday per female rat ) . a third group was contaminated with nu in drinking water at a concentration of 40 mgl ( dose about 1 mgday per female rat ) . control mothers drank noncontaminated water ( fourth group ) . after weaning on postnatal day 21 , male pups were still exposed to nu via drinking water ( 1.5 , 10 , or 40 mgl ) until they reached 9 months of age . female pups were also still exposed to nu via drinking water ( 40 mgl ) until they reached 9 months of age . health parameters , that is , body weight , water consumption , and food intake , were measured at the end of nu exposure ( at 9 months of age ) . on the first and second days , each animal was individually placed in an open field ( 45 45 cm ) and was monitored by an automated activity monitoring system ( bioseb , chaville , france ) . lateral and horizontal movements were recorded over a 15 min session , only on the first day . on the third day when the rats were acclimated to the open field , they were tested in a two - object recognition task . after a 1-hour delay , the rat was returned to the open field and allowed to explore two objects , one identical to those presented at the first session ( familiar object ) and the other different ( novel object ) , for an additional 3 min period ( second session ) . spatial working memory was assessed on the fourth day in a y - maze with three arms ( 70 cm long , 50 cm high , 10 cm wide at the bottom , and 20 cm wide at the top ) which converged at an equal angle . each rat was placed at the centre of the maze and was allowed to move freely through the maze for a 10 min test session . anxiety was assessed on day five in an elevated plus maze comprising a wooden cross at a height of 70 cm with two open ( 10 cm70 cm ) and two closed arms with walls ( 10 cm55 cm70 cm ) , arranged such that the arms of the same type were opposite to each other and connected by a common open central platform ( 5 cm5 cm ) . at the beginning of the session , the rat was placed at the centre of the maze always facing the same open arm . standard spatiotemporal measures were recorded , including the number of entries in the open and closed arms and the cumulative time spent in the different parts of the maze ( open and closed arms ) . an arm entry was recorded if all four of the animal 's paws were in the arm . the rats were individually placed in a glass cylinder ( height of 60 cm and diameter of 40 cm ) , containing enough water such that the hind legs could not reach the bottom of the cylinder but the tail could . immobility was measured during the last 5 min of the test ( the animal was judged to be immobile when it floated in an upright position and made only minimal movements to keep its head above water ) . at the end of behavioural tests , each animal was euthanised with isoflurane , placed prone on the stereotaxic instrument and the head of the rat was fixed in a holder . for this purpose a skin incision was made followed by a horizontal incision in the descending part of the trapezius muscle to reveal the arachnoid membrane . for the analysis of csf , protein of 50 l was removed by methanol precipitation using 200 l of cold methanol ( 20c ) followed by 5 min centrifugation at 14000 rpm . to check for data quality , a blank sample ( deionised water ) and a pool sample ( a mixture of all csf samples ) the samples were analysed on a dionex ultimate 3000 ( thermo fisher scientific , france ) coupled to a q - exactive plus mass spectrometer ( thermo fisher scientific , france ) . to select the most discriminant variables , we found most appropriate to examine the clustering of features with the variable score values as calculated by hierarchical cluster analysis applied to wc loadings of the pls - da model . samples ( cerebral cortex and csf ) were prepared by adding 8 ml of 70% ultrapure nitric acid and 2 ml of hydrogen peroxide . the uranium content of samples was determined using an inductively coupled plasma mass spectrometer ( icpms - vgpq , excell , thermo electron corporation ) with bismuth ( 1 gl ) as internal standard . for uranium , values were expressed as nanograms per gram of fresh tissue or nanograms per l of csf and presented as mean sem . body weight was not significantly modified in rats exposed to 1.5 , 10 , or 40 mgl nu compared with the control group ( table 1 ) . food intake was also not significantly changed in nu - exposed rats in comparison with control rats ( table 1 ) . daily water consumption did not significantly change in rats exposed to 1.5 , 10 , or 40 mgl nu , when compared with control rats ( table 1 ) . in rats exposed to 1.5 , 10 , or 40 mgl nu , no significant effect on lines crossed or rearing the medium - term memory of rats was assessed by the time spent exploring novel and familiar objects . groups exposed to 1.5 , 10 , or 40 mgl nu did not prefer the novel object to the familiar object ( figure 2(b ) ) . this indicated a loss of medium - term memory in rats exposed to nu without dose effect . the spatial working memory capacities of rats were assessed by spontaneous alternation and number of arm visits , in the y - maze . however , in rats exposed to 40 mgl nu , a significant decrease in the percentage of spontaneous alternation was observed in comparison with the control group ( 16% , p < 0.05 ) ( figure 3(b ) ) . this decrease in alternation behaviour was not associated with changes of general locomotor activity , measured as the number of arm visits ( figure 3(a ) ) . for rats exposed to 1.5 or 10 mgl nu , no significant effect was found on the percentage alternation or on the number of visits to each arm , when compared with controls ( figure 3 ) . the anxiety - like behaviour of rats was assessed by the time spent in the closed arms and the number of closed arm entries in the elevated plus maze . rats exposed to 40 mgl nu spent significantly more time in the closed arms than did the controls ( 31% , p < 0.01 ) , the rats exposed to 1.5 mgl nu ( + 18% , p < 0.05 ) , and the rats exposed to 10 mgl nu ( + 18% , p < 0.05 ) ( figure 4(b ) ) . their number of visits to the closed arms did not differ significantly from that of the other groups ( figure 4(a ) ) . for rats exposed to 1.5 or 10 mgl nu , no significant difference was observed in the time spent in or the number of visits to the closed arms compared with controls ( figure 4 ) . the depressive - like behaviour of rats was assessed by the time they spent immobile during the 5 last minutes of the test . the immobility time did not differ in rats exposed to 1.5 or 10 mgl nu in comparison with control rats ( figure 5 ) , but it increased significantly ( + 163% , p < 0.05 ) when rats were exposed to 40 mgl for 9 months ( figure 5 ) . as this parameter is usually used to evaluate depressive - like behaviour , this result indicates that the depressive - like behaviour was not affected by exposure to 1.5 or 10 mgl nu but increased after 9 months of exposure to 40 mgl nu . in females exposed to nu compared with controls ( figures 6(a1 ) and 6(c1 ) ) . the number of rearings during the open - field test , spontaneous alternation and the number of arm visits in the y - maze , the time spent in the closed arms of the elevated plus maze , and the immobility time during the forced swimming test were not significantly modified in female rats exposed to 40 mgl nu compared with control female rats ( figure 6 ) . during the object recognition test , exposed and controls groups spent significantly more time exploring the novel object than the familiar object ( figure 6(d ) ) . the variables responsible for the discrimination between control rats and rats exposed to 40 mgl nu are shown in figure 7 . using principal component analysis , pls - da and hierarchical ascendant classification , a model was created with the 86 most discriminating variables from the initial 1244 detected csf analytical features . from this model , we were able to observe and select the best discriminating variables of the 86 significant ones that discriminate control from exposed rats , for male , female , and both animals . when comparing males to females , from the top 18 control to nu discriminating variables , 7 were found exclusively related to female rats from control rats and 7 others to male rats . four variables were common for female rats and male rats , corresponding to variables discriminating exposed rats versus control rats , regardless of gender ( figure 7 ) . these 4 variables have been putatively identified as n2-succinyl - l - arginine , n4-acetylaminobutanoate , and n - methylsalsolinol , which decreased in nu - exposed rats compared to control rats , and butyric acid , which increased in nu - exposed rats ( table 2 ) . this metabolomics analysis thus showed that some metabolites differed in the csf of male versus female and the nu - exposed versus control groups , whereas others were only specific of nu exposure , irrespective of gender . uranium concentrations in the cortex of male rats exposed to 10 or 40 mgl nu were significantly increased ( resp . , + 110% and + 218% ) in comparison with control rats ( table 1 ) . no significant difference was observed between rats exposed to 1.5 mgl nu and control rats ( table 1 ) . the concentration of uranium in csf increased significantly in male rats exposed to 40 mgl nu compared with control rats ( 19.7 6.0 ngl versus 8.1 2.5 ngl , p < 0.05 ) . although there is an undeniable risk of radiological toxicity from orally ingested nu , the hazards of nu have been little investigated , especially after chronic exposure . the primary objective of this experimental study was to obtain new data to shed light on the long - term central effects of nu chronically ingested through drinking water at environmental doses . more specifically , we sought to obtain ( i ) a phenotypic brain signature associated with nu exposure and ( ii ) a comparison of the gender related response to nu to reveal any sexual dimorphism associated with exposure . the strength of the present study lies in its combination of a wide range of uranium levels ( 1.5 to 40 mgl ) , a large panel of behavioural tests ( locomotor activity , memory , anxiety , and depression ) , and the use of a highly relevant , innovative , and sensitive metabolomics approach , which yields a metabolic fingerprint relevant to nu exposure . body weight , food intake , and water consumption were evaluated as endpoints of the general toxicity of nu . they were not significantly affected by nu exposure in our experimental conditions , which is in accordance with previous studies in adult rats chronically exposed to du [ 27 , 28 ] . these results are not surprising since low doses of nu were used in the present study . to study the potential adverse effects of uranium on the neurobehaviour of rats chronically exposed to nu , we first investigated total activity . the open - field procedure involves primary motor activity , evaluated by calculating the number of line crossings , and exploratory activity , evaluated by calculating the number of rearings on the hind limbs . in our experimental conditions , motor activity and exploratory activity were not significantly modified in male rats exposed to nu , regardless of the dose . these results are in accordance with a report that there was no significant effect on motor / exploratory activity after 9-month exposure to 40 mgl du or 4%-eu in adult rats . however , the opposite results , that is , hyperactivity or hypoactivity , in terms of line crossing and rearing or the distance travelled , have also been observed in rats exposed to du [ 2931 ] . locomotor activity is closely related to the cholinergic system and exploratory rearing behaviour is evidently related to glutamatergic mechanisms . we also analysed the effects of nu on emotional behaviour using the forced swimming test , which is generally considered as an animal model of depression . forced swimming data revealed that male rats that received the highest dose of nu showed increased susceptibility to depressive - like behaviour . the results of the present study suggest also that exposure to 40 mgl nu significantly affects anxiety - like behaviour in the elevated plus maze test , one of the tests most frequently used in behavioural psychopharmacology to assess the potential anxiolytic properties of drugs . this result is consistent with previous data demonstrating a significant effect on anxiety - like behaviour in rats exposed to 4%-eu or du since birth [ 27 , 37 ] . among them , oct2 , a member of the polyspecific organic cation transporter family , is expressed notably in the limbic system , is implicated in anxiety and depression - related behaviour , and could be a target of uranium . data obtained with the y - maze test indicated that exposure of male rats to 40 mgl nu resulted in impairment of spatial working memory , as previously observed in adult rats exposed to 4%-eu for 9 months , but not in adult rats exposed to du for 9 months , suggesting that the percent of u plays a role . whatever the dose used in the present experimental study these results suggest that sensitivity to nu may differ according to the kind of memory studied . the hippocampus for working spatial memory and the hippocampus / entorhinal cortex for medium - term memory may be responsible for this differential sensitivity . our behavioural tests showed a strong gender effect , with changes more evidently in males than in females . the single study to date of the involvement of sexual dimorphism in the effects of uranium on behaviour showed increased locomotor activity in male rats , but not females , after ingestion of du . a noael ( no - observed - adverse - effect level ) threshold , less than 1.5 mgl , may be suggested on the basis of these observed behavioural effects . recently demonstrated that the brain is the organ most sensitive to chronic exposure by du ingestion . some transporters present at the blood - brain barrier , such as organic anion transporting polypeptide 1c1 ( oatp1c1 ) and monocarboxylate transporter 8 ( mct8 ) , may alter brain development , locomotion , or cognition . these effects on neurogenesis could impair synaptic plasticity and might cause cognitive dysfunction in adulthood . we used a ms - based metabolomics approach to reveal any metabolic disruption associated to nu exposure that we found highly suitable to reveal low - dose radionuclide contamination [ 13 , 14 ] . the objective of this preliminary study was to use csf metabolomics to discriminate between groups of rats exposed or not to nu and to determine whether there is a difference between males and females . we have shown here that some variables specifically discriminated either male and/or female rats exposed to nu from controls at the onset of behavioural deficits , whereas others were only nu specific , irrespective of gender . among these latter variables that discriminated exposed and nonexposed rats to nu , 4 metabolites were putatively identified as n2-succinyl - l - arginine , n4-acetylaminobutanoate , n - methylsalsolinol , and butyrate . for instance , the two first belong to the metabolic pathway of arginine and proline and n - methylsalsolinol is implicated on the balance impairment between dopamine and acetylcholine . butyric acid which can be found in csf ( database accession number hmdb00039 ) is also an end - product of ketone bodies metabolism ( kegg pathway map00650 ) , which are compounds used by brain for alternative energy production to glucose . our finding is the first evaluation of the nu - induced health risk in the case of chronic environmental exposure . it suggests that exposure to low - dose nu during development and adulthood can have an impact on behaviour and on the csf metabolome , highlighting an impact on the brain function and activity in our rat model .

the combined dynamic derivative , also called diamond- ( ) dynamic derivative ( ) , was introduced as a linear convex combination of the well - known delta and nabla dynamic derivatives on time scales . by a time scale using the delta and nabla derivatives , the notions of delta and nabla integrals were defined ( see ) . we assume , throughout this paper , that the basic notions of the time scales are well known and understood . let f : be continuous function and let a , b . then , the diamond- integral of f from a to b is defined by ( 1)abf(t)t=abf(t)t+(1)abf(t)t , aaaaaaaaaaaaaaaaaiaaaaaii01 . let f : be continuous function and let a , b . then , the diamond- integral of f from a to b is defined by ( 1)abf(t)t=abf(t)t+(1)abf(t)t , aaaaaaaaaaaaaaaaaiaaaaaii01 . it is clear that for = 1 the diamond- integral reduces to the standard delta integral and for = 0 the diamond- integral reduces to the standard nabla integral.moreover , if = , then ( 2)abf(t)t=abf(t)dt ; if = , then ( 3)abf(t)t=t = ab1f(t)+(1)t = a+1bf(t ) ; if = h , where h > 0 , then ( 4)abf(t)t = h(k = a / hb / h1f(kh)+(1)k = a / h+1b / hf(kh ) ) ; if = q , where q > 1 , then ( 5)abf(t)t=(q1)(k = logq(a)logq(b)1qkf(qk)aaaaaaiaaa+(1)k = logq(a)+1logq(b)qk1f(qk ) ) . from the above definition it is clear that for = 1 the diamond- integral reduces to the standard delta integral and for = 0 the diamond- integral reduces to the standard nabla integral . moreover , if = , then ( 2)abf(t)t=abf(t)dt ; if = , then ( 3)abf(t)t=t = ab1f(t)+(1)t = a+1bf(t ) ; if = h , where h > 0 , then ( 4)abf(t)t = h(k = a / hb / h1f(kh)+(1)k = a / h+1b / hf(kh ) ) ; if = q , where q > 1 , then ( 5)abf(t)t=(q1)(k = logq(a)logq(b)1qkf(qk)aaaaaaiaaa+(1)k = logq(a)+1logq(b)qk1f(qk ) ) . recently , the jensen inequality , the improvement of the jensen inequality , and their converses are given for time scale integrals ( see [ 24 ] ) . let be a time scale , and let a , b . then the time scale interval is denoted and defined by [ a , b] = [ a , b]. theorem 3 ( see ) . let c , d . if g c([a , b] , ( c , d ) ) , w c([a , b] , ) with a | w(t ) | t > 0 and c((c , d ) , ) is convex , then ( 6)(abg(t)|w(t)|tab|w(t)|t)ab(g(t))|w(t)|tab|w(t)|t . let c , d . if g c([a , b] , ( c , d ) ) , w c([a , b] , ) with a | w(t ) | t > 0 and c((c , d ) , ) is convex , then ( 6)(abg(t)|w(t)|tab|w(t)|t)ab(g(t))|w(t)|tab|w(t)|t . note that in this jensen inequality we have nonnegative weights . in order to give a better version of this jensen inequality on time scales , dinu then , w c( , ) is an -steffensen - popoviciu ( -sp ) weight for g on [ a , b] if ( 7)abw(t)t>0 , ab(g(t))+w(t)t0 , for every convex function c([m , m ] , ) , where ( 8)m = inft[a , b]tg(t ) , m = supt[a , b]tg(t ) . then , w c( , ) is an -steffensen - popoviciu ( -sp ) weight for g on [ a , b] if ( 7)abw(t)t>0 , ab(g(t))+w(t)t0 , for every convex function c([m , m ] , ) , where ( 8)m = inft[a , b]tg(t ) , m = supt[a , b]tg(t ) . in the following lemma he gives a characterization for -sp weight for a nondecreasing function g on time scales . then , w is an -sp weight for a nondecreasing function g c([a , b] , ) if and only if it verifies the following condition : ( 9)as(g(s)g(t))w(t)t0,sb(g(t)g(s))w(t)t0 , for every s [ a , b].if the following stronger ( but more suitable ) condition holds ( 10)0asw(t)tabw(t)t for every s[a , b]t , then w is also an -sp weight for the nondecreasing continuous function g. let w c( , ) such that aw(t)t > 0 . then , w is an -sp weight for a nondecreasing function g c([a , b] , ) if and only if it verifies the following condition : ( 9)as(g(s)g(t))w(t)t0,sb(g(t)g(s))w(t)t0 , for every s [ a , b]. if the following stronger ( but more suitable ) condition holds ( 10)0asw(t)tabw(t)t for every s[a , b]t , then w is also an -sp weight for the nondecreasing continuous function g. as given in , all positive weights are -sp weights , for any continuous function g and every . the jensen inequality on time scales , where it is allowed that the weight function takes some negative values , is given in the following theorem . let g c([a , b] , [ m , m ] ) and let w c([a , b] , ) such that aw(t)t > 0 . then , the following two statements are equivalent : ( i ) w is an -sp weight for g on [ a , b];(ii ) for every convex function c([m , m ] , ) , it holds ( 11)(abg(t)w(t)tabw(t)t)ab(g(t))w(t)tabw(t)t . let g c([a , b] , [ m , m ] ) and let w c([a , b] , ) such that aw(t)t > 0 . then , the following two statements are equivalent : ( i ) w is an -sp weight for g on [ a , b];(ii ) for every convex function c([m , m ] , ) , it holds ( 11)(abg(t)w(t)tabw(t)t)ab(g(t))w(t)tabw(t)t . w is an -sp weight for g on [ a , b] ; for every convex function c([m , m ] , ) , it holds ( 11)(abg(t)w(t)tabw(t)t)ab(g(t))w(t)tabw(t)t . remark 7 . let g be nondecreasing function . if = , then theorem 6 is equivalent to the jensen - steffensen inequality given by steffensen in ( see also [ 6 , page 57 ] ) . on the other hand if we take = in theorem 6 , we obtain the integral version of the jensen - steffensen inequality given by boas ( see also [ 6 , page 59 ] ) . if = , then theorem 6 is equivalent to the jensen - steffensen inequality given by steffensen in ( see also [ 6 , page 57 ] ) . on the other hand if we take = in theorem 6 , we obtain the integral version of the jensen - steffensen inequality given by boas ( see also [ 6 , page 59 ] ) . considering the converse of the jensen inequality , dinu gives the following definition of -hermite - hadamard ( -hh ) weight , its characterization for a nondecreasing function g on time scales , and the improvement of the converse of the jensen inequality for some negative weights . then w c( , ) is an -hermite - hadamard ( -hh ) weight for g on [ a , b] if ( 12)abw(t)t>0,ab(g(t))w(t)tabw(t)tmgmm(m)+gmmm(m ) , for every convex function c([m , m ] , ) , where ( 13)m = inft[a , b]tg(t ) , m = supt[a , b]tg(t),g=abg(t)w(t)tabw(t)t . then w c( , ) is an -hermite - hadamard ( -hh ) weight for g on [ a , b] if ( 12)abw(t)t>0,ab(g(t))w(t)tabw(t)tmgmm(m)+gmmm(m ) , for every convex function c([m , m ] , ) , where ( 13)m = inft[a , b]tg(t ) , m = supt[a , b]tg(t),g=abg(t)w(t)tabw(t)t . let w c([a , b] , ) be such that aw(t)t > 0 . then w is an -hh weight for a nondecreasing function g c([a , b] , ) if and only if it verifies the following condition : ( 14)g(b)g(s)g(b)g(a)as(g(t)g(a))w(t)t + g(s)g(a)g(b)g(a)sb(g(b)g(t))w(t)t0 for every s [ a , b]. let w c([a , b] , ) be such that aw(t)t > 0 . then w is an -hh weight for a nondecreasing function g c([a , b] , ) if and only if it verifies the following condition : ( 14)g(b)g(s)g(b)g(a)as(g(t)g(a))w(t)t + g(s)g(a)g(b)g(a)sb(g(b)g(t))w(t)t0 for every s [ a , b]. in the next result dinu gives the connection between these two classes of weights on a time scale . every -sp weight for g on [ a , b] is an -hh weight for g on [ a , b] , for all . every -sp weight for g on [ a , b] is an -hh weight for g on [ a , b] , for all . in the following two sections of our paper we give some further generalizations of the jensen - type inequalities on time scales allowing negative weights , and we also give the mean - value theorems of the lagrange and cauchy type for the functionals obtained by taking the difference of the left - hand side and right - hand side of these new inequalities . these results also generalize the results given in for continuous and discrete cases . section 4 in our paper deals with exponential convexity and logarithmic convexity of the functionals obtained in two previous sections . finally , in section 5 we present several families of exponentially convex functions which fulfil the conditions of our results . the results from sections 4 and 5 generalize the results given in for continuous and discrete cases . let m , m , where m m. consider the green function g : [ m , m][m , m ] defined by ( 15)g(x , y)={(xm)(ym)mmfor myx,(ym)(xm)mmfor xym . the function g is convex and continuous with respect to both x and y. it is well known that ( see , e.g. , [ 811 ] ) any function c([m , m ] , ) can be represented by ( 16)(x)=mxmm(m)+xmmm(m)+mmg(x , y)(y)dy , where the function g is defined in ( 15 ) . using ( 16 ) , we now derive several interesting results concerning the jensen - type inequalities . in the following theorem , we give the generalization of the jensen inequality on time scales , where negative weights are also allowed . let g c([a , b] , ) be such that g([a , b])[m , m ] . let w c([a , b] , ) be such that aw(t)t 0 and ag(t)w(t)t/aw(t)t [ m , m ] . then , the following two statements are equivalent : ( i)for every convex function c([m , m ] , )(17)(abg(t)w(t)tabw(t)t)ab(g(t))w(t)tabw(t)t holds;(ii)for all y [ m , m](18)g(abg(t)w(t)tabw(t)t , y)abg(g(t),y)w(t)tabw(t)t holds , where g : [ m , m][m , m ] is defined in ( 15).furthermore , the statements ( i ) and ( ii ) are also equivalent if we change the sign of inequality in both ( 17 ) and ( 18 ) . let g c([a , b] , ) be such that g([a , b])[m , m ] . let w c([a , b] , ) be such that aw(t)t 0 and ag(t)w(t)t/aw(t)t [ m , m ] . then , the following two statements are equivalent : ( i)for every convex function c([m , m ] , )(17)(abg(t)w(t)tabw(t)t)ab(g(t))w(t)tabw(t)t holds;(ii)for all y [ m , m](18)g(abg(t)w(t)tabw(t)t , y)abg(g(t),y)w(t)tabw(t)t holds , where g : [ m , m][m , m ] is defined in ( 15).furthermore , the statements ( i ) and ( ii ) are also equivalent if we change the sign of inequality in both ( 17 ) and ( 18 ) . for every convex function c([m , m ] , )(17)(abg(t)w(t)tabw(t)t)ab(g(t))w(t)tabw(t)t holds ; for all y [ m , m](18)g(abg(t)w(t)tabw(t)t , y)abg(g(t),y)w(t)tabw(t)t holds , where g : [ m , m][m , m ] is defined in ( 15 ) . proof(i ) ( ii ) : let ( i ) hold . as the function g( , y ) ( y [ m , m ] ) is continuous and convex , it follows that ( 18 ) holds.(ii ) ( i ) : let ( ii ) hold . then by using ( 16 ) we get ( 19)ab(g(t))w(t)tabw(t)t(abg(t)w(t)tabw(t)t ) = mm[abg(g(t),y)w(t)tabw(t)taaaaaaaii g(abg(t)w(t)tabw(t)t , y)](y)dy . if the function is also convex , then (y ) 0 for all y [ m , m ] , and hence it follows that for every convex function c([m , m ] , ) inequality ( 17 ) holds . moreover , it is not necessary to demand the existence of the second derivative of the function ( see [ 6 , page 172 ] ) . the differentiability condition can be directly eliminated by using the fact that it is possible to approximate uniformly a continuous convex function by convex polynomials.the last part of our theorem can be proved analogously . as the function g( , y ) ( y [ m , m ] ) is continuous and convex , it follows that ( 18 ) holds . then by using ( 16 ) we get ( 19)ab(g(t))w(t)tabw(t)t(abg(t)w(t)tabw(t)t ) = mm[abg(g(t),y)w(t)tabw(t)taaaaaaaii g(abg(t)w(t)tabw(t)t , y)](y)dy . if the function is also convex , then (y ) 0 for all y [ m , m ] , and hence it follows that for every convex function c([m , m ] , ) inequality ( 17 ) holds . moreover , it is not necessary to demand the existence of the second derivative of the function ( see [ 6 , page 172 ] ) . the differentiability condition can be directly eliminated by using the fact that it is possible to approximate uniformly a continuous convex function by convex polynomials . ( i)for every concave function c([m , m ] , ) the reverse inequality in ( 17 ) holds.(ii)for all y [ m , m ] inequality ( 18 ) holds.moreover , statements ( i ) and ( ii ) are also equivalent if we change the sign of inequality in both statements ( i ) and ( ii ) . ( i)for every concave function c([m , m ] , ) the reverse inequality in ( 17 ) holds.(ii)for all y [ m , m ] inequality ( 18 ) holds.moreover , statements ( i ) and ( ii ) are also equivalent if we change the sign of inequality in both statements ( i ) and ( ii ) . for every concave function c([m , m ] , ) the reverse inequality in ( 17 ) holds . for all y [ m , m ] inequality ( 18 ) holds . let m = g(a ) and m = g(b ) , and make the substitution y = g(s ) . since g is nondecreasing , we have that g(s ) 0 for all s [ a , b]. if c([m , m ] , ) is convex , then (g(s ) ) 0 for all s [ a , b]. hence , if and only if ( 21)g(abg(t)w(t)tabw(t)t , g(s))abg(g(t),g(s))w(t)tabw(t)t holds for all s [ a , b] , then for every continuous convex function inequality ( 17 ) holds . consider ( 19 ) . let m = g(a ) and m = g(b ) , and make the substitution y = g(s ) . since g is nondecreasing , we have that g(s ) 0 for all s [ a , b]. if c([m , m ] , ) is convex , then (g(s ) ) 0 for all s [ a , b]. hence , if and only if ( 21)g(abg(t)w(t)tabw(t)t , g(s))abg(g(t),g(s))w(t)tabw(t)t holds for all s [ a , b] , then for every continuous convex function inequality ( 17 ) holds . combining the result from theorem 11 with theorem 6 and lemma 5 , we get the following two corollaries . corollary 14 . let g c([a , b] , [ m , m ] ) and let w c([a , b] , ) be such that aw(t)t > 0 . then w is an -sp weight for g on [ a , b] if and only if ( 22)g(abg(t)w(t)tabw(t)t , y)abg(g(t),y)w(t)tabw(t)t holds for all y [ m , m ] , where g is defined in ( 15 ) . let g c([a , b] , [ m , m ] ) and let w c([a , b] , ) be such that aw(t)t > 0 . then w is an -sp weight for g on [ a , b] if and only if ( 22)g(abg(t)w(t)tabw(t)t , y)abg(g(t),y)w(t)tabw(t)t holds for all y [ m , m ] , where g is defined in ( 15 ) . corollary 15 . let g c([a , b] , [ m , m ] ) be nondecreasing function and w c([a , b] , ) such that aw(t)t > 0 . then ( 23)as(g(s)g(t))w(t)t0,sb(g(t)g(s))w(t)t0 hold for all s [ a , b] , if and only if ( 24)g(abg(t)w(t)tabw(t)t , y)abg(g(t),y)w(t)tabw(t)t holds for all y [ m , m ] , where g is defined in ( 15 ) . let g c([a , b] , [ m , m ] ) be nondecreasing function and w c([a , b] , ) such that aw(t)t > 0 . then ( 23)as(g(s)g(t))w(t)t0,sb(g(t)g(s))w(t)t0 hold for all s [ a , b] , if and only if ( 24)g(abg(t)w(t)tabw(t)t , y)abg(g(t),y)w(t)tabw(t)t holds for all y [ m , m ] , where g is defined in ( 15 ) . to shorten the notation , in the sequel we will use the following notation : ( 25)g=abg(t)w(t)tabw(t)t . under the assumptions of theorem 11 , we define the following functional 1(g , ) : ( 26)j1(g,)={ab(g(t))w(t)tabw(t)t(abg(t)w(t)tabw(t)t ) if y[m , m ] inequality ( 18 ) holds,(abg(t)w(t)tabw(t)t)ab(g(t))w(t)tabw(t)t if y[m , m ] the reverse inequality in ( 18 ) holds , where the function is defined on [ m , m ] . clearly , if is continuous and convex , then 1(g , ) is nonnegative . let g c([a , b] , ) and c([m , m ] , ) . [ m , m ] such that ( 27)j1(g,)=()j1(g,0 ) holds , where 0(x ) = x/2 . let g c([a , b] , ) and c([m , m ] , ) . . then there exists [ m , m ] such that ( 27)j1(g,)=()j1(g,0 ) holds , where 0(x ) = x/2 . proofsince the function is continuous and ( 28)abg(g(t),y)w(t)tabw(t)tg(abg(t)w(t)tabw(t)t , y ) does not change its positivity on [ m , m ] , applying the integral mean - value theorem on ( 19 ) we get that there exists [ m , m ] such that ( 29)ab(g(t))w(t)tabw(t)t(abg(t)w(t)tabw(t)t ) = ()mm[abg(g(t),y)w(t)tabw(t)taaaaaaaaaaaaaai g(abg(t)w(t)tabw(t)t , y)]dy holds . as in , it can be easily checked that it holds ( 30)mmg(x , y)dy=mx(xm)(ym)mmdy + xm(ym)(xm)mmdy=12(xm)(xm ) . calculating the integral on the right - hand side of ( 29 ) , we get ( 31)ab(g(t))w(t)tabw(t)t(abg(t)w(t)tabw(t)t ) = ()[1abw(t)taaaaaaaaiiaaab(mmg(g(t),y)dy)w(t)taaaaaaaaaaammg(g,y)dy1abw(t)t ] = ()[1abw(t)tab12(g(t)m)aaaaaaaaaaaaaaaaaaaaaai(g(t)m)w(t)taaaaaaaaaai12(gm)(gm)1abw(t)t ] = 12()[ab(g(t))2w(t)tabw(t)tg2 ] and the proof is completed . since the function is continuous and ( 28)abg(g(t),y)w(t)tabw(t)tg(abg(t)w(t)tabw(t)t , y ) does not change its positivity on [ m , m ] , applying the integral mean - value theorem on ( 19 ) we get that there exists [ m , m ] such that ( 29)ab(g(t))w(t)tabw(t)t(abg(t)w(t)tabw(t)t ) = ()mm[abg(g(t),y)w(t)tabw(t)taaaaaaaaaaaaaai g(abg(t)w(t)tabw(t)t , y)]dy holds . as in , it can be easily checked that it holds ( 30)mmg(x , y)dy=mx(xm)(ym)mmdy + xm(ym)(xm)mmdy=12(xm)(xm ) . calculating the integral on the right - hand side of ( 29 ) , we get ( 31)ab(g(t))w(t)tabw(t)t(abg(t)w(t)tabw(t)t ) = ()[1abw(t)taaaaaaaaiiaaab(mmg(g(t),y)dy)w(t)taaaaaaaaaaammg(g,y)dy1abw(t)t ] = ()[1abw(t)tab12(g(t)m)aaaaaaaaaaaaaaaaaaaaaai(g(t)m)w(t)taaaaaaaaaai12(gm)(gm)1abw(t)t ] = 12()[ab(g(t))2w(t)tabw(t)tg2 ] and the proof is completed . remark 17 . theorem 16 can also be proved by using the following two convex functions : ( 32)1(x)=2x2(x ) , 2(x)=(x)2x2 , where ( 33)=minx[m , m](x ) , =maxx[m , m](x ) . since 1 and 2 are continuous and convex , we have ( 34)j1(g,1)0 , j1(g,2)0 . hence , as the function is continuous , there exists [ m , m ] such that ( 27 ) holds . theorem 16 can also be proved by using the following two convex functions : ( 32)1(x)=2x2(x ) , 2(x)=(x)2x2 , where ( 33)=minx[m , m](x ) , =maxx[m , m](x ) . since 1 and 2 are continuous and convex , we have ( 34)j1(g,1)0 , j1(g,2)0 . let g c([a , b] , ) and , c([m , m ] , ) . . then there exists [ m , m ] such that ( 36)j1(g,)j1(g,)=()( ) holds , provided that the denominator on the left - hand side of ( 36 ) is nonzero . let g c([a , b] , ) and , c([m , m ] , ) . . then there exists [ m , m ] such that ( 36)j1(g,)j1(g,)=()( ) holds , provided that the denominator on the left - hand side of ( 36 ) is nonzero . prooflet be defined as the linear combination of functions and by ( 37)(x)=j1(g,)(x)j1(g,)(x ) . then c([m , m ] , ) . by applying theorem 16 on , it follows that there exists [ m , m ] such that ( 38)j1(g,)=()j1(g,0 ) . after a short calculation we get that 1(g , ) = 0 . by hypothesis 1(g , 0 ) 0 ( otherwise , we have a contradiction with 1(g , ) 0 ) , so it follows that ( 39)()=0 , which is equivalent to ( 36 ) . let be defined as the linear combination of functions and by ( 37)(x)=j1(g,)(x)j1(g,)(x ) . then c([m , m ] , ) . by applying theorem 16 on , it follows that there exists [ m , m ] such that ( 38)j1(g,)=()j1(g,0 ) . after a short calculation we get that 1(g , ) = 0 . by hypothesis 1(g , 0 ) 0 ( otherwise , we have a contradiction with 1(g , ) 0 ) , so it follows that ( 39)()=0 , which is equivalent to ( 36 ) . if the inverse of the function / exists , then ( 36 ) gives ( 40)=()1(j1(g,)j1(g, ) ) . in theorem 18 , if the inverse of the function / exists , then ( 36 ) gives ( 40)=()1(j1(g,)j1(g, ) ) . remark 20 . note that setting the function as (x ) = x/2 in theorem 18 , we get the statement of theorem 16 . note that setting the function as (x ) = x/2 in theorem 18 , we get the statement of theorem 16 . as a consequence of the above two mean - value theorems , the following corollaries easily follow . let g c([a , b] , [ m , m ] ) , , : [ m , m ] , and let w c([a , b] , ) be an -sp weight for g. let 1 be defined as in ( 26 ) . then the following two statements hold . ( i)if c([m , m ] , ) , then there exists [ m , m ] such that ( 27 ) holds.(ii)if , c([m , m ] , ) , then there exists [ m , m ] such that ( 36 ) holds . let g c([a , b] , [ m , m ] ) , , : [ m , m ] , and let w c([a , b] , ) be an -sp weight for g. let 1 be defined as in ( 26 ) . then the following two statements hold . ( i)if c([m , m ] , ) , then there exists [ m , m ] such that ( 27 ) holds.(ii)if , c([m , m ] , ) , then there exists [ m , m ] such that ( 36 ) holds . if c([m , m ] , ) , then there exists [ m , m ] such that ( 27 ) holds . if , c([m , m ] , ) , then there exists [ m , m ] such that ( 36 ) holds . proofthe statement ( i ) ( statement ( ii ) , resp . , ) directly follows from theorem 16 ( theorem 18 , resp . , ) and corollary 14 . the statement ( i ) ( statement ( ii ) , resp . , ) directly follows from theorem 16 ( theorem 18 , resp . , ) and corollary 14 . let g c([a , b] , [ m , m ] ) be monotone function , , : [ m , m ] , and w c([a , b] , ) such that aw(t)t > 0 . let ( 9 ) hold for all s [ a , b]. let 1 be defined as in ( 26 ) . then the following two statements hold . ( i)if c([m , m ] , ) , then there exists [ m , m ] such that ( 27 ) holds.(ii)if , c([m , m ] , ) , then there exists [ m , m ] such that ( 36 ) holds . let g c([a , b] , [ m , m ] ) be monotone function , , : [ m , m ] , and w c([a , b] , ) such that aw(t)t > 0 . let ( 9 ) hold for all s [ a , b]. let 1 be defined as in ( 26 ) ( i)if c([m , m ] , ) , then there exists [ m , m ] such that ( 27 ) holds.(ii)if , c([m , m ] , ) , then there exists [ m , m ] such that ( 36 ) holds . if c([m , m ] , ) , then there exists [ m , m ] such that ( 27 ) holds . if , c([m , m ] , ) , then there exists [ m , m ] such that ( 36 ) holds . proofthe statement ( i ) ( statement ( ii ) , resp . , ) directly follows from theorem 16 ( theorem 18 , resp . , ) and corollary 15 . the statement ( i ) ( statement ( ii ) , resp . , ) directly follows from theorem 16 ( theorem 18 , resp . , ) and corollary 15 . using the similar method as in previous section , in the following theorem we obtain the generalization of the converse of the jensen inequality on time scales , where negative weights are also allowed . let g c([a , b] , ) be such that g([a , b])[m , m ] and let c , d [ m , m ] ( c d ) be such that c g(t ) d for all t [ a , b]. let w c([a , b] , ) be such that aw(t)t 0 . ( i)for every convex function c([m , m ] , )(41)ab(g(t))w(t)tabw(t)tdgdc(c)+gcdc(d ) holds.(ii)for all y [ m , m](42)abg(g(t),y)w(t)tabw(t)tdgdcg(c , y)+gcdcg(d , y ) holds , where the function g : [ m , m][m , m ] is defined in ( 15).furthermore , the statements ( i ) and ( ii ) are also equivalent if we change the sign of inequality in both ( 41 ) and ( 42 ) . let g c([a , b] , ) be such that g([a , b])[m , m ] and let c , d [ m , m ] ( c d ) be such that c g(t ) d for all t [ a , b]. let w c([a , b] , ) be such that aw(t)t 0 . ( i)for every convex function c([m , m ] , )(41)ab(g(t))w(t)tabw(t)tdgdc(c)+gcdc(d ) holds.(ii)for all y [ m , m](42)abg(g(t),y)w(t)tabw(t)tdgdcg(c , y)+gcdcg(d , y ) holds , where the function g : [ m , m][m , m ] is defined in ( 15).furthermore , the statements ( i ) and ( ii ) are also equivalent if we change the sign of inequality in both ( 41 ) and ( 42 ) . for every convex function c([m , m ] , )(41)ab(g(t))w(t)tabw(t)tdgdc(c)+gcdc(d ) holds . for all y [ m , m](42)abg(g(t),y)w(t)tabw(t)tdgdcg(c , y)+gcdcg(d , y ) holds , where the function g : [ m , m][m , m ] is defined in ( 15 ) . proofthe idea of the proof is very similar to the proof of theorem 11.(i ) ( ii ) : let ( i ) hold . as the function g( , y ) ( y [ m , m ] ) is continuous and convex , it follows that ( 42 ) holds.(ii ) ( i ) : let ( ii ) hold . then by using ( 16 ) we get ( 43)dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)t = mm[abg(g(t),y)w(t)tabw(t)tdgdcg(c , y)+gcdcg(d , y)aaaaaaaa abg(g(t),y)w(t)tabw(t)t](y)dy . if the function is also convex , then (y ) 0 for all y [ m , m ] , and hence it follows that for every convex function c([m , m ] , ) the inequality ( 41 ) holds . moreover , it is not necessary to demand the existence of the second derivative of the function ( see [ 6 , page 172 ] ) . the differentiability condition can be directly eliminated by using the fact that it is possible to approximate uniformly a continuous convex function by convex polynomials.the last part of our theorem can be proved analogously . ( i ) ( ii ) : let ( i ) hold . as the function g( , y ) ( y [ m , m ] ) is continuous and convex , it follows that ( 42 ) holds . then by using ( 16 ) we get ( 43)dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)t = mm[abg(g(t),y)w(t)tabw(t)tdgdcg(c , y)+gcdcg(d , y)aaaaaaaa abg(g(t),y)w(t)tabw(t)t](y)dy . if the function is also convex , then (y ) 0 for all y [ m , m ] , and hence it follows that for every convex function c([m , m ] , ) the inequality ( 41 ) holds . moreover , it is not necessary to demand the existence of the second derivative of the function ( see [ 6 , page 172 ] ) . the differentiability condition can be directly eliminated by using the fact that it is possible to approximate uniformly a continuous convex function by convex polynomials . ( i)for every concave function c([m , m ] , ) , the reverse inequality in ( 41 ) holds.(ii)for all y [ m , m ] inequality ( 42 ) holds.moreover , statements ( i ) and ( ii ) are also equivalent if we change the sign of inequality in both statements ( i ) and ( ii ) . ( i)for every concave function c([m , m ] , ) , the reverse inequality in ( 41 ) holds.(ii)for all y [ m , m ] inequality ( 42 ) holds.moreover , statements ( i ) and ( ii ) are also equivalent if we change the sign of inequality in both statements ( i ) and ( ii ) . for every concave function c([m , m ] , ) , the reverse inequality in ( 41 ) holds . for all y [ m , m ] inequality ( 42 ) holds . remark 25 . note that in all the results in this section we allow that the mean value g goes out of the interval [ m , m ] , while in the results from the previous section we demanded that g[m , m ] . note that in all the results in this section we allow that the mean value g goes out of the interval [ m , m ] , while in the results from the previous section we demanded that g[m , m ] . setting c = m and d = m in theorem 23 , we get the following result . let g c([a , b] , ) be such that g([a , b])[m , m ] . let w c([a , b] , ) be such that aw(t)t 0 . ( i)for every convex function c([m , m ] , )(44)ab(g(t))w(t)tabw(t)tmgmm(m)+gmmm(m ) holds.(ii)for all y [ m , m](45)abg(g(t),y)w(t)tabw(t)t0 holds , where the function g : [ m , m][m , m ] is defined as in ( 15).furthermore , statements ( i ) and ( ii ) are also equivalent if we change the sign of inequality in both ( 44 ) and ( 45 ) . let g c([a , b] , ) be such that g([a , b])[m , m ] . let w c([a , b] , ) be such that aw(t)t 0 . ( i)for every convex function c([m , m ] , )(44)ab(g(t))w(t)tabw(t)tmgmm(m)+gmmm(m ) holds.(ii)for all y [ m , m](45)abg(g(t),y)w(t)tabw(t)t0 holds , where the function g : [ m , m][m , m ] is defined as in ( 15).furthermore , statements ( i ) and ( ii ) are also equivalent if we change the sign of inequality in both ( 44 ) and ( 45 ) . for every convex function c([m , m ] , )(44)ab(g(t))w(t)tabw(t)tmgmm(m)+gmmm(m ) holds . for all y [ m , m](45)abg(g(t),y)w(t)tabw(t)t0 holds , where the function g : [ m , m][m , m ] is defined as in ( 15 ) . , we get the result from lemma 9.let c = m and d = m. then ( 43 ) transforms into ( 46)mgmm(m)+gmmm(m)ab(g(t))w(t)tabw(t)t = mmabg(g(t),y)w(t)tabw(t)t(y)dy.let aw(t)t > 0 , and suppose that g is nondecreasing and that it has the first derivative . let m = g(a ) and m = g(b ) , and make the substitution y = g(s ) since g is nondecreasing , we have that g(s ) 0 for all s [ a , b]. if c([m , m ] , ) is convex , then (g(s ) ) 0 for all s [ a , b]. hence , if and only if ( 48)abg(g(t),g(s))w(t)t0 holds for all s [ a , b] , then for every continuous convex function inequality ( 44 ) holds . note that ( 48 ) is equivalent to condition ( 14 ) . as a consequence of corollary 26 let c = m and d = m. then ( 43 ) transforms into ( 46)mgmm(m)+gmmm(m)ab(g(t))w(t)tabw(t)t = mmabg(g(t),y)w(t)tabw(t)t(y)dy . let aw(t)t > 0 , and suppose that g is nondecreasing and that it has the first derivative . let m = g(a ) and m = g(b ) , and make the substitution y = g(s ) . since g is nondecreasing , we have that g(s ) 0 for all s [ a , b]. if c([m , m ] , ) is convex , then (g(s ) ) 0 for all s [ a , b]. hence , if and only if ( 48)abg(g(t),g(s))w(t)t0 holds for all s [ a , b] , then for every continuous convex function inequality ( 44 ) holds . note that ( 48 ) is equivalent to condition ( 14 ) . corollary 28 . let g c([a , b] , [ m , m ] ) and let w c([a , b] , ) be an -sp weight for g on [ a , b]. then ( 49)abg(g(t),y)w(t)tabw(t)t0 holds for all y [ m , m ] . let g c([a , b] , [ m , m ] ) and let w c([a , b] , ) be an -sp weight for g on [ a , b]. then ( 49)abg(g(t),y)w(t)tabw(t)t0 holds for all y [ m , m ] . the proof follows directly from theorem 10 and corollary 26 . under the assumptions of theorem 23 , we define the following functional 2(g , ) : ( 50)j2(g,)={dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)taaaaaaaif y[m , m ] inequality ( 42 ) holds,ab(g(t))w(t)tabw(t)tdgdc(c)gcdc(d)aaaaaaaif y[m , m ] the reverse inequality aaaaaaain ( 42 ) holds , where the function is defined on [ m , m ] . clearly , if is continuous and convex , then 2(g , ) is nonnegative . let g c([a , b] , ) and c([m , m ] , ) . then there exists [ m , m ] such that ( 51)j2(g,)=()j2(g,0 ) holds , where 0(x ) = x/2 . let g c([a , b] , ) and c([m , m ] , ) . then there exists [ m , m ] such that ( 51)j2(g,)=()j2(g,0 ) holds , where 0(x ) = x/2 . proofthe idea of the proof is very similar to the proof of theorem 16.since the function is continuous and ( 52)dgdcg(c , y)+gcdcg(d , y)abg(g(t),y)w(t)tabw(t)t does not change its positivity on [ m , m ] , applying the integral mean - value theorem on ( 43 ) we get that there exists [ m , m ] such that ( 53)dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)t = ()mm[abg(g(t),y)w(t)tabw(t)tdgdcg(c , y)+gcdcg(d , y)aaaaaaaaaaaaai abg(g(t),y)w(t)tabw(t)t]dy . calculating the integral on the right - hand side of ( 53 ) , we get ( 54)dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)t = 12()[ab(g(t))2w(t)tabw(t)tdgdcc2+gcdcd2aaaaaaaaaaaa ab(g(t))2w(t)tabw(t)t ] and we get statement ( 51 ) of our theorem . the idea of the proof is very similar to the proof of theorem 16 . since the function is continuous and ( 52)dgdcg(c , y)+gcdcg(d , y)abg(g(t),y)w(t)tabw(t)t does not change its positivity on [ m , m ] , applying the integral mean - value theorem on ( 43 ) we get that there exists [ m , m ] such that ( 53)dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)t = ()mm[abg(g(t),y)w(t)tabw(t)tdgdcg(c , y)+gcdcg(d , y)aaaaaaaaaaaaai abg(g(t),y)w(t)tabw(t)t]dy . calculating the integral on the right - hand side of ( 53 ) , we get ( 54)dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)t = 12()[ab(g(t))2w(t)tabw(t)tdgdcc2+gcdcd2aaaaaaaaaaaa ab(g(t))2w(t)tabw(t)t ] and we get statement ( 51 ) of our theorem . remark 30 . note that ( 54 ) can also be expressed as ( 55)dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)t = 12()[g(c+d)cdab(g(t))2w(t)tabw(t)t ] . note that ( 54 ) can also be expressed as ( 55)dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)t = 12()[g(c+d)cdab(g(t))2w(t)tabw(t)t ] . let g c([a , b] , ) and , c([m , m ] , ) . . then there exists [ m , m ] such that ( 56)j2(g,)j2(g,)=()( ) holds , provided that the denominator on the left - hand side of ( 56 ) is nonzero . let g c([a , b] , ) and , c([m , m ] , ) . . then there exists [ m , m ] such that ( 56)j2(g,)j2(g,)=()( ) holds , provided that the denominator on the left - hand side of ( 56 ) is nonzero . first we recall some definitions and facts about exponentially convex and logarithmically convex functions ( see , e.g. , [ 13 , 14 ] or ) which we need for our results . a function f : i (i ) is n - exponentially convex in the jensen sense on i , if ( 57)i , j=1nijf(xi+xj2)0 holds for all choices i and xi i , i = 1 , , n.a function f : i is n - exponentially convex if it is n - exponentially convex in the jensen sense and continuous on i. a function f : i (i ) is n - exponentially convex in the jensen sense on i , if ( 57)i , j=1nijf(xi+xj2)0 holds for all choices i and xi i , i = 1 , , n. a function f : i is n - exponentially convex if it is n - exponentially convex in the jensen sense and continuous on i. remark 33 . it is clear from the definition that 1-exponentially convex functions in the jensen sense are in fact nonnegative functions . also , n - exponentially convex functions in the jensen sense are k - exponentially convex in the jensen sense for every k , k n. it is clear from the definition that 1-exponentially convex functions in the jensen sense are in fact nonnegative functions . also , n - exponentially convex functions in the jensen sense are k - exponentially convex in the jensen sense for every k , k n. by definition of positive semidefinite matrices and some basic linear algebra , we have the following proposition . if f is an n - exponentially convex function in the jensen sense , then the matrix [ f((xi + xj)/2)]i , j=1 is positive , semidefinite for all k , k n. particularly , det[f((xi + xj)/2)]i , j=1 0 for all k , k n. if f is an n - exponentially convex function in the jensen sense , then the matrix [ f((xi + xj)/2)]i , j=1 is positive , semidefinite for all k , k n. particularly , det[f((xi + xj)/2)]i , j=1 0 for all k , k n. definition 35 . a function f : i is exponentially convex in the jensen sense on i , if it is n - exponentially convex in the jensen sense for all n .a function f : i is exponentially convex if it is exponentially convex in the jensen sense and continuous . a function f : i is exponentially convex in the jensen sense on i , if it is n - exponentially convex in the jensen sense for all n . a function f : i is exponentially convex if it is exponentially convex in the jensen sense and continuous . some examples of exponentially convex functions are ( see ) as follows : ( i)f : i defined by f(x ) = ce , where c 0 and k ;(ii)f : defined by f(x ) = x , where k > 0;(iii)f : defined by f(x)=e - kx , where k > 0 . some examples of exponentially convex functions are ( see ) as follows : ( i)f : i defined by f(x ) = ce , where c 0 and k ;(ii)f : defined by f(x ) = x , where k > 0;(iii)f : defined by f(x)=e - kx , where k > 0 . f : i defined by f(x ) = ce , where c 0 and k ; f : defined by f(x ) = x , where k > 0 ; f : defined by f(x)=e - kx , where k > 0 . it is known ( and easy to show ) that f : i is log - convex in the jensen sense on i , if and only if ( 58)2f(x)+2f(x+y2)+2f(y)0 holds for every , and x , y i. it follows that a positive function is log - convex in the jensen sense if and only if it is 2-exponentially convex in the jensen sense . also , using basic convexity theory , it follows that a positive function is log - convex if and only if it is 2-exponentially convex . it is known ( and easy to show ) that f : i is log - convex in the jensen sense on i , if and only if ( 58)2f(x)+2f(x+y2)+2f(y)0 holds for every , and x , y i. it follows that a positive function is log - convex in the jensen sense if and only if it is 2-exponentially convex in the jensen sense . also , using basic convexity theory , it follows that a positive function is log - convex if and only if it is 2-exponentially convex . the following lemma is equivalent to the definition of convex function ( see [ 6 , page 2 ] ) . lemma 38 . if x1 , x2 , x3 i are such that x1 < x2 < x3 , then the function f : i is convex if and only if the following inequality holds : ( 59)(x3x2)f(x1)+(x1x3)f(x2)+(x2x1)f(x3)0 . if x1 , x2 , x3 i are such that x1 < x2 < x3 , then the function f : i is convex if and only if the following inequality holds : ( 59)(x3x2)f(x1)+(x1x3)f(x2)+(x2x1)f(x3)0 . we will also need the following result ( see , e.g. , ) . if f : i is a convex function and x1 , x2 , y1 , y2 i are such that x1 y1 , x2 y2 , x1 x2 , and y1 y2 , then the following inequality is valid : ( 60)f(x2)f(x1)x2x1f(y2)f(y1)y2y1 . if f : i is a convex function and x1 , x2 , y1 , y2 i are such that x1 y1 , x2 y2 , x1 x2 , and y1 y2 , then the following inequality is valid : ( 60)f(x2)f(x1)x2x1f(y2)f(y1)y2y1 . when dealing with functions with different degree of smoothness , divided differences are found to be very useful . the second order divided difference of a function f : i at mutually different points x0 , x1 , x2 i is defined recursively by ( 61)[xi;f]=f(xi ) , i=0,1,2,[xi , xi+1;f]=f(xi+1)f(xi)xi+1xi , i=0,1,[x0,x1,x2;f]=[x1,x2;f][x0,x1;f]x2x0 . the second order divided difference of a function f : i at mutually different points x0 , x1 , x2 i is defined recursively by ( 61)[xi;f]=f(xi ) , i=0,1,2,[xi , xi+1;f]=f(xi+1)f(xi)xi+1xi , i=0,1,[x0,x1,x2;f]=[x1,x2;f][x0,x1;f]x2x0 . the value [ x0 , x1 , x2 ; f ] is independent of the order of the points x0 , x1 , and x2 . this definition may be extended to include the case in which some or all the points coincide ( see [ 6 , page 16 ] ) . x0 in ( 61 ) , we obtain ( 62)limx1x0[x0,x1,x2;f]=[x0,x0,x2;f]=f(x2)f(x0)f(x0)(x2x0)(x2x0)2,aaaaaaaaaaaaaaaaaaaaaaaaax2x0 provided that f exists , and furthermore , taking the limits xi x0 , i = 1,2 , in ( 61 ) , we obtain ( 63)limx2x0 limx1x0[x0,x1,x2;f]=[x0,x0,x0;f]=f(x0)2 provided that f exists.a function f : i is convex if and only if for every choice of three mutually different points x0 , x1 , x2 i[x0 , x1 , x2 ; f ] 0 holds . the value [ x0 , x1 , x2 ; f ] is independent of the order of the points x0 , x1 , and x2 . this definition may be extended to include the case in which some or all the points coincide ( see [ 6 , page 16 ] ) . x0 in ( 61 ) , we obtain ( 62)limx1x0[x0,x1,x2;f]=[x0,x0,x2;f]=f(x2)f(x0)f(x0)(x2x0)(x2x0)2,aaaaaaaaaaaaaaaaaaaaaaaaax2x0 provided that f exists , and furthermore , taking the limits xi x0 , i = 1,2 , in ( 61 ) , we obtain ( 63)limx2x0 limx1x0[x0,x1,x2;f]=[x0,x0,x0;f]=f(x0)2 provided that f exists . a function f : i is convex if and only if for every choice of three mutually different points x0 , x1 , x2 i[x0 , x1 , x2 ; f ] 0 holds . now , we use an idea from to give an elegant method of producing an n - exponentially convex and exponentially convex functions applying the functionals 1 and 2 on a given family of functions with the same property . let i , i = 1,2 , be linear functionals defined in ( 26 ) and ( 50 ) , respectively . let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . then i(g , ) is an n - exponentially convex function in the jensen sense on j. if the function i(g , ) is also continuous on j , then it is n - exponentially convex on j. let i , i = 1,2 , be linear functionals defined in ( 26 ) and ( 50 ) , respectively . let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . then i(g , ) is an n - exponentially convex function in the jensen sense on j. if the function i(g , ) is also continuous on j , then it is n - exponentially convex on j. proofdefine the function : i by ( 64)(x)=j , k=1njkrjk(x ) , where j , k , rj , rk j , 1 j , k n , rjk = ( rj + rk)/2 , and rjk . using the assumption that for every choice of three mutually different points x0 , x1 , x2 [ m , m] [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense on j , we obtain ( 65)[x0,x1,x2;]=j , k=1njk[x0,x1,x2;rjk]0 . therefore , is convex ( and continuous ) function on i. hence i(g , ) 0 , i = 1,2 , which implies that ( 66)j , k=1njkji(g,rjk)0 . we conclude that the function i(g , ) is n - exponentially convex on j in the jensen sense.if i(g , ) is continuous on j , then i(g , ) is n - exponentially convex by definition . define the function : i by ( 64)(x)=j , k=1njkrjk(x ) , where j , k , rj , rk j , 1 j , k n , rjk = ( rj + rk)/2 , and rjk . using the assumption that for every choice of three mutually different points x0 , x1 , x2 [ m , m] [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense on j , we obtain ( 65)[x0,x1,x2;]=j , k=1njk[x0,x1,x2;rjk]0 . therefore , is convex ( and continuous ) function on i. hence i(g , ) 0 , i = 1,2 , which implies that ( 66)j , k=1njkji(g,rjk)0 . we conclude that the function i(g , ) is n - exponentially convex on j in the jensen sense . if i(g , ) is continuous on j , then i(g , ) is n - exponentially convex by definition . let i , i = 1,2 , be linear functionals defined in ( 26 ) and ( 50 ) , respectively . let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . then i(g , ) is an exponentially convex function in the jensen sense on j. if the function i(g , ) is also continuous on j , then it is exponentially convex on j. let i , i = 1,2 , be linear functionals defined in ( 26 ) and ( 50 ) , respectively . let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . then i(g , ) is an exponentially convex function in the jensen sense on j. if the function i(g , ) is also continuous on j , then it is exponentially convex on j. corollary 44 . let i , i = 1,2 , be linear functionals defined in ( 26 ) and ( 50 ) , respectively . let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is 2-exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . ( i) i(g , ) is a 2-exponentially convex function in the jensen sense on j.(ii)if i(g , ) is continuous on j , then it is also 2-exponentially convex on j. if i(g , ) is additionally strictly positive , then it is also log - convex on j , and for r , s , t j such that r < s < t , we have ( 67)(ji(g,s))tr(ji(g,r))ts(ji(g,t))sr.(iii)if i(g , ) is strictly positive and differentiable function on j , then for every p , q , u , v j such that p u , q v , we have ( 68)mp , q(g , ji,)mu , v(g , ji, ) , i=1,2 , where ( 69)mp , q(g , ji,)={(ji(g,p)ji(g,q))1/(pq),pq;exp((d / dp)ji(g,p)ji(g,p)),p = q , for p , q . let i , i = 1,2 , be linear functionals defined in ( 26 ) and ( 50 ) , respectively . let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is 2-exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . then ( i) i(g , ) is a 2-exponentially convex function in the jensen sense on j.(ii)if i(g , ) is continuous on j , then it is also 2-exponentially convex on j. if i(g , ) is additionally strictly positive , then it is also log - convex on j , and for r , s , t j such that r < s < t , we have ( 67)(ji(g,s))tr(ji(g,r))ts(ji(g,t))sr.(iii)if i(g , ) is strictly positive and differentiable function on j , then for every p , q , u , v j such that p u , q v , we have ( 68)mp , q(g , ji,)mu , v(g , ji, ) , i=1,2 , where ( 69)mp , q(g , ji,)={(ji(g,p)ji(g,q))1/(pq),pq;exp((d / dp)ji(g,p)ji(g,p)),p = q , for p , q . i(g , ) is a 2-exponentially convex function in the jensen sense on j. if i(g , ) is continuous on j , then it is also 2-exponentially convex on j. if i(g , ) is additionally strictly positive , then it is also log - convex on j , and for r , s , t j such that r < s < t , we have ( 67)(ji(g,s))tr(ji(g,r))ts(ji(g,t))sr . if i(g , ) is strictly positive and differentiable function on j , then for every p , q , u , v j such that p u , q v , we have ( 68)mp , q(g , ji,)mu , v(g , ji, ) , i=1,2 , where ( 69)mp , q(g , ji,)={(ji(g,p)ji(g,q))1/(pq),pq;exp((d / dp)ji(g,p)ji(g,p)),p = q , for p , q . proof(i ) and the first part of ( ii ) are immediate consequences of theorem 42 . if i(g , ) is continuous and strictly positive , its log - convexity is an immediate consequence of remark 37 . now applying lemma 38 on the function f(x ) = logi(g , x ) and r , s , t j ( r < s < t ) , we get ( 70)(ts)ji(g,r)+(rt)ji(g,s)+(sr)ji(g,t)0 , which is equivalent to inequality ( 67).to prove ( iii ) , let i(g , ) be strictly positive and differentiable and therefore continuous too . by ( ii ) , the function i(g , ) is log - convex on j ; that is , the function logi(g , ) is convex on j , and by proposition 39 we obtain ( 71)logji(g,p)logji(g,q)pq logji(g,u)logji(g,v)uv for p u , q v , p q , and u v , concluding that ( 72)mp , q(g , ji,)mu , v(g , ji, ) . the cases p = q and u = v follow from ( 71 ) as limit cases . ( i ) and the first part of ( ii ) are immediate consequences of theorem 42 . if i(g , ) is continuous and strictly positive , its log - convexity is an immediate consequence of remark 37 . now applying lemma 38 on the function f(x ) = logi(g , x ) and r , s , t j ( r < s < t ) , we get ( 70)(ts)ji(g,r)+(rt)ji(g,s)+(sr)ji(g,t)0 , which is equivalent to inequality ( 67 ) . to prove ( iii ) , let i(g , ) be strictly positive and differentiable and therefore continuous too . by ( ii ) , the function i(g , ) is log - convex on j ; that is , the function logi(g , ) is convex on j , and by proposition 39 we obtain ( 71)logji(g,p)logji(g,q)pq logji(g,u)logji(g,v)uv for p u , q v , p q , and u v , concluding that ( 72)mp , q(g , ji,)mu , v(g , ji, ) . the cases p = q and u note that the results from theorem 42 , corollary 43 , and corollary 44 still hold when two of the points x0 , x1 , x2 [ m , m ] coincide , for a family of differentiable functions such that the function [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense ( exponentially convex in the jensen sense and log - convex in the jensen sense ) , and furthermore , they still hold when all three points coincide for a family of twice differentiable functions with the same property . note that the results from theorem 42 , corollary 43 , and corollary 44 still hold when two of the points x0 , x1 , x2 [ m , m ] coincide , for a family of differentiable functions such that the function [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense ( exponentially convex in the jensen sense and log - convex in the jensen sense ) , and furthermore , they still hold when all three points coincide for a family of twice differentiable functions with the same property . in this section we will vary on choice of a family = { : j } in order to construct different examples of exponentially convex functions and construct some means . it is ( d / dx)(x ) = e > 0 which shows that is convex on for every . from remark 36 , it follows that ( d / dx)(x ) is exponentially convex . therefore , [ x0 , x1 , x2 ; ] is exponentially convex ( see ) ( and so exponentially convex in the jensen sense ) . now using corollary 43 we conclude that i(g , ) , ( i = 1,2 ) are exponentially convex in the jensen sense . it is easy to verify that these mappings are continuous , so they are exponentially convex.for this family of functions , p , q(g , i , ) from ( 69 ) becomes ( 75)mp , q(g , ji,1 ) = { ( ji(g,p)ji(g,q))1/(pq),pq;exp(ji(g , idp)ji(g,p)2p),p = q0;exp(ji(g , id0)3ji(g,0)),p = q=0 , and using ( 68 ) we have that it is monotonous in parameters p and q.if i ( i = 1,2 ) are positive , using theorems 18 and 31 applied for = p 1 and = q 1 , it follows that ( 76)p , q(g , ji,1)=logmp , q(g , ji,1 ) ( i=1,2 ) satisfy p , q(g , i , 1)[m , m ] . if we set g([a , b] ) = [ m , m ] then we have that p , q(g , i , 1 ) are means ( of the function g ) . note that by ( 68 ) they are monotonous means . it is ( d / dx)(x ) = e > 0 which shows that is convex on for every . from remark 36 , it follows that ( d / dx)(x ) is exponentially convex . therefore , [ x0 , x1 , x2 ; ] is exponentially convex ( see ) ( and so exponentially convex in the jensen sense ) . now using corollary 43 we conclude that i(g , ) , ( i = 1,2 ) are exponentially convex in the jensen sense . it is easy to verify that these mappings are continuous , so they are exponentially convex . for this family of functions , p , q(g , i , ) from ( 69 ) becomes ( 75)mp , q(g , ji,1 ) = { ( ji(g,p)ji(g,q))1/(pq),pq;exp(ji(g , idp)ji(g,p)2p),p = q0;exp(ji(g , id0)3ji(g,0)),p = q=0 , and using ( 68 ) we have that it is monotonous in parameters p and q. if i ( i = 1,2 ) are positive , using theorems 18 and 31 applied for = p 1 and = q 1 , it follows that ( 76)p , q(g , ji,1)=logmp , q(g , ji,1 ) ( i=1,2 ) satisfy p , q(g , i , 1)[m , m ] . if we set g([a , b] ) = [ m , m ] then we have that p , q(g , i , 1 ) are means ( of the function g ) . it is ( d / dx)(x ) = x = e > 0 , which shows that is convex function for x > 0 . also , from remark 36 it follows that ( d / dx)(x ) is exponentially convex . therefore [ x0 , x1 , x2 ; ] is exponentially convex ( and so exponentially convex in the jensen sense ) . here we assume that [ m , m](0 , ) , so our family 2 of fulfills the conditions of corollary 43 . in this case p , q(g , i , ) from ( 69 ) becomes ( 79)mp , q(g , ji,2 ) = { ( ji(g,p)ji(g,q))1/(pq),pq;exp(12pp(p1)ji(g,0p)ji(g,p)),p = q0,1;exp(1ji(g,02)2ji(g,0)),p = q=0;exp(1ji(g,01)2ji(g,1)),p = q=1 . if i ( i = 1,2 ) are positive , by applying theorems 18 and 31 for = p 2 and = q 2 , it follows that for i = 1,2 there exist i [ m , m ] such that ( 80)ipq = ji(g,p)ji(g,q ) . since the function i i is invertible for p q , we have ( 81)m(ji(g,p)ji(g,q))1/(pq)m.also , p , q(g , i , 2 ) is continuous , symmetric , and monotonous ( by ( 68 ) ) . if we set g([a , b] ) = [ m , m ] , then we have that ( 82)m = mint[a , b]t{g(t)}(ji(g,p)ji(g,q))1/(pq)maxt[a , b]t{g(t)}=m , aaaaaaaaifor i=1,2 , which shows that p , q(g , i , 2 ) are means ( of the function g).now we impose one additional parameter r in case g([a , b] ) = [ m , m ] . for r 0 by substituting g g , p p / r , and q q / r in ( 82 ) , we get ( 83)m = mint[a , b]t{gr(t)}(ji(gr,p)ji(gr,q))r/(pq)maxt[a , b]t{gr(t)}=m , aaaaaaaaaifor i=1,2 . we define new generalized means as follows : ( 84)mp , q;r(g , ji,2)={(mp / r , q / r(gr , ji,2))1/r , r0;(mp / r , q / r(logg , ji,1)),r=0 . it is ( d / dx)(x ) = x = e > 0 , which shows that is convex function for x > 0 . also , from remark 36 it follows that ( d / dx)(x ) is exponentially convex . therefore [ x0 , x1 , x2 ; ] is exponentially convex ( and so exponentially convex in the jensen sense ) . here we assume that [ m , m](0 , ) , so our family 2 of fulfills the conditions of corollary 43 . in this case p , q(g , i , ) from ( 69 ) becomes ( 79)mp , q(g , ji,2 ) = { ( ji(g,p)ji(g,q))1/(pq),pq;exp(12pp(p1)ji(g,0p)ji(g,p)),p = q0,1;exp(1ji(g,02)2ji(g,0)),p = q=0;exp(1ji(g,01)2ji(g,1)),p = q=1 . if i ( i = 1,2 ) are positive , by applying theorems 18 and 31 for = p 2 and = q 2 , it follows that for i = 1,2 there exist i [ m , m ] such that ( 80)ipq = ji(g,p)ji(g,q ) . since the function i i is invertible for p q , we have ( 81)m(ji(g,p)ji(g,q))1/(pq)m . also , p , q(g , i , 2 ) is continuous , symmetric , and monotonous ( by ( 68 ) ) . if we set g([a , b] ) = [ m , m ] , then we have that ( 82)m = mint[a , b]t{g(t)}(ji(g,p)ji(g,q))1/(pq)maxt[a , b]t{g(t)}=m , aaaaaaaaifor i=1,2 , which shows that p , q(g , i , 2 ) are means ( of the function g ) . now we impose one additional parameter r in case g([a , b] ) = [ m , m ] . for r 0 by substituting g g , p p / r , and q q / r in ( 82 ) , we get ( 83)m = mint[a , b]t{gr(t)}(ji(gr,p)ji(gr,q))r/(pq)maxt[a , b]t{gr(t)}=m , aaaaaaaaaifor i=1,2 . we define new generalized means as follows : ( 84)mp , q;r(g , ji,2)={(mp / r , q / r(gr , ji,2))1/r , r0;(mp / r , q / r(logg , ji,1)),r=0 . these new generalized means are also monotonic . consider a family of functions ( 85)3={:(0,)(0,):(0, ) } defined by ( 86)(x)={x(log)2,1;x22,=1 . it is ( d / dx)(x ) = > 0 , which shows that is convex function for > 0 . also , from remark 36 it follows that ( d / dx)(x ) is exponentially convex . therefore [ x0 , x1 , x2 ; ] is exponentially convex ( and so exponentially convex in the jensen sense ) . here we assume that [ m , m](0 , ) , so our family 3 of fulfills the conditions of corollary 43 . in this case p , q(g , i , ) from ( 69 ) becomes ( 87)mp , q(g , ji,3 ) = { ( ji(g,p)ji(g,q))1/(pq),pq;exp(ji(g , idp)pji(g,p)2plogp),p = q1;exp(2ji(g , id1)3ji(g,1)),p = q=1 , and by ( 68 ) it is monotonous function in parameters p and q.if i ( i = 1,2 ) are positive , using theorems 18 and 31 applied for = p 3 and = q 3 , it follows that ( 88)p , q(g , ji,3)=l(p , q)logmp , q(g , ji,3 ) satisfies p , q(g , i , 3)[m , m ] . here l(p , q ) is the logarithmic mean defined by l(p , q ) = ( p q)/(logp logq ) for p q , l(p , p ) = p. consider a family of functions ( 85)3={:(0,)(0,):(0, ) } defined by ( 86)(x)={x(log)2,1;x22,=1 . it is ( d / dx)(x ) = > 0 , which shows that is convex function for > 0 . also , from remark 36 it follows that ( d / dx)(x ) is exponentially convex . therefore [ x0 , x1 , x2 ; ] is exponentially convex ( and so exponentially convex in the jensen sense ) . here we assume that [ m , m](0 , ) , so our family 3 of fulfills the conditions of corollary 43 . in this case p , q(g , i , ) from ( 69 ) becomes ( 87)mp , q(g , ji,3 ) = { ( ji(g,p)ji(g,q))1/(pq),pq;exp(ji(g , idp)pji(g,p)2plogp),p = q1;exp(2ji(g , id1)3ji(g,1)),p = q=1 , and by ( 68 ) it is monotonous function in parameters p and q. if i ( i = 1,2 ) are positive , using theorems 18 and 31 applied for = p 3 and = q 3 , it follows that ( 88)p , q(g , ji,3)=l(p , q)logmp , q(g , ji,3 ) satisfies p , q(g , i , 3)[m , m ] . here l(p , q ) is the logarithmic mean defined by l(p , q ) = ( p q)/(logp logq ) for p q , l(p , p ) = p. example 49 . consider a family of functions ( 89)4={:(0,)(0,):(0, ) } defined by ( 90)(x)=ex. it is ( d2/dx2)(x)=e - x>0 , which shows that is convex function for > 0 also , from remark 36 it follows that ( d / dx)(x ) is exponentially convex . therefore [ x0 , x1 , x2 ; ] is exponentially convex ( and so exponentially convex in the jensen sense ) . here we assume that [ m , m](0 , ) , so our family 4 of fulfills the conditions of corollary 43 . in this case p , q(g , i , ) from ( 69 ) becomes ( 91)mp , q(g , ji,4 ) = { ( ji(g,p)ji(g,q))1/(pq),pq;exp(ji(g , idp)2pji(g,p)1p),p = q , and it is monotonous function in parameters p and q by ( 68).if i ( i = 1,2 ) are positive , using theorems 18 and 31 applied for = p 4 and = q 4 , it follows that ( 92)p , q(g , ji,4)=(p+q)logmp , q(g , ji,4 ) satisfies p , q(g , i , 4)[m , m ] . consider a family of functions ( 89)4={:(0,)(0,):(0, ) } defined by ( 90)(x)=ex. it is ( d2/dx2)(x)=e - x>0 , which shows that is convex function for > 0 also , from remark 36 it follows that ( d / dx)(x ) is exponentially convex . therefore [ x0 , x1 , x2 ; ] is exponentially convex ( and so exponentially convex in the jensen sense ) . here we assume that [ m , m](0 , ) , so our family 4 of fulfills the conditions of corollary 43 . in this case p , q(g , i , ) from ( 69 ) becomes ( 91)mp , q(g , ji,4 ) = { ( ji(g,p)ji(g,q))1/(pq),pq;exp(ji(g , idp)2pji(g,p)1p),p = q , and it is monotonous function in parameters p and q by ( 68 ) . if i ( i = 1,2 ) are positive , using theorems 18 and 31 applied for = p 4 and = q 4 , it follows that ( 92)p , q(g , ji,4)=(p+q)logmp , q(g , ji,4 ) satisfies p , q(g , i , 4)[m , m ] .

we give further improvements of the jensen inequality and its converse on time scales , allowing also negative weights . these results generalize the jensen inequality and its converse for both discrete and continuous cases . further , we investigate the exponential and logarithmic convexity of the differences between the left - hand side and the right - hand side of these inequalities and present several families of functions for which these results can be applied .

1. Preliminaries 2. Improvement of the Jensen Inequality on Time Scales 3. Improvement of the Converse of the Jensen Inequality on Time Scales 4. Exponential and Logarithmic Convexity 5. Examples

the combined dynamic derivative , also called diamond- ( ) dynamic derivative ( ) , was introduced as a linear convex combination of the well - known delta and nabla dynamic derivatives on time scales . we assume , throughout this paper , that the basic notions of the time scales are well known and understood . recently , the jensen inequality , the improvement of the jensen inequality , and their converses are given for time scale integrals ( see [ 24 ] ) . in order to give a better version of this jensen inequality on time scales , dinu then , w c( , ) is an -steffensen - popoviciu ( -sp ) weight for g on [ a , b] if ( 7)abw(t)t>0 , ab(g(t))+w(t)t0 , for every convex function c([m , m ] , ) , where ( 8)m = inft[a , b]tg(t ) , m = supt[a , b]tg(t ) . in the following lemma he gives a characterization for -sp weight for a nondecreasing function g on time scales . the jensen inequality on time scales , where it is allowed that the weight function takes some negative values , is given in the following theorem . on the other hand if we take = in theorem 6 , we obtain the integral version of the jensen - steffensen inequality given by boas ( see also [ 6 , page 59 ] ) . on the other hand if we take = in theorem 6 , we obtain the integral version of the jensen - steffensen inequality given by boas ( see also [ 6 , page 59 ] ) . considering the converse of the jensen inequality , dinu gives the following definition of -hermite - hadamard ( -hh ) weight , its characterization for a nondecreasing function g on time scales , and the improvement of the converse of the jensen inequality for some negative weights . in the following two sections of our paper we give some further generalizations of the jensen - type inequalities on time scales allowing negative weights , and we also give the mean - value theorems of the lagrange and cauchy type for the functionals obtained by taking the difference of the left - hand side and right - hand side of these new inequalities . these results also generalize the results given in for continuous and discrete cases . section 4 in our paper deals with exponential convexity and logarithmic convexity of the functionals obtained in two previous sections . finally , in section 5 we present several families of exponentially convex functions which fulfil the conditions of our results . the results from sections 4 and 5 generalize the results given in for continuous and discrete cases . the function g is convex and continuous with respect to both x and y. it is well known that ( see , e.g. using ( 16 ) , we now derive several interesting results concerning the jensen - type inequalities . in the following theorem , we give the generalization of the jensen inequality on time scales , where negative weights are also allowed . moreover , it is not necessary to demand the existence of the second derivative of the function ( see [ 6 , page 172 ] ) . since g is nondecreasing , we have that g(s ) 0 for all s [ a , b]. calculating the integral on the right - hand side of ( 29 ) , we get ( 31)ab(g(t))w(t)tabw(t)t(abg(t)w(t)tabw(t)t ) = ()[1abw(t)taaaaaaaaiiaaab(mmg(g(t),y)dy)w(t)taaaaaaaaaaammg(g,y)dy1abw(t)t ] = ()[1abw(t)tab12(g(t)m)aaaaaaaaaaaaaaaaaaaaaai(g(t)m)w(t)taaaaaaaaaai12(gm)(gm)1abw(t)t ] = 12()[ab(g(t))2w(t)tabw(t)tg2 ] and the proof is completed . calculating the integral on the right - hand side of ( 29 ) , we get ( 31)ab(g(t))w(t)tabw(t)t(abg(t)w(t)tabw(t)t ) = ()[1abw(t)taaaaaaaaiiaaab(mmg(g(t),y)dy)w(t)taaaaaaaaaaammg(g,y)dy1abw(t)t ] = ()[1abw(t)tab12(g(t)m)aaaaaaaaaaaaaaaaaaaaaai(g(t)m)w(t)taaaaaaaaaai12(gm)(gm)1abw(t)t ] = 12()[ab(g(t))2w(t)tabw(t)tg2 ] and the proof is completed . then there exists [ m , m ] such that ( 36)j1(g,)j1(g,)=()( ) holds , provided that the denominator on the left - hand side of ( 36 ) is nonzero . then there exists [ m , m ] such that ( 36)j1(g,)j1(g,)=()( ) holds , provided that the denominator on the left - hand side of ( 36 ) is nonzero . by hypothesis 1(g , 0 ) 0 ( otherwise , we have a contradiction with 1(g , ) 0 ) , so it follows that ( 39)()=0 , which is equivalent to ( 36 ) . using the similar method as in previous section , in the following theorem we obtain the generalization of the converse of the jensen inequality on time scales , where negative weights are also allowed . proofthe idea of the proof is very similar to the proof of theorem 11. moreover , it is not necessary to demand the existence of the second derivative of the function ( see [ 6 , page 172 ] ) . note that in all the results in this section we allow that the mean value g goes out of the interval [ m , m ] , while in the results from the previous section we demanded that g[m , m ] . note that in all the results in this section we allow that the mean value g goes out of the interval [ m , m ] , while in the results from the previous section we demanded that g[m , m ] . setting c = m and d = m in theorem 23 , we get the following result . since g is nondecreasing , we have that g(s ) 0 for all s [ a , b]. proofthe idea of the proof is very similar to the proof of theorem 16.since the function is continuous and ( 52)dgdcg(c , y)+gcdcg(d , y)abg(g(t),y)w(t)tabw(t)t does not change its positivity on [ m , m ] , applying the integral mean - value theorem on ( 43 ) we get that there exists [ m , m ] such that ( 53)dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)t = ()mm[abg(g(t),y)w(t)tabw(t)tdgdcg(c , y)+gcdcg(d , y)aaaaaaaaaaaaai abg(g(t),y)w(t)tabw(t)t]dy . calculating the integral on the right - hand side of ( 53 ) , we get ( 54)dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)t = 12()[ab(g(t))2w(t)tabw(t)tdgdcc2+gcdcd2aaaaaaaaaaaa ab(g(t))2w(t)tabw(t)t ] and we get statement ( 51 ) of our theorem . calculating the integral on the right - hand side of ( 53 ) , we get ( 54)dgdc(c)+gcdc(d)ab(g(t))w(t)tabw(t)t = 12()[ab(g(t))2w(t)tabw(t)tdgdcc2+gcdcd2aaaaaaaaaaaa ab(g(t))2w(t)tabw(t)t ] and we get statement ( 51 ) of our theorem . then there exists [ m , m ] such that ( 56)j2(g,)j2(g,)=()( ) holds , provided that the denominator on the left - hand side of ( 56 ) is nonzero . then there exists [ m , m ] such that ( 56)j2(g,)j2(g,)=()( ) holds , provided that the denominator on the left - hand side of ( 56 ) is nonzero . a function f : i (i ) is n - exponentially convex in the jensen sense on i , if ( 57)i , j=1nijf(xi+xj2)0 holds for all choices i and xi i , i = 1 , , n.a function f : i is n - exponentially convex if it is n - exponentially convex in the jensen sense and continuous on i. a function f : i (i ) is n - exponentially convex in the jensen sense on i , if ( 57)i , j=1nijf(xi+xj2)0 holds for all choices i and xi i , i = 1 , , n. a function f : i is n - exponentially convex if it is n - exponentially convex in the jensen sense and continuous on i. remark 33 . also , n - exponentially convex functions in the jensen sense are k - exponentially convex in the jensen sense for every k , k n. by definition of positive semidefinite matrices and some basic linear algebra , we have the following proposition . a function f : i is exponentially convex in the jensen sense on i , if it is n - exponentially convex in the jensen sense for all n .a function f : i is exponentially convex if it is exponentially convex in the jensen sense and continuous . a function f : i is exponentially convex if it is exponentially convex in the jensen sense and continuous . it is known ( and easy to show ) that f : i is log - convex in the jensen sense on i , if and only if ( 58)2f(x)+2f(x+y2)+2f(y)0 holds for every , and x , y i. it follows that a positive function is log - convex in the jensen sense if and only if it is 2-exponentially convex in the jensen sense . now , we use an idea from to give an elegant method of producing an n - exponentially convex and exponentially convex functions applying the functionals 1 and 2 on a given family of functions with the same property . let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . using the assumption that for every choice of three mutually different points x0 , x1 , x2 [ m , m] [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense on j , we obtain ( 65)[x0,x1,x2;]=j , k=1njk[x0,x1,x2;rjk]0 . therefore , is convex ( and continuous ) function on i. hence i(g , ) 0 , i = 1,2 , which implies that ( 66)j , k=1njkji(g,rjk)0 . using the assumption that for every choice of three mutually different points x0 , x1 , x2 [ m , m] [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense on j , we obtain ( 65)[x0,x1,x2;]=j , k=1njk[x0,x1,x2;rjk]0 . let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . then i(g , ) is an exponentially convex function in the jensen sense on j. if the function i(g , ) is also continuous on j , then it is exponentially convex on j. let i , i = 1,2 , be linear functionals defined in ( 26 ) and ( 50 ) , respectively . let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is 2-exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . ( i) i(g , ) is a 2-exponentially convex function in the jensen sense on j. let = { : j } , where j is an interval in , be a family of functions c([m , m ] , ) such that the function [ x0 , x1 , x2 ; ] is 2-exponentially convex in the jensen sense on j for every choice of three mutually different points x0 , x1 , x2 [ m , m ] . then ( i) i(g , ) is a 2-exponentially convex function in the jensen sense on j. i(g , ) is a 2-exponentially convex function in the jensen sense on j. if i(g , ) is continuous on j , then it is also 2-exponentially convex on j. if i(g , ) is additionally strictly positive , then it is also log - convex on j , and for r , s , t j such that r < s < t , we have ( 67)(ji(g,s))tr(ji(g,r))ts(ji(g,t))sr . proof(i ) and the first part of ( ii ) are immediate consequences of theorem 42 . the cases p = q and u note that the results from theorem 42 , corollary 43 , and corollary 44 still hold when two of the points x0 , x1 , x2 [ m , m ] coincide , for a family of differentiable functions such that the function [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense ( exponentially convex in the jensen sense and log - convex in the jensen sense ) , and furthermore , they still hold when all three points coincide for a family of twice differentiable functions with the same property . note that the results from theorem 42 , corollary 43 , and corollary 44 still hold when two of the points x0 , x1 , x2 [ m , m ] coincide , for a family of differentiable functions such that the function [ x0 , x1 , x2 ; ] is n - exponentially convex in the jensen sense ( exponentially convex in the jensen sense and log - convex in the jensen sense ) , and furthermore , they still hold when all three points coincide for a family of twice differentiable functions with the same property . now using corollary 43 we conclude that i(g , ) , ( i = 1,2 ) are exponentially convex in the jensen sense . for this family of functions , p , q(g , i , ) from ( 69 ) becomes ( 75)mp , q(g , ji,1 ) = { ( ji(g,p)ji(g,q))1/(pq),pq;exp(ji(g , idp)ji(g,p)2p),p = q0;exp(ji(g , id0)3ji(g,0)),p = q=0 , and using ( 68 ) we have that it is monotonous in parameters p and q. if i ( i = 1,2 ) are positive , using theorems 18 and 31 applied for = p 1 and = q 1 , it follows that ( 76)p , q(g , ji,1)=logmp , q(g , ji,1 ) ( i=1,2 ) satisfy p , q(g , i , 1)[m , m ] . therefore [ x0 , x1 , x2 ; ] is exponentially convex ( and so exponentially convex in the jensen sense ) . consider a family of functions ( 85)3={:(0,)(0,):(0, ) } defined by ( 86)(x)={x(log)2,1;x22,=1 .

it is now firmly established that mitochondria are key players in different pathophysiological contexts ( duchen , 2004 ) . indeed , they are very efficient machines for decoding intracellular signals and in particular the calcium ( ca ) one ( clapham , 2007 ) . the mitochondrial respiratory chain , by pumping protons across the ion - impermeable inner membrane , establishes a gradient ( h ) , composed of an electrical potential ( ) and a concentration ratio ( ph ) , according to the nernst equation : h = zf + rt ln[h]i/[h]o . this has major implications for ca transport and distribution . in actively respiring mitochondria , considering that the buffering capacity is mostly provided by weak acids , the gradient is supposed to be maintained mostly as an electrical gradient across the inner membrane ( ~ 180 mv ) . this implies a strong thermodynamic force in favor of the accumulation of cations ( rizzuto et al . , 2000 ) . ca import across the outer mitochondrial membrane ( omm ) occurs through the voltage - dependent anion channels ( vdac ) ( simamura et al . , vdac is as a large voltage - gated channel , fully opened with high - conductance and weak anion - selectivity at low transmembrane potentials ( < 2030 mv ) , but switching to cation selectivity and lower conductance at higher potentials ( colombini , 2009 ; de pinto et al . , 2008 ; shoshan - barmatz et al . , ca mitochondrial traffic across the inner mitochondrial membrane ( imm ) takes place essentially through two pathways : i ) an electrophoretic uniporter that transports ca down the electrical gradient established by the respiratory chain , and ii ) a na / ca exchanger , mostly expressed in excitable cells ( muscle and brain ) , and a h / ca exchanger , that represents the prevailing route in most other tissues ( see fig . 1 ) . these electroneutral antiporters prevent the attainment of an electrochemical equilibrium ( rizzuto et al . , 2000 ) . despite intense investigations on the mitochondrial ca uniporter ( mcu ) for almost 50 years , the molecular identity of this gated channel is still a matter of debate . among the early candidates for the mcu were mitochondria - localized ryr1 ( beutner et al . , 2005 ) and the uncoupling proteins ( ucp ) 2 and 3 ( trenker et al . , 2007 ) , but such a role for these proteins is still controversial and further work will be needed to definitively elucidate the molecular identity of the channels or carriers involved . the identity of the mcu , as the product of the ccdc109a gene , has recently been reported by two recent publications ( baughman et al . , 2011 ; de stefani et al . , 2011 ) . in that respect , the report that the imm protein leucine - zipper - ef hand - containing transmembrane region ( letm1 ) , previously described as a k / h exchanger ( nowikovsky et al . , 2004 ) , could be a ca / h antiporter , catalyzing the uptake of ca into mitochondria was received with great interest ( jiang et al . , 2009 ) . these results remain a subject of intense discussion because they diverge in several issues from previous studies ; in particular , the effect of letm1 down - regulation / over - expression on mitochondrial ca transport could be a secondary effect of a decreased k / h exchange activity . finally , it is important to mention the recent description of an imm ca - binding protein named mitochondrial calcium uptake 1 ( micu1 ) , which appears essential for mitochondrial ca uptake ( perocchi et al . , 2010 ) . micu1 is a single - pass transmembrane protein and thus does not seem to participate in channel pore formation . it is possible that micu1 is part of a complex with the mitochondrial ca channel , or functions as ca buffer , or as a ca - dependent regulatory protein acting as a ca sensor ( through a pair of ca - binding ef - hand domains present in its sequence , the mutation of which eliminates the mitochondrial ca uptake ) . about the system for mitochondrial ca extrusion , the mitochondrial na / ca exchanger ( mncx ) and h / ca exchanger ( mhcx ) , have well known kinetic characteristics , are present in the imm ( bernardi , 1999b ) . recently , strong evidence has been provided that the na / ca exchanger isoform nclx is the long - sought protein responsible for the mitochondrial na - dependent ca efflux ( palty et al . , moreover , great attention has been drawn to the potential role of a large - conductance channel , commonly referred to as the permeability transition pore ( ptp ) . it is supposed to be a multiprotein complex activated in various pathophysiological conditions ( e.g. , mitochondrial ca overload ) ( see fig . 1 ) . its role in mitochondrial ca homeostasis , however , remains elusive ( bernardi and forte , 2007 ) . in a variety of cell systems ( ranging from epithelial cells to neurons ) , the cytosolic [ ca ] ( [ ca]c ) rises evoked by physiological stimulations are always paralleled by rapid mitochondrial [ ca ] ( [ ca]m ) increases , that reach values well above those of the bulk cytosol ( up to ~ 500 m in chromaffin cells ) ( giorgi et al . , 2009 ) . a ca signal originating in the cytoplasm that is propagated to mitochondria permits the transmission of an activatory signal to the energy powerhouse of the cell . here , three key metabolic enzymes ( pyruvate , -ketoglutarate , and isocitrate dehydrogenases ) are activated by ca . thus , in conjunction with the triggering of energy - consuming processes in the cytosol ( contraction , secretion , etc . ) , mitochondrial dehydrogenases are stimulated , adapting aerobic metabolism to the increased energy needs of an active cell ( rimessi et al . the alteration of the ca signals that reach mitochondria in association with different pathological conditions ( e.g. , oxidative stress ) can induce a profound alteration of organelle structure and function . as a consequence , the cell may be driven to its death ( giorgi et al . , 2008 ) . here , we review some human diseases associated with perturbations in mitochondrial homeostasis with particular emphasis on the role of ca signals . aging is not considered as an adaptative response , but rather as the price of other evolutionary selected functions , currently not completely specified ( kirkwood and austad , 2000 ) . this degeneration is characterized by a progressive loss of organ function , proposed to be at the basis of several degenerative disorders , promoted by a progressive occurrence of apoptosis in non - proliferating cells . it was recognized that mitochondrial atp synthesis is associated with reactive oxygen species ( ros ) production , including primarily superoxide ( o2 ) formation by the respiratory chain complexes i and iii ( balaban et al . , 2005 ; raha and robinson , 2000 ) . subsequently , superoxide leads to the formation of diverse oxidant products , such as peroxynitrite , hydrogen peroxide ( h2o2 ) and the highly reactive hydroxyl radical ( oh ) . mitochondria are considered the major source of ros and the extent of oxidative damage to macromolecules is generally thought to be the random consequence of physiological functions , as exemplified by lipid peroxidation , protein oxidation , and mitochondrial dna mutations ( cadenas and davies , 2000 ) . different works on the molecular routes of apoptosis have revealed the important role of mitochondria in decoding of oxidative insults , with the release of proteins such as cytochrome c ( cyt c ) or smac / diablo , which act as co - factors of effector caspases . ca overload , through a still controversial mechanism , can also lead to the opening of the ptp and swelling of the organelle in what appears to be a pivotal pathway ( duchen , 2000 ; ravagnan et al . , 2002 ) . one important implication of recent findings is that these phenomena are genetically determined at least in part and biochemically controlled by a stress - induced signal transduction pathway involving the 66-kda isoform of shc , p66shc . the relationship between mitochondria and p66shc emerged once the protein was effectively localized to the organelle and by the observation that , upon oxidative stress , part of the cytosolic pool of p66shc translocates to mitochondria ( orsini et al . the protein lacks a conventional mitochondrial targeting sequence , but its association with the mitochondrial tom / tim import complex and the mitochondrial heat shock protein mthsp70 has been reported ( orsini et al . , 2004 ) . within mitochondria , in particular in the mitochondrial intermembrane space , p66shc binds cyt c and acts as a redox - enzyme , generating h2o2 , which , in turn , induces the opening of the ptp and apoptosis . ros production by p66shc appears to be a specialized function whereby electrons are diverted from the mitochondrial electron transport chain to catalyze the partial reduction of molecular oxygen ( giorgio et al . , 2005 ) . furthermore , the ch2 domain of p66shc ( a specific n - terminus domain that distinguishes p66shc from the shorter shc isoforms , p46 and p52 ) seems to form a redox module responsible for apoptosis initiation , and can be activated through reversible tetramerization by forming two disulfide bonds ( donoghue et al . , the redox activity of p66shc is likely to be the explanation of the decrease in ros levels typical of p66shc knockout cells ( migliaccio et al . , 2006 ) , as well as an altered mitochondrial metabolism , characterized by lower oxygen consumption under basal conditions ( nemoto et al . , 2006 ) to exert its functions , p66shc has to be phosphorylated at serine 36 ( migliaccio et al . , 1999 ) : the responsible kinase was identified as pkc ( pinton et al . , 2007 ) . this phosphorylation of ser36 induces translocation of phosphorylated p66shc to mitochondria , and both h2o2 challenge and pkc activation promote binding of p66shc to pin1 , causing this translocation ( pinton et al . , 2007 ) . the enzymatic activity of pin1 is fundamental because the phosphorylated sites , once isomerized by pin1 , become targets for dephosphorylation by pp2a ( wulf et al . , 2005 ) . this solves the apparent paradox showing the importance of p66shc phosphorylation and the dephosphorylated state of p66shc within mitochondria . thus , the signaling pathway involving pkc , pin1 and pp2a controls the mitochondrial import of p66shc where the translocated protein can exert its oxidoreductase activity , generating h2o2 and inducing the opening of the ptp . all these events are finely linked to an altered mitochondrial ca homeostasis , revealing a primary mitochondrial perturbation both in structure and function . in fact , oxidative stress causes a drastic reduction of [ ca]m in mef cells , whereas knockouts for p66shc or pin1 are mostly insensitive to h2o2 treatment . moreover , the simple over - expression of pkc induces a strong decrease of mitochondrial ca uptake , underlining how mitochondrial ca alteration is one of the first events of oxidoreductase activity of p66shc . translocation of p66shc to mitochondria is a characteristic event of some kinds of tumors , such as prostate cancer . mef cells from p66shc knockout mice were more resistant to peitc ( an apoptosis - inhibitor ) and the same treatment increased both ser36 phosphorylation and p66shc - pin1 binding in pc-3 and lncap human cancer prostate cells ( xiao and singh , 2010 ) . moreover , growth stimulation of prostate cancer cells with 5-dihydrotestosterone ( dht ) is accompanied by increased p66shc levels , ros production , translocation of p66shc into mitochondria and an augmented interaction with cyt c ( veeramani et al . , 2008 ) . among other deleterious consequences described in this article , mitochondrial impairment has also been associated with the pathogenesis of some neurodegenerative and neuroinflammatory disorders . many lines of evidence suggest that both mitochondrial dna mutations and increased oxidative stress are major contributors in aging a process rendering the organism prone to neurodegeneration . particularly interesting , it has been demonstrated in many of the most frequently occurring diseases that mitochondria interact with proteins crucial to the development the pathology . in these interactions , ca homeostasis often plays a key role ( see fig . 2 ) . this brings hope for effective therapies that target mitochondrial processes and interaction sites of mitochondria and disease - related proteins . the most frequent age - related neurodegenerative disorder alzheimer 's disease ( ad ) is manifested by an impairment in cognitive abilities , particularly responsible for memory functions . at the molecular level , ad pathogenesis is associated with the accumulation of a number of proteins , including reelin and presenilins , which leads to the overproduction of -amyloid ( a ) . the -amyloid toxicity is further supported by the inability of neurons to properly regulate intracellular ca levels ( mattson , 2007 ; thibault et al . , 2007 ) . harmful consequences of a activity include increased ca influx into neurons , rendering them prone to excitotoxicity and apoptosis ( bezprozvanny and mattson , 2008 ) . also the interaction of a with other cations , such as fe and cu , leads to the generation of oh which causes membrane lipid peroxidation and , in turn , impairs the function of plasma membrane ca atpase ( pmca ) , a ca pump responsible for ca extrusion from the cell interior to the extracellular compartment . moreover , lipid peroxidation due to destabilization of membrane composition causes depolarization of plasma membrane and leads to the opening of nmdar and voltage - gated ca channels , additionally intensifying an influx of toxic amounts of ca into the cytoplasm ( berridge , 2010 ) . finally , mutants of presenilins lower the [ ca ] of intracellular store by increasing ca leakage and reducing ca uptake ( brunello et al . , 2009 ) . ca homeostasis can also be affected by ros due to the deterioration of membranes forming the intracellular ca stores ( e.g. , endoplasmic reticulum , er ) . it has also been demonstrated that a accumulates in mitochondria decreasing the activity of complexes iii and iv of the respiratory chain ( anandatheerthavarada and devi , 2007 ) . through such a decrease in mitochondrial function , a propels a vicious cycle in which an elevation in cytosolic ca levels , oxidative stress , and decreased atp synthesis , all induce a further ca overload and an even higher oxidative stress ( lin and beal , 2006 ) . taken together these findings indicate that suppression or blocking of ca influx could in effect promote the survival of degenerating neurons . several preliminary trials of l - type ca and nmdar channel blockers have been carried out with some success ; however , crucial obstacles still need to be overcome before the therapy can be considered successful ( mattson , 2007 ) . the most frequent movement disorder parkinson 's disease ( pd ) is caused by widespread neurodegeneration in the brain and a significant selective loss of nigrostriatal dopaminergic neurons ( thomas and beal , 2010 ) . processes underlying pd pathogenesis , especially in its non - familial occurrence , remain unclear . in both cases , the engagement of l - type ca channels is crucial in spontaneous pacemaking ( hausser et al . , 2004 ) , while at the same time it may contribute to elevated toxicity of mitochondrial toxins to nigrostriatal dopaminergic neurons . in this way , ca homeostasis is a regulator of their selective vulnerability ( chan et al . , 2009 ; surmeier et al . , 2010 ) . it has been proposed that the reduction in expression of the transient receptor potential cation ( trpc1 ) channel may be involved in dopaminergic neurodegeneration . trpc1 could also be neuroprotective against pd - inducing agents ( bollimuntha et al . , 2005 ) . huntington 's disease ( hd ) is characterized by severe motor , cognitive , and psychiatric symptoms . the genetic etiology of hd has been established to be a cag - triplet mutation within the huntingtin gene , inherited in an autosomal dominant fashion . possible links between mitochondrial dysfunctions and changes in ca homeostasis and hd pathology have been recently reviewed ( celsi et al . , 2009 ) . a number of authors report that this disease , prevalently initiated by neurological insults , is related to shifts in ca homeostasis . an increase in extracellular glutamate concentration sustains long - term distortions in ca signaling , a characteristic recognized as the epileptic phenotype ( delorenzo et al . a number of mitochondrial , as well as nuclear , dna mutations have been implicated with epileptic phenotypes . changes in electron transport chain activity increase metabolic demand in neurons and lead to distortions in ca homeostasis increasing its dendritic level . the number and distribution of mitochondria varies from one tissue to another , depending from the tissue 's dependence on oxidative phosphorylation for energy provision . for this reason , mitochondrial diseases ( mids ) predominantly manifest themselves in tissue / organs with high - energy requirements , and are aggravated by fever , infection , stress , toxic agents , or certain drugs ( finsterer , 2006 ) . mids are mainly due to mutations in mitochondrial or nuclear dna ( mtdna and ndna , respectively ) , resulting in the altered function of the respiratory chain or oxidative phosphorylation ( oxphos ) ( reinecke et al . , 2009 ) . the respiratory chain machinery consists of approximately 80 proteins , of which 13 are encoded by mtdna and all others are encoded by ndna ( dimauro , 2004 ; finsterer , 2010 ) . interplay between the mitochondrial and nuclear genomes is evident in disorders of nuclear- and mtdna - encoded subunits of the oxphos complexes ( smeitink et al . , deficiencies in oxphos result in immediate and downstream metabolic , structural , and functional effects . atp production , and consequently atp / adp homeostasis , is disturbed in oxphos deficiencies ( smeitink et al . , 2006 ) . the mitochondrial genome possesses a very high mutation rate , 10- to 17-fold higher than that observed in ndna . protective histones are also lacking , and although mtdna repair systems do exist ( de souza - pinto et al . , 2009 ) , they are not sufficient to counteract the oxidative damage sustained by the mitochondrial genome ( tuppen et al . , 2010 ) . at the same time , pathogenic ndna mutations are likely to be more numerous than pathogenic mtdna mutations ( finsterer , 2010 ) . point mutations have been correlated with numerous diseases , such as leigh syndrome , barth syndrome , neuropathies , cardiomyopathies , hepatopathy , nephropathy , multisystemic disease , and neurodegenerative diseases , including pd , ad and amyotrophic lateral sclerosis . ( dimauro , 2004 ; finsterer , 2008 ; guy et al . , 2002 ) . given the multi - systemic feature of mitochondrial cytopathies , different pharmacological approaches are required , but most of the proposed therapies are usually of scarce effectiveness ( zaffanello and zamboni , 2005 ) . non - specific drug therapy consists of different pharmacological agents , divided according to the type of action into antioxidants and lactate lowering agents ( which remove noxious metabolites ) , electron transfer mediators ( which bypass the defective site ) , alternative energy providers , cofactors , and other agents ( finsterer , 2010 ) , such as coenzyme q10 , ascorbic acid , vitamin e , lipoic acid , riboflavin , thiamin , niacin , and vitamin k ( phylloquinone and menadione ) . the general objective is to increase mitochondrial atp production and to slow ( or arrest ) the progression of the clinical symptoms ( marriage et al . , 2003 ) . in order to correct the energy imbalance in mids , it would be interesting to introduce drugs able to act on intracellular ca homeostasis since a strong relationship exists between ca and mitochondrial atp synthesis ( see fig . 2 ) . this ca content is actively maintained by the sarcoplasmic / endoplasmic ca atpase ( serca ) located on membranes of the er that constantly pump cytosolic ca into the lumen , at the expense of atp . this atp is mostly provided by mitochondria through the close contact sites between er and mitochondria ( giorgi et al . , 2009 ) . it has been reported , especially for defects on respiratory complex i , that cells from patients with mitochondrial diseases show reduced mitochondrial membrane potential together with reduced atp synthesis . this causes a reduced mitochondrial ca uptake under physiological stimulations ( visch et al . , 2006 ) . since ca uptake by mitochondria promotes krebs cycle activity and atp synthesis ( jouaville et al . , 1999 ) , it could be hypothesized that a negative feedback is created that maintains low intracellular ca levels and atp contents in cells derived from mids patients . in this scenario , it would be interesting to study the molecules capable of increasing er or mitochondrial ca levels . it has already been proposed that the pharmacological inhibition of the mitochondrial sodium ca exchanger ( that extrudes ca from mitochondria after physiological stimulation , recovering the basal [ ca ] ) is able to restore mitochondrial atp synthesis in cells harboring mitochondrial dna mutations ( brini et al . mitochondrial ca uptake is also a signaling event in the apoptotic cascade , especially in oxidative stress conditions . indeed , sustained mitochondrial ca elevations are able to induce the opening of the ptp , almost without stimulating the krebs cycle . since oxidative stress appears to be a common feature in many mids , therapeutical approaches based on ca handling should be considered together with antioxidant drugs or blockers of the ptp . the latter , especially cyclosporin a ( csa ) , are able to inhibit cytochrome c release induced by a number of apoptotic stimuli ( ow et al . , 2008 ; walter et al . , 1998 ) and cyclosporins are used as immunosuppressants ( probably independently from their mitochondrial activity ) and diverse studies support a protective role for csa in diseases with associated mitochondrial dysfunctions . myoblasts from patients affected by ullrich congenital muscular dystrophy display several mitochondrial alterations and a higher susceptibility to apoptosis due to the inappropriate opening of the ptp . in these conditions , it has been observed that csa inhibits apoptosis engagement ( merlini et al . , 2008 ) . a multitude of recent studies suggest that mitochondria play critical roles in both the life and death of terminally differentiated cardiac myocytes . mitochondria malfunction has been implicated in various cardiac pathologies , including ischemia reperfusion ( i / r ) injury , cardiomyopathy , and congestive heart failure ( gustafsson and gottlieb , 2008 ) . in healthy myocytes , mitochondria occupy a large portion of the cell and are located between the myofibrils and just below the sarcolemma ( andrienko et al . , 2003 ) . pathophysiological conditions that can open the ptp are characteristically present during myocardial i / r ( di lisa and bernardi , 2006 ) ( see fig . 2 ) . as such , an increasing number of pharmacological approaches are currently being developed to try to interfere with these parameters . cardioprotective effects of strategies that limit mitochondrial ca accumulation , have been demonstrated ( miyamae et al . , 1996 ) . for instance , treatment of hearts with ruthenium red ( rr ) or ru360 , inhibitors of the ca uniporter , reduced infarct size in hearts subjected to i / r ( garcia - rivas gde et al . , 2006 ; park et al . , 1990 ) . in addition , ru360 treatment dramatically decreased [ ca]m and inhibited ptp opening , which was associated with improved recovery of heart performance ( goldberg and bursey , 1992 ) . mitochondrial membrane potential is another critical regulator of ca accumulation : depolarization reduces the driving force for ca uptake by mitochondria and thereby prevents ca overload ; this prompted researchers to consider the decrease of the potential as a possible mechanism of cellular protection . this objective is attained with uncoupler agents such as 2 - 4-dinitrophenol ( dnp ) and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone ( fccp ) ( brennan et al . , 2006 ) , and mitochondrial atp - sensitive k ( mitokatp ) channel openers , such as diazoxide ( garlid et al . , 1997 ; iwai et al . , 2000 ) or nicorandil , ( carreira et al . , 2008 ; iwai et al . , 2002 ) , which showed a cardioprotective effect in different models of i / r ( ishii et al . , 2005 ; kitakaze et al . pharmacological openers of the mitokatp channel have been developed for the treatment of angina pectoris ( nicorandil ) and hypertension ( cromakalim and pinacidil ) ( grover et al . cardiac mitokatp channels were also identified as important mediators of protection during ischemic preconditioning ( ipc ) , the most potent method for reducing reperfusion injury ( fryer et al . , 2000 ; murry et al . , 1986 ) , and thus represent a promising drug target . oxidative stress has also been associated with loss of cells in heart failure , i / r injury , and doxorubicin - induced cardiomyopathy , whereas reducing ros production is cardioprotective ( gustafsson and gottlieb , 2008 ) . moreover , there is an increased production of ros in failing hearts after myocardial infarction and consequently lipid peroxidation , decrease of mtdna copy number , and a reduced oxidative capacity due to lower activity of electron transfer enzymes ( ide et al . , 2001 ) . antioxidants , such as vitamin e ( sethi et al . , 2000 ) , ginkgo biloba extract ( seif - el - nasr and el - fattah , 1995 ) , trans - resveratrol ( goh et al . , 2007 ; ray et al . , 1999 ) , that remove radicals from cells , protect against i / r injury ( reeve et al . , 2005 ) . the use of novel mitochondria - targeted antioxidants has gained much interest ( victor and rocha , 2007 ) . mitoq , based on the endogenous mitochondrial ubiquinone coenzyme q , is such a compound ; a recent study demonstrated that feeding rats with mitoq significantly limited cardiac reperfusion injury ( adlam et al . , 2005 ) . moreover , inhibition of the ptp through direct targeting of its putative components ( i.e. , the adenine nucleotide translocator ( ant ) and cyclophilin d ( cyp - d ) ) may represent an effective therapeutic approach in protecting the heart against various cardiac pathologies ( reviewed in ( bernardi and forte , 2007 ; halestrap and pasdois , 2009 ) ) . csa has been demonstrated to be cardioprotective in isolated cardiomyocytes subjected to anoxia and reoxygenation ( griffiths et al . , 2000 ; nazareth et al . , 1991 ) , in langendorff - perfused hearts subjected to global i / r ( griffiths and halestrap , 1993 ) , as well as in animals subjected to coronary artery ligation and reperfusion ( hausenloy et al . , 2003 ) . one problem with the use of csa is that it can induce additional effects through inhibition of the ca - regulated protein phosphatase calcineurin , that has direct effects on heart function ( periasamy , 2002 ) and also undesirable immunosuppressive activity ( schreiber and crabtree , 1992 ) . to overcome this , other cyclosporine derivatives , such as the immunosuppressant sanglifehrin a ( sfa ) , as well as non - immunosuppressants [ n - methyl - ala]csa , [ n - methyl - val]csa , debio 025 , nim 811 and unil025 , were found to exhibit similar properties in terms of blocking pore opening , while being devoid of calcineurin inhibitory activity ( clarke et al . , 2002 ; gomez et al . in addition , studies on isolated mitochondria and cultured cardiomyocytes have confirmed a protective role of the ant inhibitor bongkrekic acid , via inhibition of pore opening ( akao et al . , recently , schaller et al . , ( 2010 ) have discovered a new mitochondrial - targeted cardioprotective drug , tro40303 ( 3,5-seco-4-nor - cholestan-5-one oxime-3-ol ) , that binds specifically to the mitochondrial translocator protein ( tspo , formerly known as peripheral benzodiazepine receptor ) and inhibits ptp opening triggered by oxidative stress . another interesting result was obtained with the free radical scavenger edavarone , which was shown to inhibit ptp opening and to prevent cardiac reperfusion injury ( tsujita et al . , 2006 ) . finally , the bcl-2 family proteins , which control mitochondrial membrane permeabilization and ca homeostasis , also play a central role in regulating apoptosis in the cardiovascular system ( gustafsson and gottlieb , 2007 ) . many of the pro - apoptotic bcl-2 proteins have been implicated in the pathogenesis of various cardiac diseases , including myocardial hypertrophy , infarction , and heart failure ( condorelli et al . , 1999 ; jung et al . , , anti - apoptotic interventions targeting the bcl-2 protein family provide opportunities for possible anti - ischemic therapies ( reed , 2001 ) , either through a gain of anti - apoptotic function or loss of pro - apoptotic function . the pharmacological strategy inhibiting mitochondrial outer membrane permeability , and thus apoptosis , by manipulation of the bcl-2 family to protect the myocardium against i / r injury is very recent but has already provided interesting results ( hetz et al . , 2005 ) , supporting the idea that clinical benefits may be obtained in the near future . from the above - described evidence , therapeutic interventions designed to prevent mitochondrial damage hold major promise as a novel strategy for reducing cardiac injury , the leading cause of death in western societies . effective therapies developed along these lines will thus represent a major advance in health care . diabetes mellitus is a disorder resulting from defects of both insulin secretion and action . to date , the cellular pathophysiology of diabetes - induced impairments , remain controversial . conceptually , the leading mechanism driving cellular pathology is believed to be associated with the accumulation of extracellular glucose , and generation of ros , thereby triggering pathological outcomes . there is abundant evidence suggesting a central role of mitochondria in diabetes , insulin resistance , insulin secretion and in complications derived from diabetes ( patti and corvera , 2010 ) . however , it is still difficult ( and controversial ) to establish whether the alterations in mitochondrial function are the cause or the effect of the disease . typically , the number and size of mitochondria is reduced in the muscle of diabetics , there is reduced mitochondrial biogenesis and switching of metabolic substrate use that is linked directly to mitochondrial enzymes ( chabi et al . , 2005 ) . mitochondria are an integral part of the insulin system found in the islet cells of the pancreas . ultrastructural examination of -cells has suggested that mitochondria are often in close proximity to the secretory insulin granules . this may facilitate metabolism secretion coupling , as atp is a major permissive factor for movement of insulin granules and for priming of exocytosis ( maechler and wollheim , 2001 ) . when blood glucose is high , the rate of glycolysis in -cells will increase and , as a consequence , the atp production is accelerated to provide the energy for insulin exocytosis . glucose - stimulated insulin release from -cells involves a complex series of signaling pathways . as in other exocytotic fusion events , ca is an essential trigger of insulin granule exocytosis ( rorsman and renstrom , 2003 ) . the ca signal resulting from mitochondrial activation completes the chain of events between glucose uptake into -cells and insulin exocytosis . a common motif in these cascades is the elevation of intracellular ca both in the cytosol and within the mitochondria . mitochondria take up ca through the mitochondrial ca uniporter in response to the glucose - induced rise in cytoplasmic ca , and release ca through the mncx ( bernardi , 1999a ) . [ ca]c rises may originate from ca influx at the plasma membrane or ca mobilization from the er . local ca influx or mobilization creates microdomains , local increases of ca exceeding the bulk [ ca ] in the cytosol ( giorgi et al . , 2009 ) . for both pathways , the amplitude and the duration of the [ ca]c rise may determine to what extent -cell mitochondria are activated . depending on the combination of secretagogues , the number and localization of such ca domains most certainly vary ( rizzuto and pozzan , 2006 ; rutter et al . , 2006 ) . the diabetic state is related to mitochondrial dysfunction and generally characterized by insufficient supply of energy or defects in the insulin signaling pathway . it has been shown that a reduction in mitochondrial ca accumulation is responsible for a reduction in insulin secretion ( wiederkehr and wollheim , 2008 ) , suggesting that modulation of mitochondrial ca may be a therapeutic target . restoration of the mitochondrial ca signal using an inhibitor of the mnce was shown to improve atp generation and insulin secretion ( lee et al . , 2003 ; visch et al . , 2004 ) , highlighting the exchanger as a potential new target for diabetes drug discovery . among the most convincing links between mitochondrial dysfunction and diabetes indeed , diabetes mellitus is very common in patients affected by mitochondrial diseases ( also referred to as mitochondrial encephalomyopathies , characterized by genetic disorders of mtdna , such as merrf and melas ) , while and a maternally - inherited form of diabetes is associated to mtdna mutations ( ballinger et al . , 1992 ) . accordingly , with the above suggested pivotal role of ca homeostasis in the cellular pathogenesis of diabetes , agonist - induced ca rises and atp generation were blunted and could be restored by applying an inhibitor of the mitochondrial ca efflux ( brini et al . , 1999 ) . finally , but not less important , an abnormal ca homeostasis and impaired mitochondrial function diabetic conditions affect er ca homeostasis in sensory neurons by lowering the er ca content due to reduced serca pump activity , inducing a mild condition of er stress that results in the decrease of nerve conductance velocity ( verkhratsky and fernyhough , 2008 ) . in conclusion , the signaling mechanisms that decode changes in nutrient supply are complex and not completely understood , but a control in intracellular [ ca ] , in particular at the mitochondrial level , appears to be of great significance for further research . mitochondria are the powerhouse of the cell being the place where oxidative metabolism takes place , rendering mitochondria crucial both in health and disease . many ( if not all ) of the mitochondrial activities are driven in a ca - dependent manner . the biochemical properties of the proteins involved in mitochondrial ca homeostasis have been known for over four decades , but only recently have we begun to unravel the dynamics of this cation at the molecular and mechanistic levels ( hajnoczky and csordas , 2010 ) . currently , specific drugs that act on mitochondrial ca homeostasis are being developed and these may open the way to new biochemically designed therapeutic approaches in the treatment of several disorders .

mitochondria are crucial in different intracellular pathways of signal transduction . mitochondria are capable of decoding a variety of extracellular stimuli into markedly different intracellular actions , ranging from energy production to cell death . the fine modulation of mitochondrial calcium ( ca2 + ) homeostasis plays a fundamental role in many of the processes involving this organelle . when mitochondrial ca2 + homeostasis is compromised , different pathological conditions can occur , depending on the cell type involved . recent data have shed light on the molecular identity of the main proteins involved in the handling of mitochondrial ca2 + traffic , opening fascinating and ambitious new avenues for mitochondria - based pharmacological strategies .

Introduction Mitochondria and aging Mitochondria and neurodegenerative diseases Mitochondrial diseases Mitochondria as drug targets in the treatment of cardiovascular diseases Mitochondria, Ca Conclusions

it is now firmly established that mitochondria are key players in different pathophysiological contexts ( duchen , 2004 ) . indeed , they are very efficient machines for decoding intracellular signals and in particular the calcium ( ca ) one ( clapham , 2007 ) . this implies a strong thermodynamic force in favor of the accumulation of cations ( rizzuto et al . ca import across the outer mitochondrial membrane ( omm ) occurs through the voltage - dependent anion channels ( vdac ) ( simamura et al . , 2008 ; shoshan - barmatz et al . despite intense investigations on the mitochondrial ca uniporter ( mcu ) for almost 50 years , the molecular identity of this gated channel is still a matter of debate . among the early candidates for the mcu were mitochondria - localized ryr1 ( beutner et al . , 2005 ) and the uncoupling proteins ( ucp ) 2 and 3 ( trenker et al . , 2007 ) , but such a role for these proteins is still controversial and further work will be needed to definitively elucidate the molecular identity of the channels or carriers involved . the identity of the mcu , as the product of the ccdc109a gene , has recently been reported by two recent publications ( baughman et al . , 2009 ) . finally , it is important to mention the recent description of an imm ca - binding protein named mitochondrial calcium uptake 1 ( micu1 ) , which appears essential for mitochondrial ca uptake ( perocchi et al . about the system for mitochondrial ca extrusion , the mitochondrial na / ca exchanger ( mncx ) and h / ca exchanger ( mhcx ) , have well known kinetic characteristics , are present in the imm ( bernardi , 1999b ) . 1 ) . its role in mitochondrial ca homeostasis , however , remains elusive ( bernardi and forte , 2007 ) . in a variety of cell systems ( ranging from epithelial cells to neurons ) , the cytosolic [ ca ] ( [ ca]c ) rises evoked by physiological stimulations are always paralleled by rapid mitochondrial [ ca ] ( [ ca]m ) increases , that reach values well above those of the bulk cytosol ( up to ~ 500 m in chromaffin cells ) ( giorgi et al . a ca signal originating in the cytoplasm that is propagated to mitochondria permits the transmission of an activatory signal to the energy powerhouse of the cell . thus , in conjunction with the triggering of energy - consuming processes in the cytosol ( contraction , secretion , etc . ) the alteration of the ca signals that reach mitochondria in association with different pathological conditions ( e.g. as a consequence , the cell may be driven to its death ( giorgi et al . here , we review some human diseases associated with perturbations in mitochondrial homeostasis with particular emphasis on the role of ca signals . this degeneration is characterized by a progressive loss of organ function , proposed to be at the basis of several degenerative disorders , promoted by a progressive occurrence of apoptosis in non - proliferating cells . mitochondria are considered the major source of ros and the extent of oxidative damage to macromolecules is generally thought to be the random consequence of physiological functions , as exemplified by lipid peroxidation , protein oxidation , and mitochondrial dna mutations ( cadenas and davies , 2000 ) . different works on the molecular routes of apoptosis have revealed the important role of mitochondria in decoding of oxidative insults , with the release of proteins such as cytochrome c ( cyt c ) or smac / diablo , which act as co - factors of effector caspases . ca overload , through a still controversial mechanism , can also lead to the opening of the ptp and swelling of the organelle in what appears to be a pivotal pathway ( duchen , 2000 ; ravagnan et al . one important implication of recent findings is that these phenomena are genetically determined at least in part and biochemically controlled by a stress - induced signal transduction pathway involving the 66-kda isoform of shc , p66shc . the relationship between mitochondria and p66shc emerged once the protein was effectively localized to the organelle and by the observation that , upon oxidative stress , part of the cytosolic pool of p66shc translocates to mitochondria ( orsini et al . , 2004 ) . within mitochondria , in particular in the mitochondrial intermembrane space , p66shc binds cyt c and acts as a redox - enzyme , generating h2o2 , which , in turn , induces the opening of the ptp and apoptosis . , 2005 ) . , the redox activity of p66shc is likely to be the explanation of the decrease in ros levels typical of p66shc knockout cells ( migliaccio et al . , 2006 ) , as well as an altered mitochondrial metabolism , characterized by lower oxygen consumption under basal conditions ( nemoto et al . , 2006 ) to exert its functions , p66shc has to be phosphorylated at serine 36 ( migliaccio et al . the enzymatic activity of pin1 is fundamental because the phosphorylated sites , once isomerized by pin1 , become targets for dephosphorylation by pp2a ( wulf et al . thus , the signaling pathway involving pkc , pin1 and pp2a controls the mitochondrial import of p66shc where the translocated protein can exert its oxidoreductase activity , generating h2o2 and inducing the opening of the ptp . moreover , the simple over - expression of pkc induces a strong decrease of mitochondrial ca uptake , underlining how mitochondrial ca alteration is one of the first events of oxidoreductase activity of p66shc . moreover , growth stimulation of prostate cancer cells with 5-dihydrotestosterone ( dht ) is accompanied by increased p66shc levels , ros production , translocation of p66shc into mitochondria and an augmented interaction with cyt c ( veeramani et al . particularly interesting , it has been demonstrated in many of the most frequently occurring diseases that mitochondria interact with proteins crucial to the development the pathology . in these interactions , ca homeostasis often plays a key role ( see fig . 2 ) . the most frequent age - related neurodegenerative disorder alzheimer 's disease ( ad ) is manifested by an impairment in cognitive abilities , particularly responsible for memory functions . at the molecular level , ad pathogenesis is associated with the accumulation of a number of proteins , including reelin and presenilins , which leads to the overproduction of -amyloid ( a ) . also the interaction of a with other cations , such as fe and cu , leads to the generation of oh which causes membrane lipid peroxidation and , in turn , impairs the function of plasma membrane ca atpase ( pmca ) , a ca pump responsible for ca extrusion from the cell interior to the extracellular compartment . , 2009 ) . it has also been demonstrated that a accumulates in mitochondria decreasing the activity of complexes iii and iv of the respiratory chain ( anandatheerthavarada and devi , 2007 ) . through such a decrease in mitochondrial function , a propels a vicious cycle in which an elevation in cytosolic ca levels , oxidative stress , and decreased atp synthesis , all induce a further ca overload and an even higher oxidative stress ( lin and beal , 2006 ) . several preliminary trials of l - type ca and nmdar channel blockers have been carried out with some success ; however , crucial obstacles still need to be overcome before the therapy can be considered successful ( mattson , 2007 ) . the most frequent movement disorder parkinson 's disease ( pd ) is caused by widespread neurodegeneration in the brain and a significant selective loss of nigrostriatal dopaminergic neurons ( thomas and beal , 2010 ) . in both cases , the engagement of l - type ca channels is crucial in spontaneous pacemaking ( hausser et al . , 2004 ) , while at the same time it may contribute to elevated toxicity of mitochondrial toxins to nigrostriatal dopaminergic neurons . in this way , ca homeostasis is a regulator of their selective vulnerability ( chan et al . , 2009 ; surmeier et al . it has been proposed that the reduction in expression of the transient receptor potential cation ( trpc1 ) channel may be involved in dopaminergic neurodegeneration . , 2005 ) . a number of mitochondrial , as well as nuclear , dna mutations have been implicated with epileptic phenotypes . the number and distribution of mitochondria varies from one tissue to another , depending from the tissue 's dependence on oxidative phosphorylation for energy provision . for this reason , mitochondrial diseases ( mids ) predominantly manifest themselves in tissue / organs with high - energy requirements , and are aggravated by fever , infection , stress , toxic agents , or certain drugs ( finsterer , 2006 ) . mids are mainly due to mutations in mitochondrial or nuclear dna ( mtdna and ndna , respectively ) , resulting in the altered function of the respiratory chain or oxidative phosphorylation ( oxphos ) ( reinecke et al . , 2009 ) . interplay between the mitochondrial and nuclear genomes is evident in disorders of nuclear- and mtdna - encoded subunits of the oxphos complexes ( smeitink et al . the mitochondrial genome possesses a very high mutation rate , 10- to 17-fold higher than that observed in ndna . , 2009 ) , they are not sufficient to counteract the oxidative damage sustained by the mitochondrial genome ( tuppen et al . , 2010 ) . point mutations have been correlated with numerous diseases , such as leigh syndrome , barth syndrome , neuropathies , cardiomyopathies , hepatopathy , nephropathy , multisystemic disease , and neurodegenerative diseases , including pd , ad and amyotrophic lateral sclerosis . given the multi - systemic feature of mitochondrial cytopathies , different pharmacological approaches are required , but most of the proposed therapies are usually of scarce effectiveness ( zaffanello and zamboni , 2005 ) . non - specific drug therapy consists of different pharmacological agents , divided according to the type of action into antioxidants and lactate lowering agents ( which remove noxious metabolites ) , electron transfer mediators ( which bypass the defective site ) , alternative energy providers , cofactors , and other agents ( finsterer , 2010 ) , such as coenzyme q10 , ascorbic acid , vitamin e , lipoic acid , riboflavin , thiamin , niacin , and vitamin k ( phylloquinone and menadione ) . the general objective is to increase mitochondrial atp production and to slow ( or arrest ) the progression of the clinical symptoms ( marriage et al . , 2003 ) . this ca content is actively maintained by the sarcoplasmic / endoplasmic ca atpase ( serca ) located on membranes of the er that constantly pump cytosolic ca into the lumen , at the expense of atp . this atp is mostly provided by mitochondria through the close contact sites between er and mitochondria ( giorgi et al . , 2009 ) . since ca uptake by mitochondria promotes krebs cycle activity and atp synthesis ( jouaville et al . , 1999 ) , it could be hypothesized that a negative feedback is created that maintains low intracellular ca levels and atp contents in cells derived from mids patients . in this scenario , it would be interesting to study the molecules capable of increasing er or mitochondrial ca levels . it has already been proposed that the pharmacological inhibition of the mitochondrial sodium ca exchanger ( that extrudes ca from mitochondria after physiological stimulation , recovering the basal [ ca ] ) is able to restore mitochondrial atp synthesis in cells harboring mitochondrial dna mutations ( brini et al . mitochondrial ca uptake is also a signaling event in the apoptotic cascade , especially in oxidative stress conditions . indeed , sustained mitochondrial ca elevations are able to induce the opening of the ptp , almost without stimulating the krebs cycle . since oxidative stress appears to be a common feature in many mids , therapeutical approaches based on ca handling should be considered together with antioxidant drugs or blockers of the ptp . , 2008 ; walter et al . myoblasts from patients affected by ullrich congenital muscular dystrophy display several mitochondrial alterations and a higher susceptibility to apoptosis due to the inappropriate opening of the ptp . in healthy myocytes , mitochondria occupy a large portion of the cell and are located between the myofibrils and just below the sarcolemma ( andrienko et al . pathophysiological conditions that can open the ptp are characteristically present during myocardial i / r ( di lisa and bernardi , 2006 ) ( see fig . 2 ) . as such , an increasing number of pharmacological approaches are currently being developed to try to interfere with these parameters . cardioprotective effects of strategies that limit mitochondrial ca accumulation , have been demonstrated ( miyamae et al . , 1996 ) . for instance , treatment of hearts with ruthenium red ( rr ) or ru360 , inhibitors of the ca uniporter , reduced infarct size in hearts subjected to i / r ( garcia - rivas gde et al . mitochondrial membrane potential is another critical regulator of ca accumulation : depolarization reduces the driving force for ca uptake by mitochondria and thereby prevents ca overload ; this prompted researchers to consider the decrease of the potential as a possible mechanism of cellular protection . , 2006 ) , and mitochondrial atp - sensitive k ( mitokatp ) channel openers , such as diazoxide ( garlid et al . , 2000 ) or nicorandil , ( carreira et al . , 2002 ) , which showed a cardioprotective effect in different models of i / r ( ishii et al . pharmacological openers of the mitokatp channel have been developed for the treatment of angina pectoris ( nicorandil ) and hypertension ( cromakalim and pinacidil ) ( grover et al . , 2000 ; murry et al . , 1986 ) , and thus represent a promising drug target . , 2001 ) . the use of novel mitochondria - targeted antioxidants has gained much interest ( victor and rocha , 2007 ) . mitoq , based on the endogenous mitochondrial ubiquinone coenzyme q , is such a compound ; a recent study demonstrated that feeding rats with mitoq significantly limited cardiac reperfusion injury ( adlam et al . moreover , inhibition of the ptp through direct targeting of its putative components ( i.e. one problem with the use of csa is that it can induce additional effects through inhibition of the ca - regulated protein phosphatase calcineurin , that has direct effects on heart function ( periasamy , 2002 ) and also undesirable immunosuppressive activity ( schreiber and crabtree , 1992 ) . in addition , studies on isolated mitochondria and cultured cardiomyocytes have confirmed a protective role of the ant inhibitor bongkrekic acid , via inhibition of pore opening ( akao et al . finally , the bcl-2 family proteins , which control mitochondrial membrane permeabilization and ca homeostasis , also play a central role in regulating apoptosis in the cardiovascular system ( gustafsson and gottlieb , 2007 ) . many of the pro - apoptotic bcl-2 proteins have been implicated in the pathogenesis of various cardiac diseases , including myocardial hypertrophy , infarction , and heart failure ( condorelli et al . the pharmacological strategy inhibiting mitochondrial outer membrane permeability , and thus apoptosis , by manipulation of the bcl-2 family to protect the myocardium against i / r injury is very recent but has already provided interesting results ( hetz et al . , 2005 ) , supporting the idea that clinical benefits may be obtained in the near future . conceptually , the leading mechanism driving cellular pathology is believed to be associated with the accumulation of extracellular glucose , and generation of ros , thereby triggering pathological outcomes . however , it is still difficult ( and controversial ) to establish whether the alterations in mitochondrial function are the cause or the effect of the disease . typically , the number and size of mitochondria is reduced in the muscle of diabetics , there is reduced mitochondrial biogenesis and switching of metabolic substrate use that is linked directly to mitochondrial enzymes ( chabi et al . mitochondria are an integral part of the insulin system found in the islet cells of the pancreas . ultrastructural examination of -cells has suggested that mitochondria are often in close proximity to the secretory insulin granules . glucose - stimulated insulin release from -cells involves a complex series of signaling pathways . a common motif in these cascades is the elevation of intracellular ca both in the cytosol and within the mitochondria . local ca influx or mobilization creates microdomains , local increases of ca exceeding the bulk [ ca ] in the cytosol ( giorgi et al . for both pathways , the amplitude and the duration of the [ ca]c rise may determine to what extent -cell mitochondria are activated . depending on the combination of secretagogues , the number and localization of such ca domains most certainly vary ( rizzuto and pozzan , 2006 ; rutter et al . , 2006 ) . the diabetic state is related to mitochondrial dysfunction and generally characterized by insufficient supply of energy or defects in the insulin signaling pathway . it has been shown that a reduction in mitochondrial ca accumulation is responsible for a reduction in insulin secretion ( wiederkehr and wollheim , 2008 ) , suggesting that modulation of mitochondrial ca may be a therapeutic target . restoration of the mitochondrial ca signal using an inhibitor of the mnce was shown to improve atp generation and insulin secretion ( lee et al . , 1992 ) . accordingly , with the above suggested pivotal role of ca homeostasis in the cellular pathogenesis of diabetes , agonist - induced ca rises and atp generation were blunted and could be restored by applying an inhibitor of the mitochondrial ca efflux ( brini et al . finally , but not less important , an abnormal ca homeostasis and impaired mitochondrial function diabetic conditions affect er ca homeostasis in sensory neurons by lowering the er ca content due to reduced serca pump activity , inducing a mild condition of er stress that results in the decrease of nerve conductance velocity ( verkhratsky and fernyhough , 2008 ) . in conclusion , the signaling mechanisms that decode changes in nutrient supply are complex and not completely understood , but a control in intracellular [ ca ] , in particular at the mitochondrial level , appears to be of great significance for further research . mitochondria are the powerhouse of the cell being the place where oxidative metabolism takes place , rendering mitochondria crucial both in health and disease . many ( if not all ) of the mitochondrial activities are driven in a ca - dependent manner . the biochemical properties of the proteins involved in mitochondrial ca homeostasis have been known for over four decades , but only recently have we begun to unravel the dynamics of this cation at the molecular and mechanistic levels ( hajnoczky and csordas , 2010 ) . currently , specific drugs that act on mitochondrial ca homeostasis are being developed and these may open the way to new biochemically designed therapeutic approaches in the treatment of several disorders .

recruitment , which began in january 1999 and ended in may 2001 , took place in eight hospital - based antenatal centers in scotland . a total of 250 women with type 1 diabetes consented to participate in the study ( a 94% participation rate of those enrolled in and planning to deliver in the centers ) , and cord blood samples were obtained from 200 subjects ( 80% ) . no differences in gestation at delivery , maternal age at delivery , years of diabetes , fetal sex , or maternal a1c ( where available ) were found between those with and without cord samples . a detailed sampling protocol was placed in all centers with local training to ensure standardization . given the effects of delayed cord clamping on stereological parameters ( 16 ) and to maximize cord blood available for collection , on immediate delivery of the fetus , two disposable cord clamps were placed at 10 cm from the umbilicus and a further two disposable cord clamps were placed at 30 cm from the umbilicus . this allowed an isolated loop of cord to be sampled for cord blood , facilitating the short median collection time for cord samples and a constant volume of fetal blood in the placenta . on delivery of the placenta , a fifth clamp was placed at 1 cm from the chorionic plate , with trimming of the cord to that level . to minimize effects of sample hemolysis on insulin levels , samples were included only if collected from the cord within 20 min and frozen within 60 min . the 200 samples were therefore restricted to those in whom 1 ) there was no evidence of hemolysis of cord blood ( 17 excluded ) ; 2 ) cord blood had been collected within 20 min ( 12 exclusions : median [ interquartile range ] collection time for remaining samples 2 min [ 17 ] ) ; 3 ) cord blood centrifuged and plasma frozen within 60 min ( 17 exclusions : time from collection to freezing for remaining samples , 17 min [ 1126 ] ) ; 4 ) antenatal glucocorticoids not administered in the 24 h before birth ( 15 excluded ) ; 5 ) children delivered before 33 weeks ' gestation ( 5 excluded ) ; and finally 6 ) placental tissue sampled appropriately at time of birth ( 59 excluded ) . a convenience sample of control mothers , with no history of obstetric or metabolic disease and with negative routine screening for gestational diabetes ( national guidelines available at http://www.sign.ac.uk/guidelines/fulltext/55/section8.html ) , were recruited from routine obstetric follow - up clinics after the 34th week of pregnancy in the same centers at the same time . of 145 women who gave initial consent , cord samples were attempted in 75 and obtained in 70 . data on clinical outcome , including caesarean section , intercurrent medical conditions , and hypertensive conditions of pregnancy , were obtained by case note review . this date was derived from dates of last menstrual period ( lmp ) , where available , or by ultrasound if there was either conflict with dates as assessed by lmp ( > 6 days ) or lmp was unavailable . weight was measured at birth and , for offspring born between 33 and 42 weeks of gestation , further expressed as an sd score ( 17 ) . skinfold thickness at subscapular and triceps was measured using holtain calipers by pediatricians at each site and using a centrally agreed protocol , which was available in writing at the time of measurement . there were no significant differences in baseline demographic or biochemical measures between those with and without skinfold measurements in either control subjects or ot1 dm ( data not shown ) . all mothers gave informed consent , and the local ethical committees approved protocols . on completion of the cord - sampling procedure and after delivery of the placenta , the placental membranes were trimmed , the umbilical cord shortened to within 1 cm of the chorionic plate , and any large maternal clots removed . placentae were then weighed to the nearest 1 g. the feto - placental index ( birth weight [ grams ] divided by placental weight [ grams ] ) was calculated for each delivery . specifically , the placenta was cut with a sharp knife into a series of parallel slices 12 cm thick . the slices were then diced into smaller blocks 23 cm wide and fixed by immersion in 4% formaldehyde for 24 h and subsequently embedded in paraffin wax . these were used to estimate the volume fractions and surface areas as previously described , with avoidance of section edges ( 10 ) . all estimates were made at the light microscope level by a combination of point and intersect counting and computer - assisted length measurements using the cast system ( version 2.0 ; olympus , glostrup , denmark ) . fields of view were selected in a systematic random fashion and analyzed as previously described ( 10 ) ( fig . volume densities of intermediate and terminal villi and of the villous trophoblast and capillaries were estimated by test point counting . villous and capillary surface densities were estimated by intersect counting . for each placenta , 200 events were counted across 20 systematic random fields of view per section . to convert volume and surface densities into absolute values ( in cm and cm ) , the volume of each placenta was taken as the reference volume . volumes were calculated from trimmed placental weights assuming a specific gravity of 1.05 g cm ( 18,19 ) . the intersections of the test lines with the villous surface also provided random start points for the measurement of orthogonal intercepts across the villous membrane , from the microvilli to the nearest capillary lumen . harmonic means of the intercept lengths were calculated and then converted to diffusion distances . given that there are standard values for physiological constants of oxygen in red cells , plasma , and tissues , including the villous membrane , the overall morphometric diffusing capacity for oxygen of the villous membrane of each placenta was able to be derived from the formula described by laga et al . this is based on the fick equation and provides an estimate of the maximal theoretical diffusion capacity of the placenta . intersections between the test lines acted as test points and were used for estimating the volume fraction of the villous components by the point counting technique . intersections between the test lines and the villous ( circles ) or capillary ( squares ) surfaces were counted to estimate the surface densities . intersections of the test lines with the villous surface also acted as random start points for the estimation of villous membrane thickness by measurement of orthogonal intercepts . ( a high - quality color digital representation of this figure is available in the online issue . ) changes of growth or adaptation of villi and the ivs were assessed by the isomorphy coefficient . briefly , this represents villous surface area , s , raised to the power 3/2 , and divided by the volume , v , of either the villous or intervillous compartment ( 20 ) and is designed to measure disproportionate growth of villous surface area compared with villous volume . similarly , the coefficients for capillary surface area relative to villous and capillary volume were also derived by s / v . the villous elaboration index , i = s / v , was also calculated for each case . this index gives similar information to the villous isomorphy coefficient , with a high value representing increased elaboration of the villous surface ( 8) . plasma insulin , 32 - 33 split proinsulin , proinsulin , leptin , igf - i , adiponectin , plasma total cholesterol , triglycerides , nonesterified fatty acid , vldl cholesterol , ldl cholesterol , hdl cholesterol , c - reactive protein ( crp ) , intercellular adhesion molecule ( icam)-1 , and interleukin ( il)-6 were assayed as previously described ( 13,2124 ) . in particular , igf - i was assayed by chemiluminescence immunoassay ( nichols institute diagnostics , san juan capistrano , ca ) using standards referenced to the world health organization 1st international reference reagent 1988 ( igf-1 87/518 ) . all lipid assays were carried out at the biochemistry department of glasgow royal infirmary , which is a centers for disease control and prevention ( atlanta , ga ) reference laboratory and accredited by clinical pathology accreditation u.k . data were analyzed using standard software ( stata 10 ; stata , college station , tx ) . in several cases ( insulin , leptin , triglycerides , vldl , nonesterified fatty acids , total - to - hdl cholesterol ratio , crp , icam-1 , and il-6 ) , measures were not normally distributed , and unadjusted values are presented as medians ( interquartile range ) and , for normally distributed variables , means sd . intergroup differences were assessed by unpaired t test after checking homogeneity of variance by means of the f test , anova , or , where further predictor variables were included , by general linear models . stepwise logistic regression was performed using an of p 0.1 for adding or removing predictors from the model . recruitment , which began in january 1999 and ended in may 2001 , took place in eight hospital - based antenatal centers in scotland . a total of 250 women with type 1 diabetes consented to participate in the study ( a 94% participation rate of those enrolled in and planning to deliver in the centers ) , and cord blood samples were obtained from 200 subjects ( 80% ) . no differences in gestation at delivery , maternal age at delivery , years of diabetes , fetal sex , or maternal a1c ( where available ) were found between those with and without cord samples . a detailed sampling protocol was placed in all centers with local training to ensure standardization . given the effects of delayed cord clamping on stereological parameters ( 16 ) and to maximize cord blood available for collection , on immediate delivery of the fetus , two disposable cord clamps were placed at 10 cm from the umbilicus and a further two disposable cord clamps were placed at 30 cm from the umbilicus . this allowed an isolated loop of cord to be sampled for cord blood , facilitating the short median collection time for cord samples and a constant volume of fetal blood in the placenta . on delivery of the placenta , a fifth clamp was placed at 1 cm from the chorionic plate , with trimming of the cord to that level . to minimize effects of sample hemolysis on insulin levels , samples were included only if collected from the cord within 20 min and frozen within 60 min . the 200 samples were therefore restricted to those in whom 1 ) there was no evidence of hemolysis of cord blood ( 17 excluded ) ; 2 ) cord blood had been collected within 20 min ( 12 exclusions : median [ interquartile range ] collection time for remaining samples 2 min [ 17 ] ) ; 3 ) cord blood centrifuged and plasma frozen within 60 min ( 17 exclusions : time from collection to freezing for remaining samples , 17 min [ 1126 ] ) ; 4 ) antenatal glucocorticoids not administered in the 24 h before birth ( 15 excluded ) ; 5 ) children delivered before 33 weeks ' gestation ( 5 excluded ) ; and finally 6 ) placental tissue sampled appropriately at time of birth ( 59 excluded ) . a convenience sample of control mothers , with no history of obstetric or metabolic disease and with negative routine screening for gestational diabetes ( national guidelines available at http://www.sign.ac.uk/guidelines/fulltext/55/section8.html ) , were recruited from routine obstetric follow - up clinics after the 34th week of pregnancy in the same centers at the same time . of 145 women who gave initial consent , cord samples were attempted in 75 and obtained in 70 . data on clinical outcome , including caesarean section , intercurrent medical conditions , and hypertensive conditions of pregnancy , were obtained by case note review . this date was derived from dates of last menstrual period ( lmp ) , where available , or by ultrasound if there was either conflict with dates as assessed by lmp ( > 6 days ) or lmp was unavailable . weight was measured at birth and , for offspring born between 33 and 42 weeks of gestation , further expressed as an sd score ( 17 ) . skinfold thickness at subscapular and triceps was measured using holtain calipers by pediatricians at each site and using a centrally agreed protocol , which was available in writing at the time of measurement . there were no significant differences in baseline demographic or biochemical measures between those with and without skinfold measurements in either control subjects or ot1 dm ( data not shown ) . on completion of the cord - sampling procedure and after delivery of the placenta , the placental membranes were trimmed , the umbilical cord shortened to within 1 cm of the chorionic plate , and any large maternal clots removed . placentae were then weighed to the nearest 1 g. the feto - placental index ( birth weight [ grams ] divided by placental weight [ grams ] ) was calculated for each delivery . specifically , the placenta was cut with a sharp knife into a series of parallel slices 12 cm thick . the slices were then diced into smaller blocks 23 cm wide and fixed by immersion in 4% formaldehyde for 24 h and subsequently embedded in paraffin wax . these were used to estimate the volume fractions and surface areas as previously described , with avoidance of section edges ( 10 ) . all estimates were made at the light microscope level by a combination of point and intersect counting and computer - assisted length measurements using the cast system ( version 2.0 ; olympus , glostrup , denmark ) . fields of view were selected in a systematic random fashion and analyzed as previously described ( 10 ) ( fig . volume densities of intermediate and terminal villi and of the villous trophoblast and capillaries were estimated by test point counting . villous and capillary surface densities were estimated by intersect counting . for each placenta , 200 events were counted across 20 systematic random fields of view per section . to convert volume and surface densities into absolute values ( in cm and cm ) , the volume of each placenta was taken as the reference volume . volumes were calculated from trimmed placental weights assuming a specific gravity of 1.05 g cm ( 18,19 ) . the intersections of the test lines with the villous surface also provided random start points for the measurement of orthogonal intercepts across the villous membrane , from the microvilli to the nearest capillary lumen . harmonic means of the intercept lengths were calculated and then converted to diffusion distances . given that there are standard values for physiological constants of oxygen in red cells , plasma , and tissues , including the villous membrane , the overall morphometric diffusing capacity for oxygen of the villous membrane of each placenta was able to be derived from the formula described by laga et al . this is based on the fick equation and provides an estimate of the maximal theoretical diffusion capacity of the placenta . intersections between the test lines acted as test points and were used for estimating the volume fraction of the villous components by the point counting technique . intersections between the test lines and the villous ( circles ) or capillary ( squares ) surfaces were counted to estimate the surface densities . intersections of the test lines with the villous surface also acted as random start points for the estimation of villous membrane thickness by measurement of orthogonal intercepts . ( a high - quality color digital representation of this figure is available in the online issue . ) changes of growth or adaptation of villi and the ivs were assessed by the isomorphy coefficient . briefly , this represents villous surface area , s , raised to the power 3/2 , and divided by the volume , v , of either the villous or intervillous compartment ( 20 ) and is designed to measure disproportionate growth of villous surface area compared with villous volume . similarly , the coefficients for capillary surface area relative to villous and capillary volume were also derived by s / v . the villous elaboration index , i = s / v , was also calculated for each case . this index gives similar information to the villous isomorphy coefficient , with a high value representing increased elaboration of the villous surface ( 8) . plasma insulin , 32 - 33 split proinsulin , proinsulin , leptin , igf - i , adiponectin , plasma total cholesterol , triglycerides , nonesterified fatty acid , vldl cholesterol , ldl cholesterol , hdl cholesterol , c - reactive protein ( crp ) , intercellular adhesion molecule ( icam)-1 , and interleukin ( il)-6 were assayed as previously described ( 13,2124 ) . in particular , igf - i was assayed by chemiluminescence immunoassay ( nichols institute diagnostics , san juan capistrano , ca ) using standards referenced to the world health organization 1st international reference reagent 1988 ( igf-1 87/518 ) . all lipid assays were carried out at the biochemistry department of glasgow royal infirmary , which is a centers for disease control and prevention ( atlanta , ga ) reference laboratory and accredited by clinical pathology accreditation u.k . data were analyzed using standard software ( stata 10 ; stata , college station , tx ) . in several cases ( insulin , leptin , triglycerides , vldl , nonesterified fatty acids , total - to - hdl cholesterol ratio , crp , icam-1 , and il-6 ) , measures were not normally distributed , and unadjusted values are presented as medians ( interquartile range ) and , for normally distributed variables , means sd . intergroup differences were assessed by unpaired t test after checking homogeneity of variance by means of the f test , anova , or , where further predictor variables were included , by general linear models . stepwise logistic regression was performed using an of p 0.1 for adding or removing predictors from the model . maternal and fetal characteristics of this cohort have been previously described and are included in table 1 . maternal type 1 diabetes was associated with marked increases in standardized birth weight and absolute values of cord insulin , proinsulin , and split 32 - 33 insulin ( 21 ) ; cortisol ; leptin ; and crp , with reductions in adiponectin , hdl cholesterol , nonesterified fatty acids , and il-6 ( table 1 ) . there was a trend toward increased placental weight in ot1 dm strengthened by adjustment for sex and gestation at delivery ( p = 0.06 ) . characteristics of mothers with type 1 diabetes and their singleton offspring versus control mothers and children data are means sd , n ( % ) , or medians ( interquartile range ) . unadjusted values are given as means sd or medians ( interquartile range ) . * p = value of significance in unpaired t , , or mann - whitney test , as appropriate . birth weights given as unadjusted p value for difference dependent on maternal diabetes status adjusted for gestational age at delivery . z weight is sd score compared with standard values for gestational age , sex , and maternal parity . analysis of placental stereological data demonstrated that intervillous space volume fraction and absolute intervillous space volume were increased in ot1 dm ( table 2 ) . adjustment for mode of delivery , sex , gestational age at birth , and smoking status strengthened this difference for both ( p = 0.028 and p = 0.008 , respectively ) . the contribution of trophoblast to villous structure was reduced in ot1 dm ( p = 0.01 ) ; however , this was not associated with an overall reduction in trophoblast volume or villous surface area ( table 2 ) . absolute values of capillary volume and surface area were similarly unaltered by the presence of maternal diabetes . placental stereology of mothers with type 1 diabetes versus control mothers * p = value of significance in unpaired t test . assessment of the complexity of the villous trees using the villous isomorphy coefficient and the elaboration index demonstrated that branching was reduced in ot1 dm ( p = 0.025 and p = 0.019 , respectively ) . with respect to capillary development , the ratio of capillary surface area relative to capillary volume was also reduced in ot1 dm ( p = 0.01 ) . the villous isomorphy coefficient and elaboration index were unrelated to gestational age , mode of delivery , sex , and smoking status . capillary development , as related to capillary surface area relative to capillary volume , however , was related to gestational age and adjustment attenuated the ot1dm - specific difference ( p = 0.24 ) . assessment of the relationship of birth weight to placental structure demonstrated a strong association with placental weight ( control subjects , r = 0.62 , p < 0.0001 ; ot1 dm , r = 0.62 , p < 0.0001 ) , with additional strong correlations of birth weight to the individual placental components , including villous volume ( control subjects , r = 0.34 , p = 0.03 ; ot1 dm , r = 0.24 , p = 0.03 ) , intervillous space volume ( control subjects , r = 0.58 , p = 0.001 ; ot1 dm , r = 0.44 , p < 0.0001 ) , and nonparenchymal volume ( control subjects , r = 0.24 , p = 0.13 ; ot1 dm , r = 0.42 , p < 0.0001 ) in control subjects and ot1 dm . all relationships were strengthened after standardizing birth weight for gestational age and sex ( p < 0.05 for controls and ot1 dm for all ) . capillary volume ( r = 0.23 , p = 0.03 ) and capillary surface area ( r = 0.21 , p = 0.05 ) were associated with standardized birth weight in ot1 dm only . for the subset with neonatal anthropometry , total skinfold thickness was associated with placental weight ( control subjects , r = 0.36 , p = 0.15 ; ot1 dm , r = 0.31 , p = 0.05 ) and capillary volume ( control subjects , r = 0.13 , p = 0.62 ; ot1 dm , r = 0.32 , p = 0.046 ) , with no relationship seen with other placental components . analysis of the associations of fetal hormonal axes to placental compartments demonstrated that insulin , in addition to known associations with birth weight ( r = 0.42 , p < 0.0001 ) and placental weight ( r = 0.44 , p < 0.0001 ) in ot1 dm , demonstrated ot1dm - specific correlations to villous volume ( r = 0.23 , p = 0.03 ) , intervillous space volume ( r = 0.25 , p = 0.02 ) , nonparenchymal volume ( r = 0.23 , p = 0.03 ) , and capillary volume ( r = 0.27 , p = 0.009 ) . cord igf - i levels were associated with birth weight in control subjects and ot1 dm ( control subjects , r = 0.32 , p = 0.04 ; ot1 dm , r = 0.48 , p < 0.0001 ) but only correlated with placental weight and substructural indexes in ot1 dm , specifically , placental weight ( r = 0.49 , p < 0.0001 ) , villous volume ( r = 0.23 , p = 0.03 ) , intervillous space volume ( r = 0.35 , p < 0.001 ) , and nonparenchymal volume ( r = 0.29 , p = 0.005 ) , with a weaker relationship to capillary volume ( r = 0.18 , p = 0.08 ) . cord leptin values demonstrated associations with birth weight ( control subjects , r = 0.48 , p = 0.002 ; ot1 dm , r = 0.40 , p < 0.0001 ) and placental weight ( control subjects , r = 0.40 , p = 0.01 ; ot1 dm , r = 0.33 , p = 0.0019 ) in control subjects and ot1 dm but were only related to intervillous space volume ( control subjects , r = 0.30 , p = 0.07 ; ot1 dm , r = 0.32 , p = 0.002 ) . assessment of maternal glycemic state in ot1 dm demonstrated that maternal a1c at 2634 weeks was associated with birth weight ( r = 0.27 , p = 0.03 ) , placental weight ( r = 0.41 , p = 0.0009 ) , intervillous volume ( r = 0.38 , p = 0.0024 ) , and nonparenchymal volume ( r = 0.25 , p = 0.05 ) . first- and third - trimester maternal a1c demonstrated similar relationships with intervillous volume ( r = 0.34 , p = 0.059 ; r = 0.31 , p = 0.03 , respectively ) but not with any of the other placental parameters including placental weight . cord adiponectin and cortisol were unrelated to birth weight or placental parameters , and no consistent associations were observed for fetal inflammatory indexes , fetal hematocrit or fetal lipids , and placental composition . isomorphy coefficients were also unrelated to birth weight , placental weight , or cord analytes in control subjects or ot1 dm . in stepwise regression models ( table 3 ) , cord igf - i was associated with intervillous volume in control subjects and ot1 dm , with further contributions of igf - i to nonparenchymal volume , trophoblast volume , capillary volume , villous surface area , and theoretical diffusion capacity in ot1 dm ( p < 0.05 for all ) . in contrast , insulin was only positively associated with capillary surface area , an association that was restricted to ot1 dm ( p = 0.017 ) . inclusion of maternal a1c at 2634 weeks attenuated this association with insulin , and igf - i became the sole associate of capillary surface area ( = 0.87 0.4 , p = 0.022 , r = 8.9% ) . notably , a1c at 2634 weeks was not related to any other placental parameters and did not alter the noted associations of igf - i demonstrated in table 3 and indeed made igf - i the sole associate of nonparenchymal volume in ot1 dm ( = 5.2 2.7 , p = 0.06 , r = 6.1% ) . for the isomorphic variables , insulin was associated with capillary surface area relative to both villous volume and capillary volume ( table 3 ) , an effect that was partially explained by maternal a1c . multivariate analysis of independent correlates of placental structure in ot1 dm and control subjects stepwise regression with log(insulin ) , log(leptin ) , adiponectin , igf - i , mode of delivery , and sex was performed with an of p 0.1 for adding or removing predictors from the model . variance ( % ) explained by the model . to assess the role of fetal hypoxia in determining placental structure , this did not attenuate the associations of igf - i with placental structure in ot1 dm . however , in control subjects , fetal hematocrit demonstrated positive associations with intervillous volume ( p = 0.023 ) , which was independent of sex ( p = 0.06 ) , and leptin ( p = 0.036 ) , with igf - i dropping out of the model . fetal hematocrit was also associated in control subjects with capillary volume ( p = 0.043 ) , which was independent of the previously observed relationship with leptin ( p = 0.013 ) ( table 3 ) . maternal and fetal characteristics of this cohort have been previously described and are included in table 1 . maternal type 1 diabetes was associated with marked increases in standardized birth weight and absolute values of cord insulin , proinsulin , and split 32 - 33 insulin ( 21 ) ; cortisol ; leptin ; and crp , with reductions in adiponectin , hdl cholesterol , nonesterified fatty acids , and il-6 ( table 1 ) . there was a trend toward increased placental weight in ot1 dm strengthened by adjustment for sex and gestation at delivery ( p = 0.06 ) . characteristics of mothers with type 1 diabetes and their singleton offspring versus control mothers and children data are means sd , n ( % ) , or medians ( interquartile range ) . unadjusted values are given as means sd or medians ( interquartile range ) . * p = value of significance in unpaired t , , or mann - whitney test , as appropriate . birth weights given as unadjusted p value for difference dependent on maternal diabetes status adjusted for gestational age at delivery . z weight is sd score compared with standard values for gestational age , sex , and maternal parity . analysis of placental stereological data demonstrated that intervillous space volume fraction and absolute intervillous space volume were increased in ot1 dm ( table 2 ) . adjustment for mode of delivery , sex , gestational age at birth , and smoking status strengthened this difference for both ( p = 0.028 and p = 0.008 , respectively ) . the contribution of trophoblast to villous structure was reduced in ot1 dm ( p = 0.01 ) ; however , this was not associated with an overall reduction in trophoblast volume or villous surface area ( table 2 ) . absolute values of capillary volume and surface area were similarly unaltered by the presence of maternal diabetes . placental stereology of mothers with type 1 diabetes versus control mothers * p = value of significance in unpaired t test . assessment of the complexity of the villous trees using the villous isomorphy coefficient and the elaboration index demonstrated that branching was reduced in ot1 dm ( p = 0.025 and p = 0.019 , respectively ) . with respect to capillary development , the ratio of capillary surface area relative to capillary volume was also reduced in ot1 dm ( p = 0.01 ) . the villous isomorphy coefficient and elaboration index were unrelated to gestational age , mode of delivery , sex , and smoking status . capillary development , as related to capillary surface area relative to capillary volume , however , was related to gestational age and adjustment attenuated the ot1dm - specific difference ( p = 0.24 ) . assessment of the relationship of birth weight to placental structure demonstrated a strong association with placental weight ( control subjects , r = 0.62 , p < 0.0001 ; ot1 dm , r = 0.62 , p < 0.0001 ) , with additional strong correlations of birth weight to the individual placental components , including villous volume ( control subjects , r = 0.34 , p = 0.03 ; ot1 dm , r = 0.24 , p = 0.03 ) , intervillous space volume ( control subjects , r = 0.58 , p = 0.001 ; ot1 dm , r = 0.44 , p < 0.0001 ) , and nonparenchymal volume ( control subjects , r = 0.24 , p = 0.13 ; ot1 dm , r = 0.42 , p < 0.0001 ) in control subjects and ot1 dm . all relationships were strengthened after standardizing birth weight for gestational age and sex ( p < 0.05 for controls and ot1 dm for all ) . capillary volume ( r = 0.23 , p = 0.03 ) and capillary surface area ( r = 0.21 , p = 0.05 ) were associated with standardized birth weight in ot1 dm only . for the subset with neonatal anthropometry , total skinfold thickness was associated with placental weight ( control subjects , r = 0.36 , p = 0.15 ; ot1 dm , r = 0.31 , p = 0.05 ) and capillary volume ( control subjects , r = 0.13 , p = 0.62 ; ot1 dm , r = 0.32 , p = 0.046 ) , with no relationship seen with other placental components . analysis of the associations of fetal hormonal axes to placental compartments demonstrated that insulin , in addition to known associations with birth weight ( r = 0.42 , p < 0.0001 ) and placental weight ( r = 0.44 , p < 0.0001 ) in ot1 dm , demonstrated ot1dm - specific correlations to villous volume ( r = 0.23 , p = 0.03 ) , intervillous space volume ( r = 0.25 , p = 0.02 ) , nonparenchymal volume ( r = 0.23 , p = 0.03 ) , and capillary volume ( r = 0.27 , p = 0.009 ) . cord igf - i levels were associated with birth weight in control subjects and ot1 dm ( control subjects , r = 0.32 , p = 0.04 ; ot1 dm , r = 0.48 , p < 0.0001 ) but only correlated with placental weight and substructural indexes in ot1 dm , specifically , placental weight ( r = 0.49 , p < 0.0001 ) , villous volume ( r = 0.23 , p = 0.03 ) , intervillous space volume ( r = 0.35 , p < 0.001 ) , and nonparenchymal volume ( r = 0.29 , p = 0.005 ) , with a weaker relationship to capillary volume ( r = 0.18 , p = 0.08 ) . cord leptin values demonstrated associations with birth weight ( control subjects , r = 0.48 , p = 0.002 ; ot1 dm , r = 0.40 , p < 0.0001 ) and placental weight ( control subjects , r = 0.40 , p = 0.01 ; ot1 dm , r = 0.33 , p = 0.0019 ) in control subjects and ot1 dm but were only related to intervillous space volume ( control subjects , r = 0.30 , p = 0.07 ; ot1 dm , r = 0.32 , p = 0.002 ) . assessment of maternal glycemic state in ot1 dm demonstrated that maternal a1c at 2634 weeks was associated with birth weight ( r = 0.27 , p = 0.03 ) , placental weight ( r = 0.41 , p = 0.0009 ) , intervillous volume ( r = 0.38 , p = 0.0024 ) , and nonparenchymal volume ( r = 0.25 , p = 0.05 ) . first- and third - trimester maternal a1c demonstrated similar relationships with intervillous volume ( r = 0.34 , p = 0.059 ; r = 0.31 , p = 0.03 , respectively ) but not with any of the other placental parameters including placental weight . cord adiponectin and cortisol were unrelated to birth weight or placental parameters , and no consistent associations were observed for fetal inflammatory indexes , fetal hematocrit or fetal lipids , and placental composition . isomorphy coefficients were also unrelated to birth weight , placental weight , or cord analytes in control subjects or ot1 dm . in stepwise regression models ( table 3 ) , cord igf - i was associated with intervillous volume in control subjects and ot1 dm , with further contributions of igf - i to nonparenchymal volume , trophoblast volume , capillary volume , villous surface area , and theoretical diffusion capacity in ot1 dm ( p < 0.05 for all ) . in contrast , insulin was only positively associated with capillary surface area , an association that was restricted to ot1 dm ( p = 0.017 ) . inclusion of maternal a1c at 2634 weeks attenuated this association with insulin , and igf - i became the sole associate of capillary surface area ( = 0.87 0.4 , p = 0.022 , r = 8.9% ) . notably , a1c at 2634 weeks was not related to any other placental parameters and did not alter the noted associations of igf - i demonstrated in table 3 and indeed made igf - i the sole associate of nonparenchymal volume in ot1 dm ( = 5.2 2.7 , p = 0.06 , r = 6.1% ) . for the isomorphic variables , insulin was associated with capillary surface area relative to both villous volume and capillary volume ( table 3 ) , an effect that was partially explained by maternal a1c . multivariate analysis of independent correlates of placental structure in ot1 dm and control subjects stepwise regression with log(insulin ) , log(leptin ) , adiponectin , igf - i , mode of delivery , and sex was performed with an of p 0.1 for adding or removing predictors from the model . variance ( % ) explained by the model . to assess the role of fetal hypoxia in determining placental structure , this did not attenuate the associations of igf - i with placental structure in ot1 dm . however , in control subjects , fetal hematocrit demonstrated positive associations with intervillous volume ( p = 0.023 ) , which was independent of sex ( p = 0.06 ) , and leptin ( p = 0.036 ) , with igf - i dropping out of the model . fetal hematocrit was also associated in control subjects with capillary volume ( p = 0.043 ) , which was independent of the previously observed relationship with leptin ( p = 0.013 ) ( table 3 ) . to our knowledge , this is the largest series examining placental stereology in a contemporary cohort of women with type 1 diabetes . it gives an important opportunity both to examine the influence of maternal diabetes on placental structure and to assess whether hormones in the fetal circulation ( particularly insulin and leptin , which are markedly raised in this cohort ) correlate with placental substructure . we find that , overall , there are relatively few systematic differences between placentas from mothers with diabetes and control subjects . in particular , the key measures that might be expected to influence substrate diffusion across the placenta ( villous surface area , capillary surface area , villous membrane thickness ) are not altered . in keeping with this , the placental morphometric diffusing capacity was also not different between diabetic and control pregnancies . historically , a range of abnormalities have been described in the placenta in type 1 diabetes , including increased placental size in conjunction with fetal macrosomia . in the oldest series , gross pathology including an increased rate of placental infarcts was described , as well as villous immaturity . in the series from the 1990s , microscopic morphology was often described as normal , particularly where maternal diabetes was well controlled ( 25 ) . increases in capillary volume and surface area ( 6,7 ) , villous surface area ( 8) , increased total diffusive conductance ( 9 ) , and intervillous and trophoblast volume ( 7,10 ) were described and their presence or absence often ascribed to the degree of maternal diabetes control . in our series , we did not observe these changes with the exception of an increase in intervillous space volume , which classically indicates a deficiency of terminal villi and persisting villous immaturity . consistent with this and previous reports ( 20 ) , we observe a reduction in the villous coefficient and elaboration index , measures of the complexity of the villous tree that would impinge upon the intervillous space , suggesting that the observed villous growth is anisomorphic . importantly , however , despite this reduction in villous development , there is no overall impact on villous volume or surface area , primarily due to an increased total placental volume in ot1 dm . one might speculate that the reduction in villous development and increased placental growth are compensatory changes , but it is not possible to determine the primary direction of this relationship . appropriately , we demonstrate that individual placental components , including villous volume , all correlate with birth weight . despite these limited changes , the inconsistent nature of the studies to date , combined with the major findings of our series , supports the view that there are minimal changes in placental structure inherent in contemporary diabetic pregnancy . this is of interest , as the overall hormonal environment on the fetal side of the circulation is markedly abnormal , with median fetal insulin and leptin levels four and three times higher , respectively , than control subjects . it is therefore striking that these significant increases in insulin and leptin do not appear to be driving any consistent change in placental structure . only igf - i displayed a consistent relationship with the volumes of the intervillous space , villous trophoblast , and placental capillaries . insulin has classically been proposed as a mediator of enhanced placental development in type 1 diabetes and was thought to underlie the reported increases in proliferation rates of trophoblast , villous stromal cells , and villous capillaries . however , insulin is not usually transported across the villous membrane , and its effects may be different on the fetal and maternal side of the placenta . in the first trimester , insulin receptors are localized to the intervillous surface of the villous trophoblast , with expression predominantly on the surfaces of sprouting segments of the villous tree ( 26 ) . in contrast , by the third trimester , the highest immunoreactivity for the insulin receptor is found in the fetal villous endothelium , in particular , in segments with capillary sprouting ( 27 ) . further analysis of the transcriptional profile and phosphorylation status of first- and third - trimester isolated trophoblast and endothelial cells in response to hyperinsulinemia suggests that there is a spatio - temporal shift in insulin response ( 28 ) , leading to the hypothesis that there is a predominant effect of maternal insulin in early pregnancy and fetal insulin in later pregnancy ( 27 ) . fetal insulin levels would be predicted to contribute to control of villous differentiation in accordance with fetal growth and nutritional needs in later pregnancy . in the current study , we demonstrate that fetal insulin is an independent associate of capillary surface area and of capillary surface area relative to villous and capillary volume . our observed relationship with capillary development would be consistent with insulin receptor localization to endothelial cells and the ability of fetal insulin to enhance capillary development , including longitudinal growth within a fixed villous volume ( 6 ) . it is notable , however , that there appears to be little impact of fetal insulin on trophoblast volumes independent of the effects of igf - i . in this analysis , it is possible that fetal hyperinsulinemia earlier in pregnancy is acting ; however , if hyperinsulinemia is established earlier in pregnancy , it is likely that cord insulin will also be increased . this is apparent in the strong relationship of cord insulin and igf-1 to measures such as birth weight ( 13 ) and our biologically plausible association of fetal insulin with placental capillary volumes . the relationship of circulating fetal igf - i to many of the placental components is striking . cord igf - i is strongly associated with birth weight and placental weight ( 12 ) , and igf - i deletion or reduced receptor expression in humans are both associated with a reduction in birth weight and placental weight ( 29,30 ) . conversely , prolonged administration of exogenous igf - i to growth - restricted fetuses substantially increases body and placental weight ( 31 ) . in short - term studies , exogenous fetal igf - i increases placental amino acid transfer and uptake and decreases proteolysis , facilitating organ - specific and placental growth ( 32 ) . these relationships are in contrast to those observed in mice , suggesting species specificity , where deletion of igf - i or its receptor igf - ir is associated with a significant reduction in fetal but not placental weight ( 33 ) . therefore , although igf - i may directly be enhancing placental growth , via receptors expressed in trophoblast and endothelium ( 3436 ) , alternative indirect mediators like adiponectin , which have been implicated in the matching of fetal and placental weight , may contribute ( 11,14 ) . however , cord levels of igf - i and adiponectin are not correlated , and adiponectin was not an independent associate in the multivariate models . fetal lipids , in particular hdl , is associated with igf-1 in fetal and adult life ( 37,38 ) . however , we did not demonstrate any relationship of fetal lipids with placental parameters ( data not shown ) despite recent recognition that placental endothelial cells efficiently transport cholesterol ( 39 ) . similarly , although inflammatory signals , including il-6 , can increase igf - i expression ( 40 ) , we did not observe an independent effect of inflammatory mediators on placental parameters ( data not shown ) . lastly , inclusion of fetal hematocrit , an index of fetal hypoxia , did not alter these relationships but did demonstrate the expected positive association with capillary volume in multivariate models . overall , our results would support the hypothesis that , in humans , igf - i has a direct effect on placental development . leptin has also been proposed as a regulator of placental growth . in human trophoblast cells in vitro , exogenous leptin treatment has mitogenic and antiapoptotic effects , while inhibition of endogenous placental leptin expression reduces cell proliferation and increases apoptotic cell number and caspase-3 activity ( 41 ) . furthermore , leptin stimulates activity of the amino acid transporter system a in human placental villous fragments at term ( 42,43 ) . lastly , leptin can induce angiogenesis in primary cultures of endothelial cells ( 44 ) . despite these effects , leptin infusion does not induce significant placental growth in sheep ( 45 ) , and in the current study , leptin did not show consistent relationships with placental components in the control subjects . this limited effect may reflect differential leptin receptor expression , as although leptin receptors have been localized to umbilical endothelial cells and trophoblast ( 46,47 ) , they have not been demonstrated on placental endothelium , which may explain our observed lack of association of fetal leptin with stereological parameters . although leptin exerted a small influence on intervillous space volume independent of igf - i in the ot1 dm placentas , leptin is markedly raised in ot1 dm , suggesting that the ability of leptin to induce placental angiogenesis and growth may be limited . it is notable , however , that maternal diabetes and indeed maternal a1c are not related to most of the placental stereological parameters with the exception of intervillous space volume . this would suggest that excess maternal nutrient supply may not be primarily responsible for changes in placental development , and , where present , such changes and increases in placental weight in general follow increased fetal weight . a notable feature of this study is the close matching of fetal growth with placental size , including placental components . it would appear that the fetus is capable of modulating placental transport in response to metabolic demands and nutrient supply . the signals that facilitate this feedback loop are largely unknown ; however , this study suggests that igf - i , and to a lesser extent insulin and leptin , all contribute and facilitate varying degrees of morphological alternation . in conclusion , we demonstrate that maternal type 1 diabetes is associated with minimal changes in placental structure and that the changes that do occur relate principally to villous maturity . second , we identify that fetal igf-1 is the principal correlate of placental substructure and that the effect of insulin is limited to capillary development . lastly , we identify that fetal leptin has a limited positive effect on placental angiogenesis and , in conjunction with igf - i and insulin , contributes to the metabolic feedback from the fetus to the placenta regarding its metabolic demands .

objectivealteration of placental structure may influence fetal overgrowth and complications of maternal diabetes . we examined the placenta in a cohort of offspring of mothers with type 1 diabetes ( ot1 dm ) to assess structural changes and determine whether these were related to maternal a1c , fetal hematocrit , fetal hormonal , or metabolic axes.research design and methodsplacental samples were analyzed using stereological techniques to quantify volumes and surface areas of key placental components in 88 ot1 dm and 39 control subjects , and results related to maternal a1c and umbilical cord analytes ( insulin , leptin , adiponectin , igf - i , hematocrit , lipids , c - reactive protein , and interleukin-6).resultsintervillous space volume was increased in ot1 dm ( ot1 dm 250 81 cm3 vs. control 217 65 cm3 ; p = 0.02 ) with anisomorphic growth of villi ( p = 0.025 ) . the placentas showed a trend to increased weight ( ot1 dm 690 19 g ; control 641 22 g ; p = 0.08 ) , but villous , nonparenchymal , trophoblast , and capillary volumes did not differ . villous surface area , capillary surface area , membrane thickness , and calculated morphometric diffusing capacity were also similar in type 1 diabetic and control subjects . a1c at 2634 weeks associated with birth weight ( r = 0.27 , p = 0.03 ) , placental weight ( r = 0.41 , p = 0.0009 ) , and intervillous space volume ( r = 0.38 , p = 0.0024 ) . in multivariate analysis of cord parameters in ot1 dm , fetal igf - i emerged as a significant correlate of most components ( intervillous space , villous , trophoblast , and capillary volumes , all p < 0.01 ) . by contrast , fetal insulin was only independently associated with capillary surface area ( positive , r2 = 6.7% ; p = 0.02).conclusionsthere are minimal placental structural differences between ot1 dm and control subjects . fetal igf - i but not fetal insulin emerges as a key correlate of placental substructural volumes , thereby facilitating feedback to the placenta regarding fetal metabolic demand .

RESEARCH DESIGN AND METHODS Recruitment and collection of cord blood. Stereological analysis. Cord blood assays. Statistical analysis. RESULTS Fetal cord analytes and placental structure in OT1DM versus control subjects. Placental composition, relationship with birth weight, and adiposity. Placental composition and relationship with fetal hormones. DISCUSSION

plasma insulin , 32 - 33 split proinsulin , proinsulin , leptin , igf - i , adiponectin , plasma total cholesterol , triglycerides , nonesterified fatty acid , vldl cholesterol , ldl cholesterol , hdl cholesterol , c - reactive protein ( crp ) , intercellular adhesion molecule ( icam)-1 , and interleukin ( il)-6 were assayed as previously described ( 13,2124 ) . plasma insulin , 32 - 33 split proinsulin , proinsulin , leptin , igf - i , adiponectin , plasma total cholesterol , triglycerides , nonesterified fatty acid , vldl cholesterol , ldl cholesterol , hdl cholesterol , c - reactive protein ( crp ) , intercellular adhesion molecule ( icam)-1 , and interleukin ( il)-6 were assayed as previously described ( 13,2124 ) . maternal type 1 diabetes was associated with marked increases in standardized birth weight and absolute values of cord insulin , proinsulin , and split 32 - 33 insulin ( 21 ) ; cortisol ; leptin ; and crp , with reductions in adiponectin , hdl cholesterol , nonesterified fatty acids , and il-6 ( table 1 ) . there was a trend toward increased placental weight in ot1 dm strengthened by adjustment for sex and gestation at delivery ( p = 0.06 ) . characteristics of mothers with type 1 diabetes and their singleton offspring versus control mothers and children data are means sd , n ( % ) , or medians ( interquartile range ) . analysis of placental stereological data demonstrated that intervillous space volume fraction and absolute intervillous space volume were increased in ot1 dm ( table 2 ) . the contribution of trophoblast to villous structure was reduced in ot1 dm ( p = 0.01 ) ; however , this was not associated with an overall reduction in trophoblast volume or villous surface area ( table 2 ) . assessment of the complexity of the villous trees using the villous isomorphy coefficient and the elaboration index demonstrated that branching was reduced in ot1 dm ( p = 0.025 and p = 0.019 , respectively ) . with respect to capillary development , the ratio of capillary surface area relative to capillary volume was also reduced in ot1 dm ( p = 0.01 ) . assessment of the relationship of birth weight to placental structure demonstrated a strong association with placental weight ( control subjects , r = 0.62 , p < 0.0001 ; ot1 dm , r = 0.62 , p < 0.0001 ) , with additional strong correlations of birth weight to the individual placental components , including villous volume ( control subjects , r = 0.34 , p = 0.03 ; ot1 dm , r = 0.24 , p = 0.03 ) , intervillous space volume ( control subjects , r = 0.58 , p = 0.001 ; ot1 dm , r = 0.44 , p < 0.0001 ) , and nonparenchymal volume ( control subjects , r = 0.24 , p = 0.13 ; ot1 dm , r = 0.42 , p < 0.0001 ) in control subjects and ot1 dm . capillary volume ( r = 0.23 , p = 0.03 ) and capillary surface area ( r = 0.21 , p = 0.05 ) were associated with standardized birth weight in ot1 dm only . for the subset with neonatal anthropometry , total skinfold thickness was associated with placental weight ( control subjects , r = 0.36 , p = 0.15 ; ot1 dm , r = 0.31 , p = 0.05 ) and capillary volume ( control subjects , r = 0.13 , p = 0.62 ; ot1 dm , r = 0.32 , p = 0.046 ) , with no relationship seen with other placental components . analysis of the associations of fetal hormonal axes to placental compartments demonstrated that insulin , in addition to known associations with birth weight ( r = 0.42 , p < 0.0001 ) and placental weight ( r = 0.44 , p < 0.0001 ) in ot1 dm , demonstrated ot1dm - specific correlations to villous volume ( r = 0.23 , p = 0.03 ) , intervillous space volume ( r = 0.25 , p = 0.02 ) , nonparenchymal volume ( r = 0.23 , p = 0.03 ) , and capillary volume ( r = 0.27 , p = 0.009 ) . cord igf - i levels were associated with birth weight in control subjects and ot1 dm ( control subjects , r = 0.32 , p = 0.04 ; ot1 dm , r = 0.48 , p < 0.0001 ) but only correlated with placental weight and substructural indexes in ot1 dm , specifically , placental weight ( r = 0.49 , p < 0.0001 ) , villous volume ( r = 0.23 , p = 0.03 ) , intervillous space volume ( r = 0.35 , p < 0.001 ) , and nonparenchymal volume ( r = 0.29 , p = 0.005 ) , with a weaker relationship to capillary volume ( r = 0.18 , p = 0.08 ) . cord leptin values demonstrated associations with birth weight ( control subjects , r = 0.48 , p = 0.002 ; ot1 dm , r = 0.40 , p < 0.0001 ) and placental weight ( control subjects , r = 0.40 , p = 0.01 ; ot1 dm , r = 0.33 , p = 0.0019 ) in control subjects and ot1 dm but were only related to intervillous space volume ( control subjects , r = 0.30 , p = 0.07 ; ot1 dm , r = 0.32 , p = 0.002 ) . assessment of maternal glycemic state in ot1 dm demonstrated that maternal a1c at 2634 weeks was associated with birth weight ( r = 0.27 , p = 0.03 ) , placental weight ( r = 0.41 , p = 0.0009 ) , intervillous volume ( r = 0.38 , p = 0.0024 ) , and nonparenchymal volume ( r = 0.25 , p = 0.05 ) . first- and third - trimester maternal a1c demonstrated similar relationships with intervillous volume ( r = 0.34 , p = 0.059 ; r = 0.31 , p = 0.03 , respectively ) but not with any of the other placental parameters including placental weight . isomorphy coefficients were also unrelated to birth weight , placental weight , or cord analytes in control subjects or ot1 dm . in stepwise regression models ( table 3 ) , cord igf - i was associated with intervillous volume in control subjects and ot1 dm , with further contributions of igf - i to nonparenchymal volume , trophoblast volume , capillary volume , villous surface area , and theoretical diffusion capacity in ot1 dm ( p < 0.05 for all ) . in contrast , insulin was only positively associated with capillary surface area , an association that was restricted to ot1 dm ( p = 0.017 ) . inclusion of maternal a1c at 2634 weeks attenuated this association with insulin , and igf - i became the sole associate of capillary surface area ( = 0.87 0.4 , p = 0.022 , r = 8.9% ) . notably , a1c at 2634 weeks was not related to any other placental parameters and did not alter the noted associations of igf - i demonstrated in table 3 and indeed made igf - i the sole associate of nonparenchymal volume in ot1 dm ( = 5.2 2.7 , p = 0.06 , r = 6.1% ) . for the isomorphic variables , insulin was associated with capillary surface area relative to both villous volume and capillary volume ( table 3 ) , an effect that was partially explained by maternal a1c . multivariate analysis of independent correlates of placental structure in ot1 dm and control subjects stepwise regression with log(insulin ) , log(leptin ) , adiponectin , igf - i , mode of delivery , and sex was performed with an of p 0.1 for adding or removing predictors from the model . to assess the role of fetal hypoxia in determining placental structure , this did not attenuate the associations of igf - i with placental structure in ot1 dm . however , in control subjects , fetal hematocrit demonstrated positive associations with intervillous volume ( p = 0.023 ) , which was independent of sex ( p = 0.06 ) , and leptin ( p = 0.036 ) , with igf - i dropping out of the model . fetal hematocrit was also associated in control subjects with capillary volume ( p = 0.043 ) , which was independent of the previously observed relationship with leptin ( p = 0.013 ) ( table 3 ) . maternal type 1 diabetes was associated with marked increases in standardized birth weight and absolute values of cord insulin , proinsulin , and split 32 - 33 insulin ( 21 ) ; cortisol ; leptin ; and crp , with reductions in adiponectin , hdl cholesterol , nonesterified fatty acids , and il-6 ( table 1 ) . there was a trend toward increased placental weight in ot1 dm strengthened by adjustment for sex and gestation at delivery ( p = 0.06 ) . characteristics of mothers with type 1 diabetes and their singleton offspring versus control mothers and children data are means sd , n ( % ) , or medians ( interquartile range ) . analysis of placental stereological data demonstrated that intervillous space volume fraction and absolute intervillous space volume were increased in ot1 dm ( table 2 ) . the contribution of trophoblast to villous structure was reduced in ot1 dm ( p = 0.01 ) ; however , this was not associated with an overall reduction in trophoblast volume or villous surface area ( table 2 ) . assessment of the complexity of the villous trees using the villous isomorphy coefficient and the elaboration index demonstrated that branching was reduced in ot1 dm ( p = 0.025 and p = 0.019 , respectively ) . with respect to capillary development , the ratio of capillary surface area relative to capillary volume was also reduced in ot1 dm ( p = 0.01 ) . assessment of the relationship of birth weight to placental structure demonstrated a strong association with placental weight ( control subjects , r = 0.62 , p < 0.0001 ; ot1 dm , r = 0.62 , p < 0.0001 ) , with additional strong correlations of birth weight to the individual placental components , including villous volume ( control subjects , r = 0.34 , p = 0.03 ; ot1 dm , r = 0.24 , p = 0.03 ) , intervillous space volume ( control subjects , r = 0.58 , p = 0.001 ; ot1 dm , r = 0.44 , p < 0.0001 ) , and nonparenchymal volume ( control subjects , r = 0.24 , p = 0.13 ; ot1 dm , r = 0.42 , p < 0.0001 ) in control subjects and ot1 dm . capillary volume ( r = 0.23 , p = 0.03 ) and capillary surface area ( r = 0.21 , p = 0.05 ) were associated with standardized birth weight in ot1 dm only . for the subset with neonatal anthropometry , total skinfold thickness was associated with placental weight ( control subjects , r = 0.36 , p = 0.15 ; ot1 dm , r = 0.31 , p = 0.05 ) and capillary volume ( control subjects , r = 0.13 , p = 0.62 ; ot1 dm , r = 0.32 , p = 0.046 ) , with no relationship seen with other placental components . analysis of the associations of fetal hormonal axes to placental compartments demonstrated that insulin , in addition to known associations with birth weight ( r = 0.42 , p < 0.0001 ) and placental weight ( r = 0.44 , p < 0.0001 ) in ot1 dm , demonstrated ot1dm - specific correlations to villous volume ( r = 0.23 , p = 0.03 ) , intervillous space volume ( r = 0.25 , p = 0.02 ) , nonparenchymal volume ( r = 0.23 , p = 0.03 ) , and capillary volume ( r = 0.27 , p = 0.009 ) . cord igf - i levels were associated with birth weight in control subjects and ot1 dm ( control subjects , r = 0.32 , p = 0.04 ; ot1 dm , r = 0.48 , p < 0.0001 ) but only correlated with placental weight and substructural indexes in ot1 dm , specifically , placental weight ( r = 0.49 , p < 0.0001 ) , villous volume ( r = 0.23 , p = 0.03 ) , intervillous space volume ( r = 0.35 , p < 0.001 ) , and nonparenchymal volume ( r = 0.29 , p = 0.005 ) , with a weaker relationship to capillary volume ( r = 0.18 , p = 0.08 ) . cord leptin values demonstrated associations with birth weight ( control subjects , r = 0.48 , p = 0.002 ; ot1 dm , r = 0.40 , p < 0.0001 ) and placental weight ( control subjects , r = 0.40 , p = 0.01 ; ot1 dm , r = 0.33 , p = 0.0019 ) in control subjects and ot1 dm but were only related to intervillous space volume ( control subjects , r = 0.30 , p = 0.07 ; ot1 dm , r = 0.32 , p = 0.002 ) . assessment of maternal glycemic state in ot1 dm demonstrated that maternal a1c at 2634 weeks was associated with birth weight ( r = 0.27 , p = 0.03 ) , placental weight ( r = 0.41 , p = 0.0009 ) , intervillous volume ( r = 0.38 , p = 0.0024 ) , and nonparenchymal volume ( r = 0.25 , p = 0.05 ) . first- and third - trimester maternal a1c demonstrated similar relationships with intervillous volume ( r = 0.34 , p = 0.059 ; r = 0.31 , p = 0.03 , respectively ) but not with any of the other placental parameters including placental weight . isomorphy coefficients were also unrelated to birth weight , placental weight , or cord analytes in control subjects or ot1 dm . in stepwise regression models ( table 3 ) , cord igf - i was associated with intervillous volume in control subjects and ot1 dm , with further contributions of igf - i to nonparenchymal volume , trophoblast volume , capillary volume , villous surface area , and theoretical diffusion capacity in ot1 dm ( p < 0.05 for all ) . in contrast , insulin was only positively associated with capillary surface area , an association that was restricted to ot1 dm ( p = 0.017 ) . inclusion of maternal a1c at 2634 weeks attenuated this association with insulin , and igf - i became the sole associate of capillary surface area ( = 0.87 0.4 , p = 0.022 , r = 8.9% ) . notably , a1c at 2634 weeks was not related to any other placental parameters and did not alter the noted associations of igf - i demonstrated in table 3 and indeed made igf - i the sole associate of nonparenchymal volume in ot1 dm ( = 5.2 2.7 , p = 0.06 , r = 6.1% ) . for the isomorphic variables , insulin was associated with capillary surface area relative to both villous volume and capillary volume ( table 3 ) , an effect that was partially explained by maternal a1c . multivariate analysis of independent correlates of placental structure in ot1 dm and control subjects stepwise regression with log(insulin ) , log(leptin ) , adiponectin , igf - i , mode of delivery , and sex was performed with an of p 0.1 for adding or removing predictors from the model . to assess the role of fetal hypoxia in determining placental structure , this did not attenuate the associations of igf - i with placental structure in ot1 dm . however , in control subjects , fetal hematocrit demonstrated positive associations with intervillous volume ( p = 0.023 ) , which was independent of sex ( p = 0.06 ) , and leptin ( p = 0.036 ) , with igf - i dropping out of the model . fetal hematocrit was also associated in control subjects with capillary volume ( p = 0.043 ) , which was independent of the previously observed relationship with leptin ( p = 0.013 ) ( table 3 ) . in particular , the key measures that might be expected to influence substrate diffusion across the placenta ( villous surface area , capillary surface area , villous membrane thickness ) are not altered . increases in capillary volume and surface area ( 6,7 ) , villous surface area ( 8) , increased total diffusive conductance ( 9 ) , and intervillous and trophoblast volume ( 7,10 ) were described and their presence or absence often ascribed to the degree of maternal diabetes control . only igf - i displayed a consistent relationship with the volumes of the intervillous space , villous trophoblast , and placental capillaries . cord igf - i is strongly associated with birth weight and placental weight ( 12 ) , and igf - i deletion or reduced receptor expression in humans are both associated with a reduction in birth weight and placental weight ( 29,30 ) . these relationships are in contrast to those observed in mice , suggesting species specificity , where deletion of igf - i or its receptor igf - ir is associated with a significant reduction in fetal but not placental weight ( 33 ) . despite these effects , leptin infusion does not induce significant placental growth in sheep ( 45 ) , and in the current study , leptin did not show consistent relationships with placental components in the control subjects . although leptin exerted a small influence on intervillous space volume independent of igf - i in the ot1 dm placentas , leptin is markedly raised in ot1 dm , suggesting that the ability of leptin to induce placental angiogenesis and growth may be limited .

heart failure is a chronic , progressive disease affecting approximately 12% of all the adult population , increasing to more than 10% in the population segment over 70 years ; its incidence rose with the extension of life - span and the aging of the population [ 13 ] . its prognosis is poor , the mortality is higher than that of some cancers and the morbidity is marked by a low quality of life and frequent rehospitalizations . death occurs in months to years from the onset , but in an unpredictable manner [ 410 ] . its evolution is the result of the overexpression of a number of biologically active molecules with negative influence on the cardiovascular system , with chronic activation of signaling pathways secondary to inadequate tissue perfusion . the compensatory mechanisms described until now are the activation of the adrenergic and renin - angiotensin systems ( raas ) with the purpose of maintaining the cardiac output but with long - term deleterious effects . balancing these systems , there are other molecular pathways with key roles in the physiology of the cardiovascular system and which are now the principal subject of research in this field . the gene of apelin is located on the long arm of x chromosome ; the apelin is syntethized as preproapelin ( 77 aminoacids ) which is then cleaved by an angiotensin - converting enzyme to shorter active fragments ( c - terminal peptides : apelin-13 , -16 , -17 , -19 , -36 ) . it was discovered in 1998 from bovine stomach extracts and named apelin : apj endogenous ligand . the most active of all is apelin-13 , the shorter peptide , with its pyroglutamated form representing the principally biological active ligand ; its activity is 8 and 60 folds higher than apelin-17 and apelin-36 , respectively . apelin s receptor , apj , identified in 1993 by homologous cloning in the human genome project , was initially considered an orphan until the discovery of apelin in 1998 . apj has 31% sequence homology with angiotensin ii type 1 receptor ( at1r ) and shares its tissue distribution , but it can not bind angiotensin ii ( ang ii ) . it is coupled with gi protein , and probably gq protein too , and it is desensitized differently by different fragments of apelin . the shorter fragments activate the receptor for a short period of time because it is rapidly internalized in the cellular membrane , returning to the surface within an hour and becoming available for activation again . in contrast , the longer fragments of apelin dissociate slowly from the receptor , blocking it in an active state for a longer period of time and lowering the recycling rate . at cellular level , the apelin activates the proteinkinase b ( akt ) , phosphatidil - inositol-3 kinase ( pi3k ) and the extracellular signal - regulated kinases ( erk ) pathway , which are involved in controlling apoptosis and cellular migration and proliferation . the apelin and its receptor can be found in a variety of tissues ( central nervous system , enterochromaffin - like gastric cells , pancreatic islet cells , osteoblasts , t - lymphocytes , adipose tissue ) , with higher concentrations in the lungs , cardiovascular system and spleen . it is synthesized locally in the endothelium and it is found in the cellular organelles as endoplasmic reticulum , golgi apparatus and secretory vesicles . later studies showed that the most important site of apelin - apj expression and action is the cardiovascular system , especially the vascular endothelium , vascular smooth muscle cells , endocardial endothelium , and in a lesser extend , the myocardium . this explains the higher concentration in the lungs and spleen , paralleling the level of vascularization [ 2025 ] . in the atrial tissue the apelin concentration is 200 folds higher than in the ventricular tissue and it appears to be correlated to the plasma level . although we do not know the exact source of the plasma apelin , it may be generated from the atrium . in some other tissues as the kidney and adrenal gland it is expressed only in the blood vessels , although its effects on these tissues are complex and cover multiple sites . apelin is a diuretic , but its effects on the kidneys are not limited only to the renal tissue . apelin is synthesized in the brain and inhibits the secretion of vasopresin from the neurons , favoring water excretion ; also it acts on the renal microcirculation and probably on the tubular function to validate its diuretic effect [ 2830 ] . the apelin / apj system counter - regulates the raas by antagonizing the activity of ang ii , its major effector . describe this mechanism as a result of direct , physical interaction between the apj and atr1 receptors , dimerization and sending atr1 into a low affinity state , thus decreasing its interaction with ang ii . apelin induces this physical interaction and increases the density of apj as compared to atr1 ( by increasing its expression and its availability at the membrane level ) , but the density of both receptors were not modified by the exogenous ang ii in this study . this may be the explanation for the development of cardiovascular diseases in the presence of dysfunctional apelin / apj system that can not balance the overactivated , injurious raas . also , recent studies demonstrated a relationship between apelin / apj system and the angiotensin converting enzyme type 2 ( ace2 ) which has a key role in counterbalancing the raas by catalyzing the transformation of ang ii into angiotensin 17 ( ang 17 ) , a molecule with opposing effects to ang ii . these findings stand for the theory than apelin / apj is a beneficial molecular pathway in the physiology of cardiovascular system . the apelin / apj system has many physiologic effects on water balance , glycemic control , nutritional behavior , immunity , but its principal target is the cardiovascular system . latest studies have showed that apelin / apj system has a key role in its normal function and in the development of vascular and heart diseases such as atherosclerosis , coronary heart disease , heart failure , systemic and pulmonary arterial hypertension and ischemic - reperfusion lesion . its action seems to be mainly autocrine and paracrine , the ligand expression paralelling the receptor expression , but its plasma concentration also corresponds to a circulating hormone . plasma concentration is about 10 g / ml with a half time of less than 5 minutes ; its slow , sustained , inotropic effect is validated in a subnanomolar concentration ( ec50=33 pmol / l ) , being the most potent endogenous inotropic molecule , overcoming the adrenomedulin and endothelin . in the experimental studies conducted so far the effect reached its maximum in 2030 min - completely different from mimetics which act within seconds - and last longer ; it was valid even when the no production was blocked , and the endothelin , the ang ii and receptors were inhibited . the undelying mechanism for apelin s inotropy is the activation of phospholipase c , proteinkinase c , na - h sarcolemmal exchange ( nhe ) and na - ca exchange ( ncx ) pathways , without involving the l - type ca - channels and voltage - activated k - channels and without inducing myocardial hypertrophy . describe that even after inhibition of both nhe and ncx , the inotropic effect of apelin continues in a proportion of about 40% ; this fact suggests the presence of additional , unclear , mechanism for inotropism . in the study of dai et al . results showed that the inotropic effect of apelin is due mostly to an increase in ca availability in the myocardial cell and not to a sensitization of myofilaments ; the mechanism is not yet completely understood . apelin - apj receptor interaction at the vascular level induces vasodilation through no release from endothelial cells ( in arteries and veins ) with consequent decrease in systemic vascular resistance , lowering the pre- and the afterload of the left ventricle and improving its filling pattern . the apj receptor is present in the vascular smooth muscle and it stimulates its contraction as was demonstrated in experimental condition on endothelial denuded vein . the vasoconstricion induced by apelin on denuded vessels and the vasodilation produced in the presence of normal functioning endothelium suggest the complexity of apelin s role on the circulatory system , the fine tuning of the vascular tone and the easily disturbed apelin s effects in the presence of injured endothelium . the studies conducted so far showed the importance of apelin / apj pathway in the development of heart failure and the fact that its downregulation in the myocardial cell contributes to the structural and functional alteration of the heart . in experimental models of iwanaga and colleagues ( dahl salt - sensitive rats with left ventricular hypertrophy and secondary heart failure ) , the cardiac apelin and apj receptor were down - regulated in the lv dysfunction stage , associated with an up - regulation of ace2 ( the breakdown enzyme for apelin peptides ) , which may contribute to further lowering the plasma apelin , which is already downregulated . in their study , the rats were treated with angiotensin receptor blocker ( arb ) , metalloproteinase ( mmp ) inhibitor and a betablocker , and although there was functional improvement in all groups , only in the arb group the apelin and apj expression increased ; this is a strong proof of the fact that the beneficial effects of acei / arbs are based on the modulation of apelin . further , the expression of apelin was downregulated after infusion of ang ii , both in the pressor and the subpressor dose , effect that was prevented with the at1r blockade , sustaining furthermore the interrelation between the raas and the apelin / apj system . similar results about the tissue expression of apelin and its marn were obtained by wang and colleagues on dogs with heart failure , but levels of apj ( and its marn ) were equivalent to normal dogs . this difference may be due to the characteristics of the species used in the experiments , but further investigation is needed . used as an exogenous vasoactive agent , apelin showed a unique combination of inotropic and vasodilatory properties of apelin , which leads to a rise of cardiac output and to a decrease of systemic vascular resistance without significant arterial hypotension and tachycardia , regarding the dose . this study brings additional proof about the importance of apelin and its receptor in the development of heart failure and the potential favorable effect of treatment with exogenous apelin in patients with systolic dysfunction , and it is backed - up by other , recent studies . in a study on genetic apelin deficiency in mice ( apelin ) the results led to the conclusions that apelin is a crucial peptide for mantaining the systolic function of the aging heart in the conditions of chronic pressure overload . szokodi et al also found low levels of apelin marn in rats myocardium exposed to mechanical stretch and chronic pressure overload . wang and colleagues used similar experimental models in 2013 ( apelin and apelin ) and observed an aggravated postinfarction remodeling , neovascularization and impaired functional recovery of apelin deficient mice . the lack of apelin compromises the activation of prosurvival akt / pi3k and erk pathway , both in vitro and in vivo , thus amplifying the myocardial damage and lowering the global cardiac performance . more recent studies that used apelin analogues confirmed these results and demonstrated their capacity of lowering the myocardial ishemic / reperfusion injury in vivo . zhang et al . in 2013 experimented the effects of exogenous apelin on rats with myocardial infarction induced through left anterior artery ( lad ) ligation ; their results showed exquisite effects of apelin on microcirculation and angiogenesis by stimulating the migration of endothelial cells to ischemic regions , enhanced healing and new vessel formation . also , apelin decreased the vascular permeability in the ischemic region with possible effects on postischemic edema and inflammation . these findings can be extrapolated to ischemic heart disease with consequent heart failure , suggesting the potential benefit of apelin treatment , both short and long term . in 2012 a study using exogenous apelin on dahl salt - sensitive rats with end - stage heart failure showed an improvement of lv dysfunction and remodeling , a lowering of the oxidative stress ( through inhibition of nadph oxidase ) , a regulation of the akt / enos pathways and apelin / apj expression in the rat hearts after apelin treatment . this study showed the correlation of apelin with the oxidative damage and gave the premise for the potential benefit of the apelin treatment in the end - stage heart failure . exogenous apelin showed benefit also upon the myocardial hypertrophy and fibrosis ( secondary to increased afterload ) through inhibition of fibroblasts tgf--induced differentiation into myofibroblasts and diminishing the fibrosis process . the already formed myocardial hypertrophy and fibrosis were reduced and the lv dysfunction was prevented . the studies published so far bring evidence for down - regulation of apelin and its receptor during the process of myocardial remodeling ( hypertensive or ischemic ) with consequent reduction of its inotropic , antifibrotic and cytoprotective effects . the treatment with apelin restores the position of apj within the membrane and revives the mechanism of inotropy , both in failing and normal hearts . the exogenous apelin has benefic effects on the cardiac dysfunction secondary to other causes too , besides hypertensive , ischemic or idiopapathic cardiomyopathy . in humans the studies conducted so far led to conflicting results : although some research showed no significant difference of apelin plasma level between idc patients and normal controls , larger studies have showed that apelin rises in early , mild to moderate heart failure , and lowers significantly in severe , end - stage heart failure . plasma apelin increases in early heart failure and lowers in the final stage but did not correlate with nt - probnp and , with the information we have at this moment , it can not be used as a diagnostic tool , nor as a prognostic marker [ 5456 ] . also , plasma apelin appears not to be correlated with age , sex , body mass index , etiology of heart failure and renal function . in patients with heart failure plasma levels of apelin ( from peripheral venous puncture ) is reduced compared to controls , regarding of nyha class and the severity of systolic dysfunction . chandrasekaran and colleagues measured the level of apelin in three different sites ( coronary sinus , aorta and renal vein ) in patients with heart failure ( of different nyha class ) and controls . they found a significant reduction of apelin in the coronary sinus of heart failure patients as comparedto controls and a reduction of apelin in the aorta of controls in comparison with the coronary sinus that was not seen in heart failure patients . also , the heart failure patients had no apelin gradient between the three sites as was seen in control patients . left ventricular apelin marn increases in chronic heart failure secondary to ischemic heart disease or idiopathic dilated cardiomyopathy ( idc ) . also , in idc the apj receptor marn is significantly decreased , although the plasma apelin was no different from the healthy controls as was demonstrated by another study . in 2010 piktin et al analyzed samples of human myocardial tissue collected from cardiac transplant recipients ( for dilated cardiomyopathy and ischemic heart disease ) and found extreme down - regulation of apj in all samples with unchanged apelin levels , which may be the explanation for the progression of the cardiac failure , the apelin having no receptors to manifest its inotropic effect . it appears that the mechanical stretch is one of the stimuli for down - regulation of apj receptor , with secondary up - regulation after mechanical offloading of the left ventricle ( after left ventricle assisting device implantation ) in chen et al . cardiac resynchronization , another method of improving the mechanic and electric properties of a failing heart , had a good influence over the plasmatic apelin at nine months after implantation of the device . it is already well - known that wall stress and activation of raas augment in heart failure and these pathways are feasible of altering the apelin - apj expression . also , the ang ii negatively influence the level of apj in the cardiomyocites . the studies conducted so far showed the importance of apelin / apj pathway in the development of heart failure and the fact that its downregulation in the myocardial cell contributes to the structural and functional alteration of the heart . in experimental models of iwanaga and colleagues ( dahl salt - sensitive rats with left ventricular hypertrophy and secondary heart failure ) , the cardiac apelin and apj receptor were down - regulated in the lv dysfunction stage , associated with an up - regulation of ace2 ( the breakdown enzyme for apelin peptides ) , which may contribute to further lowering the plasma apelin , which is already downregulated . in their study , the rats were treated with angiotensin receptor blocker ( arb ) , metalloproteinase ( mmp ) inhibitor and a betablocker , and although there was functional improvement in all groups , only in the arb group the apelin and apj expression increased ; this is a strong proof of the fact that the beneficial effects of acei / arbs are based on the modulation of apelin . further , the expression of apelin was downregulated after infusion of ang ii , both in the pressor and the subpressor dose , effect that was prevented with the at1r blockade , sustaining furthermore the interrelation between the raas and the apelin / apj system . similar results about the tissue expression of apelin and its marn were obtained by wang and colleagues on dogs with heart failure , but levels of apj ( and its marn ) were equivalent to normal dogs . this difference may be due to the characteristics of the species used in the experiments , but further investigation is needed . used as an exogenous vasoactive agent , apelin showed a unique combination of inotropic and vasodilatory properties of apelin , which leads to a rise of cardiac output and to a decrease of systemic vascular resistance without significant arterial hypotension and tachycardia , regarding the dose . this study brings additional proof about the importance of apelin and its receptor in the development of heart failure and the potential favorable effect of treatment with exogenous apelin in patients with systolic dysfunction , and it is backed - up by other , recent studies . in a study on genetic apelin deficiency in mice ( apelin ) the results led to the conclusions that apelin is a crucial peptide for mantaining the systolic function of the aging heart in the conditions of chronic pressure overload . szokodi et al also found low levels of apelin marn in rats myocardium exposed to mechanical stretch and chronic pressure overload . wang and colleagues used similar experimental models in 2013 ( apelin and apelin ) and observed an aggravated postinfarction remodeling , neovascularization and impaired functional recovery of apelin deficient mice . the lack of apelin compromises the activation of prosurvival akt / pi3k and erk pathway , both in vitro and in vivo , thus amplifying the myocardial damage and lowering the global cardiac performance . more recent studies that used apelin analogues confirmed these results and demonstrated their capacity of lowering the myocardial ishemic / reperfusion injury in vivo . zhang et al . in 2013 experimented the effects of exogenous apelin on rats with myocardial infarction induced through left anterior artery ( lad ) ligation ; their results showed exquisite effects of apelin on microcirculation and angiogenesis by stimulating the migration of endothelial cells to ischemic regions , enhanced healing and new vessel formation . also , apelin decreased the vascular permeability in the ischemic region with possible effects on postischemic edema and inflammation . these findings can be extrapolated to ischemic heart disease with consequent heart failure , suggesting the potential benefit of apelin treatment , both short and long term . in 2012 a study using exogenous apelin on dahl salt - sensitive rats with end - stage heart failure showed an improvement of lv dysfunction and remodeling , a lowering of the oxidative stress ( through inhibition of nadph oxidase ) , a regulation of the akt / enos pathways and apelin / apj expression in the rat hearts after apelin treatment . this study showed the correlation of apelin with the oxidative damage and gave the premise for the potential benefit of the apelin treatment in the end - stage heart failure . exogenous apelin showed benefit also upon the myocardial hypertrophy and fibrosis ( secondary to increased afterload ) through inhibition of fibroblasts tgf--induced differentiation into myofibroblasts and diminishing the fibrosis process . the already formed myocardial hypertrophy and fibrosis were reduced and the lv dysfunction was prevented . the studies published so far bring evidence for down - regulation of apelin and its receptor during the process of myocardial remodeling ( hypertensive or ischemic ) with consequent reduction of its inotropic , antifibrotic and cytoprotective effects . the treatment with apelin restores the position of apj within the membrane and revives the mechanism of inotropy , both in failing and normal hearts . the exogenous apelin has benefic effects on the cardiac dysfunction secondary to other causes too , besides hypertensive , ischemic or idiopapathic cardiomyopathy . in humans the studies conducted so far led to conflicting results : although some research showed no significant difference of apelin plasma level between idc patients and normal controls , larger studies have showed that apelin rises in early , mild to moderate heart failure , and lowers significantly in severe , end - stage heart failure . plasma apelin increases in early heart failure and lowers in the final stage but did not correlate with nt - probnp and , with the information we have at this moment , it can not be used as a diagnostic tool , nor as a prognostic marker [ 5456 ] . also , plasma apelin appears not to be correlated with age , sex , body mass index , etiology of heart failure and renal function . in patients with heart failure plasma levels of apelin ( from peripheral venous puncture ) is reduced compared to controls , regarding of nyha class and the severity of systolic dysfunction . chandrasekaran and colleagues measured the level of apelin in three different sites ( coronary sinus , aorta and renal vein ) in patients with heart failure ( of different nyha class ) and controls . they found a significant reduction of apelin in the coronary sinus of heart failure patients as comparedto controls and a reduction of apelin in the aorta of controls in comparison with the coronary sinus that was not seen in heart failure patients . also , the heart failure patients had no apelin gradient between the three sites as was seen in control patients . left ventricular apelin marn increases in chronic heart failure secondary to ischemic heart disease or idiopathic dilated cardiomyopathy ( idc ) . also , in idc the apj receptor marn is significantly decreased , although the plasma apelin was no different from the healthy controls as was demonstrated by another study . in 2010 piktin et al analyzed samples of human myocardial tissue collected from cardiac transplant recipients ( for dilated cardiomyopathy and ischemic heart disease ) and found extreme down - regulation of apj in all samples with unchanged apelin levels , which may be the explanation for the progression of the cardiac failure , the apelin having no receptors to manifest its inotropic effect . it appears that the mechanical stretch is one of the stimuli for down - regulation of apj receptor , with secondary up - regulation after mechanical offloading of the left ventricle ( after left ventricle assisting device implantation ) in chen et al . cardiac resynchronization , another method of improving the mechanic and electric properties of a failing heart , had a good influence over the plasmatic apelin at nine months after implantation of the device . it is already well - known that wall stress and activation of raas augment in heart failure and these pathways are feasible of altering the apelin - apj expression . also , the ang ii negatively influence the level of apj in the cardiomyocites . apelin / apj is a crucial molecular pathway in the protection of the heart from the injuries caused by hemodynamic overload or structural damage , regardless of the cause , and thus temporary preventing the development of heart failure . apelin is a counter - regulator of the raas and keeps its activation in a balance . intense and prolonged raas activation disable the apelin cardioprotection and puts the system into withdrawal . experimental administration of apelin in heart failure showed unique combination of inotropism and vasodilation effects with no short - term deleterious effects . this is the premise for the potential benefit of apelin in heart failure , but further studies are needed .

heart failure is a chronic , progressive disease in which the overexpression of biologically active molecules and neurohomonal activation are the key factors of the evolution and natural history . the apelin - apj system is a newly discovered molecular pathway and the raas counterbalance is its principal effect . the apelin is a potent inotrope , vasodilator and diuretic with crucial cardioprotective effects against angiotensin and aldosterone injuries . intense and prolonged raas induces the downregulation of the apelin and its receptor at myocardial level and cancels their protection . compared to the vasoactive agents used in the treatment of acute heart failure , exogen apelin has unique intropic and vasodilatory effects without deleterious consequences , being a promising therapeutic option .

Introduction Physiological effect of apelin/APJ receptor pathway on the cardiovascular system The role of apelin/APJ system in heart failure Experimental animal studies Human studies Conclusions

heart failure is a chronic , progressive disease affecting approximately 12% of all the adult population , increasing to more than 10% in the population segment over 70 years ; its incidence rose with the extension of life - span and the aging of the population [ 13 ] . its prognosis is poor , the mortality is higher than that of some cancers and the morbidity is marked by a low quality of life and frequent rehospitalizations . its evolution is the result of the overexpression of a number of biologically active molecules with negative influence on the cardiovascular system , with chronic activation of signaling pathways secondary to inadequate tissue perfusion . the compensatory mechanisms described until now are the activation of the adrenergic and renin - angiotensin systems ( raas ) with the purpose of maintaining the cardiac output but with long - term deleterious effects . balancing these systems , there are other molecular pathways with key roles in the physiology of the cardiovascular system and which are now the principal subject of research in this field . the gene of apelin is located on the long arm of x chromosome ; the apelin is syntethized as preproapelin ( 77 aminoacids ) which is then cleaved by an angiotensin - converting enzyme to shorter active fragments ( c - terminal peptides : apelin-13 , -16 , -17 , -19 , -36 ) . apelin s receptor , apj , identified in 1993 by homologous cloning in the human genome project , was initially considered an orphan until the discovery of apelin in 1998 . the shorter fragments activate the receptor for a short period of time because it is rapidly internalized in the cellular membrane , returning to the surface within an hour and becoming available for activation again . at cellular level , the apelin activates the proteinkinase b ( akt ) , phosphatidil - inositol-3 kinase ( pi3k ) and the extracellular signal - regulated kinases ( erk ) pathway , which are involved in controlling apoptosis and cellular migration and proliferation . the apelin and its receptor can be found in a variety of tissues ( central nervous system , enterochromaffin - like gastric cells , pancreatic islet cells , osteoblasts , t - lymphocytes , adipose tissue ) , with higher concentrations in the lungs , cardiovascular system and spleen . it is synthesized locally in the endothelium and it is found in the cellular organelles as endoplasmic reticulum , golgi apparatus and secretory vesicles . later studies showed that the most important site of apelin - apj expression and action is the cardiovascular system , especially the vascular endothelium , vascular smooth muscle cells , endocardial endothelium , and in a lesser extend , the myocardium . this explains the higher concentration in the lungs and spleen , paralleling the level of vascularization [ 2025 ] . in the atrial tissue the apelin concentration is 200 folds higher than in the ventricular tissue and it appears to be correlated to the plasma level . in some other tissues as the kidney and adrenal gland it is expressed only in the blood vessels , although its effects on these tissues are complex and cover multiple sites . apelin is a diuretic , but its effects on the kidneys are not limited only to the renal tissue . apelin is synthesized in the brain and inhibits the secretion of vasopresin from the neurons , favoring water excretion ; also it acts on the renal microcirculation and probably on the tubular function to validate its diuretic effect [ 2830 ] . the apelin / apj system counter - regulates the raas by antagonizing the activity of ang ii , its major effector . apelin induces this physical interaction and increases the density of apj as compared to atr1 ( by increasing its expression and its availability at the membrane level ) , but the density of both receptors were not modified by the exogenous ang ii in this study . this may be the explanation for the development of cardiovascular diseases in the presence of dysfunctional apelin / apj system that can not balance the overactivated , injurious raas . also , recent studies demonstrated a relationship between apelin / apj system and the angiotensin converting enzyme type 2 ( ace2 ) which has a key role in counterbalancing the raas by catalyzing the transformation of ang ii into angiotensin 17 ( ang 17 ) , a molecule with opposing effects to ang ii . these findings stand for the theory than apelin / apj is a beneficial molecular pathway in the physiology of cardiovascular system . the apelin / apj system has many physiologic effects on water balance , glycemic control , nutritional behavior , immunity , but its principal target is the cardiovascular system . latest studies have showed that apelin / apj system has a key role in its normal function and in the development of vascular and heart diseases such as atherosclerosis , coronary heart disease , heart failure , systemic and pulmonary arterial hypertension and ischemic - reperfusion lesion . plasma concentration is about 10 g / ml with a half time of less than 5 minutes ; its slow , sustained , inotropic effect is validated in a subnanomolar concentration ( ec50=33 pmol / l ) , being the most potent endogenous inotropic molecule , overcoming the adrenomedulin and endothelin . in the experimental studies conducted so far the effect reached its maximum in 2030 min - completely different from mimetics which act within seconds - and last longer ; it was valid even when the no production was blocked , and the endothelin , the ang ii and receptors were inhibited . results showed that the inotropic effect of apelin is due mostly to an increase in ca availability in the myocardial cell and not to a sensitization of myofilaments ; the mechanism is not yet completely understood . apelin - apj receptor interaction at the vascular level induces vasodilation through no release from endothelial cells ( in arteries and veins ) with consequent decrease in systemic vascular resistance , lowering the pre- and the afterload of the left ventricle and improving its filling pattern . the apj receptor is present in the vascular smooth muscle and it stimulates its contraction as was demonstrated in experimental condition on endothelial denuded vein . the vasoconstricion induced by apelin on denuded vessels and the vasodilation produced in the presence of normal functioning endothelium suggest the complexity of apelin s role on the circulatory system , the fine tuning of the vascular tone and the easily disturbed apelin s effects in the presence of injured endothelium . the studies conducted so far showed the importance of apelin / apj pathway in the development of heart failure and the fact that its downregulation in the myocardial cell contributes to the structural and functional alteration of the heart . in experimental models of iwanaga and colleagues ( dahl salt - sensitive rats with left ventricular hypertrophy and secondary heart failure ) , the cardiac apelin and apj receptor were down - regulated in the lv dysfunction stage , associated with an up - regulation of ace2 ( the breakdown enzyme for apelin peptides ) , which may contribute to further lowering the plasma apelin , which is already downregulated . in their study , the rats were treated with angiotensin receptor blocker ( arb ) , metalloproteinase ( mmp ) inhibitor and a betablocker , and although there was functional improvement in all groups , only in the arb group the apelin and apj expression increased ; this is a strong proof of the fact that the beneficial effects of acei / arbs are based on the modulation of apelin . further , the expression of apelin was downregulated after infusion of ang ii , both in the pressor and the subpressor dose , effect that was prevented with the at1r blockade , sustaining furthermore the interrelation between the raas and the apelin / apj system . similar results about the tissue expression of apelin and its marn were obtained by wang and colleagues on dogs with heart failure , but levels of apj ( and its marn ) were equivalent to normal dogs . this difference may be due to the characteristics of the species used in the experiments , but further investigation is needed . used as an exogenous vasoactive agent , apelin showed a unique combination of inotropic and vasodilatory properties of apelin , which leads to a rise of cardiac output and to a decrease of systemic vascular resistance without significant arterial hypotension and tachycardia , regarding the dose . this study brings additional proof about the importance of apelin and its receptor in the development of heart failure and the potential favorable effect of treatment with exogenous apelin in patients with systolic dysfunction , and it is backed - up by other , recent studies . in a study on genetic apelin deficiency in mice ( apelin ) the results led to the conclusions that apelin is a crucial peptide for mantaining the systolic function of the aging heart in the conditions of chronic pressure overload . wang and colleagues used similar experimental models in 2013 ( apelin and apelin ) and observed an aggravated postinfarction remodeling , neovascularization and impaired functional recovery of apelin deficient mice . these findings can be extrapolated to ischemic heart disease with consequent heart failure , suggesting the potential benefit of apelin treatment , both short and long term . in 2012 a study using exogenous apelin on dahl salt - sensitive rats with end - stage heart failure showed an improvement of lv dysfunction and remodeling , a lowering of the oxidative stress ( through inhibition of nadph oxidase ) , a regulation of the akt / enos pathways and apelin / apj expression in the rat hearts after apelin treatment . this study showed the correlation of apelin with the oxidative damage and gave the premise for the potential benefit of the apelin treatment in the end - stage heart failure . the already formed myocardial hypertrophy and fibrosis were reduced and the lv dysfunction was prevented . the studies published so far bring evidence for down - regulation of apelin and its receptor during the process of myocardial remodeling ( hypertensive or ischemic ) with consequent reduction of its inotropic , antifibrotic and cytoprotective effects . the exogenous apelin has benefic effects on the cardiac dysfunction secondary to other causes too , besides hypertensive , ischemic or idiopapathic cardiomyopathy . in humans the studies conducted so far led to conflicting results : although some research showed no significant difference of apelin plasma level between idc patients and normal controls , larger studies have showed that apelin rises in early , mild to moderate heart failure , and lowers significantly in severe , end - stage heart failure . plasma apelin increases in early heart failure and lowers in the final stage but did not correlate with nt - probnp and , with the information we have at this moment , it can not be used as a diagnostic tool , nor as a prognostic marker [ 5456 ] . also , plasma apelin appears not to be correlated with age , sex , body mass index , etiology of heart failure and renal function . in patients with heart failure plasma levels of apelin ( from peripheral venous puncture ) is reduced compared to controls , regarding of nyha class and the severity of systolic dysfunction . chandrasekaran and colleagues measured the level of apelin in three different sites ( coronary sinus , aorta and renal vein ) in patients with heart failure ( of different nyha class ) and controls . they found a significant reduction of apelin in the coronary sinus of heart failure patients as comparedto controls and a reduction of apelin in the aorta of controls in comparison with the coronary sinus that was not seen in heart failure patients . also , the heart failure patients had no apelin gradient between the three sites as was seen in control patients . left ventricular apelin marn increases in chronic heart failure secondary to ischemic heart disease or idiopathic dilated cardiomyopathy ( idc ) . in 2010 piktin et al analyzed samples of human myocardial tissue collected from cardiac transplant recipients ( for dilated cardiomyopathy and ischemic heart disease ) and found extreme down - regulation of apj in all samples with unchanged apelin levels , which may be the explanation for the progression of the cardiac failure , the apelin having no receptors to manifest its inotropic effect . it appears that the mechanical stretch is one of the stimuli for down - regulation of apj receptor , with secondary up - regulation after mechanical offloading of the left ventricle ( after left ventricle assisting device implantation ) in chen et al . it is already well - known that wall stress and activation of raas augment in heart failure and these pathways are feasible of altering the apelin - apj expression . the studies conducted so far showed the importance of apelin / apj pathway in the development of heart failure and the fact that its downregulation in the myocardial cell contributes to the structural and functional alteration of the heart . in experimental models of iwanaga and colleagues ( dahl salt - sensitive rats with left ventricular hypertrophy and secondary heart failure ) , the cardiac apelin and apj receptor were down - regulated in the lv dysfunction stage , associated with an up - regulation of ace2 ( the breakdown enzyme for apelin peptides ) , which may contribute to further lowering the plasma apelin , which is already downregulated . in their study , the rats were treated with angiotensin receptor blocker ( arb ) , metalloproteinase ( mmp ) inhibitor and a betablocker , and although there was functional improvement in all groups , only in the arb group the apelin and apj expression increased ; this is a strong proof of the fact that the beneficial effects of acei / arbs are based on the modulation of apelin . further , the expression of apelin was downregulated after infusion of ang ii , both in the pressor and the subpressor dose , effect that was prevented with the at1r blockade , sustaining furthermore the interrelation between the raas and the apelin / apj system . similar results about the tissue expression of apelin and its marn were obtained by wang and colleagues on dogs with heart failure , but levels of apj ( and its marn ) were equivalent to normal dogs . this difference may be due to the characteristics of the species used in the experiments , but further investigation is needed . used as an exogenous vasoactive agent , apelin showed a unique combination of inotropic and vasodilatory properties of apelin , which leads to a rise of cardiac output and to a decrease of systemic vascular resistance without significant arterial hypotension and tachycardia , regarding the dose . this study brings additional proof about the importance of apelin and its receptor in the development of heart failure and the potential favorable effect of treatment with exogenous apelin in patients with systolic dysfunction , and it is backed - up by other , recent studies . in a study on genetic apelin deficiency in mice ( apelin ) the results led to the conclusions that apelin is a crucial peptide for mantaining the systolic function of the aging heart in the conditions of chronic pressure overload . also , apelin decreased the vascular permeability in the ischemic region with possible effects on postischemic edema and inflammation . these findings can be extrapolated to ischemic heart disease with consequent heart failure , suggesting the potential benefit of apelin treatment , both short and long term . in 2012 a study using exogenous apelin on dahl salt - sensitive rats with end - stage heart failure showed an improvement of lv dysfunction and remodeling , a lowering of the oxidative stress ( through inhibition of nadph oxidase ) , a regulation of the akt / enos pathways and apelin / apj expression in the rat hearts after apelin treatment . this study showed the correlation of apelin with the oxidative damage and gave the premise for the potential benefit of the apelin treatment in the end - stage heart failure . the already formed myocardial hypertrophy and fibrosis were reduced and the lv dysfunction was prevented . the studies published so far bring evidence for down - regulation of apelin and its receptor during the process of myocardial remodeling ( hypertensive or ischemic ) with consequent reduction of its inotropic , antifibrotic and cytoprotective effects . the treatment with apelin restores the position of apj within the membrane and revives the mechanism of inotropy , both in failing and normal hearts . the exogenous apelin has benefic effects on the cardiac dysfunction secondary to other causes too , besides hypertensive , ischemic or idiopapathic cardiomyopathy . in humans the studies conducted so far led to conflicting results : although some research showed no significant difference of apelin plasma level between idc patients and normal controls , larger studies have showed that apelin rises in early , mild to moderate heart failure , and lowers significantly in severe , end - stage heart failure . plasma apelin increases in early heart failure and lowers in the final stage but did not correlate with nt - probnp and , with the information we have at this moment , it can not be used as a diagnostic tool , nor as a prognostic marker [ 5456 ] . also , plasma apelin appears not to be correlated with age , sex , body mass index , etiology of heart failure and renal function . in patients with heart failure plasma levels of apelin ( from peripheral venous puncture ) is reduced compared to controls , regarding of nyha class and the severity of systolic dysfunction . they found a significant reduction of apelin in the coronary sinus of heart failure patients as comparedto controls and a reduction of apelin in the aorta of controls in comparison with the coronary sinus that was not seen in heart failure patients . also , the heart failure patients had no apelin gradient between the three sites as was seen in control patients . left ventricular apelin marn increases in chronic heart failure secondary to ischemic heart disease or idiopathic dilated cardiomyopathy ( idc ) . in 2010 piktin et al analyzed samples of human myocardial tissue collected from cardiac transplant recipients ( for dilated cardiomyopathy and ischemic heart disease ) and found extreme down - regulation of apj in all samples with unchanged apelin levels , which may be the explanation for the progression of the cardiac failure , the apelin having no receptors to manifest its inotropic effect . it appears that the mechanical stretch is one of the stimuli for down - regulation of apj receptor , with secondary up - regulation after mechanical offloading of the left ventricle ( after left ventricle assisting device implantation ) in chen et al . cardiac resynchronization , another method of improving the mechanic and electric properties of a failing heart , had a good influence over the plasmatic apelin at nine months after implantation of the device . it is already well - known that wall stress and activation of raas augment in heart failure and these pathways are feasible of altering the apelin - apj expression . also , the ang ii negatively influence the level of apj in the cardiomyocites . apelin / apj is a crucial molecular pathway in the protection of the heart from the injuries caused by hemodynamic overload or structural damage , regardless of the cause , and thus temporary preventing the development of heart failure . apelin is a counter - regulator of the raas and keeps its activation in a balance . intense and prolonged raas activation disable the apelin cardioprotection and puts the system into withdrawal . this is the premise for the potential benefit of apelin in heart failure , but further studies are needed .

the dopamine-3 ( d3 ) receptor subtype has been identified as an important target for agents currently in clinical use for the treatment of a variety of neurological diseases , including schizophrenia , parkinson s disease , and depression . however , all of the clinically approved drugs targeting the d3 receptor have a very limited selectivity over d2 receptors and other off - targets . considerable effort has been devoted in the past decade to the design of potent and selective d3 ligands . although it was initially challenging to design highly selective d3 ligands , due to the high degree of sequence homology between the d2 and d3 receptors , recent sar studies have demonstrated the feasibility of such a design . for example , upon the basis of pramipexole ( 1 ) , a potent d3 agonist with only modest selectivity over the d2 receptor , we designed and prepared compounds cj-1638 ( 2 ) and cj-1639 ( 3 ) , which are potent and selective d3 agonists ( figure 1 ) . compounds 2 and 3 bind to the d3 receptor , with ki values < 1 nm and display > 1000-fold selectivity over both the d1 and d2 receptors . further , with the determination of the d3 receptor crystal structure and derived computational models , the small molecule sars have been fortified . not only have highly d3 receptor selective ligands been discovered , but the roles of the orthosteric site and a secondary binding pocket have further defined the drug protein interactions responsible for affinity , selectivity , and efficacy . potent and selective d3 antagonists may have a therapeutic potential for the treatment of drug addictions and relate disorders . herein we report the design and sar study of a new class of d3 antagonists . analysis of several classes of known d3 antagonists shows that their structures can be divided into three regions as shown in figure 2 : the headgroup ( in blue ) , the linker ( in black ) , and the tail ( in red ) . the headgroup consists of a basic amine tethered to an aromatic group ( typically phenyl ) , substituted with a hydrogen bonding acceptor ( a nitrile in 4(3 ) or a methoxyl group in 6(20 ) ) , or one or two small hydrophobic groups in 5(21,22 ) and 7 . we first selected a new head group with these features for the design and development of a new class of d3 antagonists . chemical structures of previous dopamine d3 receptor antagonists . among potential head groups under consideration , we found that tranylcypromine ( 8 , figure 3 ) was attractive because , like pramipexole ( 1 ) and 7 , it contains a structurally rigid phenethylamine moiety . because the affinities of tranylcypromine for the dopamine receptors are not known , we first tested the commercially available , racemic tranylcypromine in our dopamine receptor binding assays using rat brain . our data showed that tranylcypromine has a weak affinity for the d3 receptor with a ki value = 12.8 m and displays 4-fold selectivity over the d2 receptor ( table 1 ) . although tranylcypromine is a weak d3 ligand and has a very limited selectivity over the d2 receptor , it was used as a starting point for our sar study , which has ultimately yielded a class of potent and selective d3 antagonists . our previous study showed that the propyl substituent on the amine group in pramipexole enhances the binding affinity to the d3 receptors by at least 1 order of magnitude . we therefore investigated whether substitution of a propyl group on the primary amine in tranylcypromine would improve the binding affinity to the d3 receptor . chiral resolution of racemic 2-phenyl - cyclopropanamine according to the reported method , followed by addition of the propyl group afforded two stereoisomers ( 1s,2r)-9 and ( 1r,2s)-9 . binding data showed that both ( 1s,2r)-9 and ( 1r,2s)-9 bind to the rat d3 receptor with ki values of 1.2 m and display approximately 80-fold selectivity over the d2 receptor ( table 1 ) . thus , addition of a propyl group to the primary amine in tranylcypromine indeed improves the binding affinity for the d3 receptor , as well as the selectivity over the d2 receptor . our previous study showed that introduction of appropriate linker and tail groups in compounds 2 and 3 onto the amine group in pramipexole significantly enhanced its selectivity for the rat d3 receptor over that for the d2 receptor . accordingly , we synthesized ( 1s,2r)-10 and ( 1r,2s)-10 with the same linker and tail groups used in compound 2 appended onto the amine group in compounds ( 1s,2r)-9 and ( 1r,2s)-9 . compounds ( 1s,2r)-10 and ( 1r,2s)-10 have ki values of 108 and 44 nm to the rat d3 receptor , respectively , representing a 1020-fold improvement over ( 1s,2r)-9 and ( 1r,2s)-9 . however , in contrast to the marked improved selectivity of compound 2 over pramipexole for the d3 receptor over the d2 receptor observed in our previous study , both ( 1s,2r)-10 and ( 1r,2s)-10 only have modest 2030-fold selectivity over the rat d2 receptor . addition of a hydrophobic group such as cl to the phenyl ring has been shown to enhance the binding affinity of antagonists to the d3 receptor . we have therefore synthesized three compounds ( 11 , 12 , and 13 ) in which a cl substitution was installed in the para- , ortho- , or meta - position of the phenyl ring of 10 . because ( 1s,2r)-10 and ( 1r,2s)-10 do not differ markedly in their binding affinity to the rat d3 receptor or their selectivity over the rat d2 receptor , we first synthesized and evaluated the racemic forms of 1113 . ( )-12 , and ( )-13 have ki values of 4.6 , 19 , and 28 nm , respectively , to the rat d3 receptor . ( )-11 and ( )-13 also display high ( > 10000 times ) selectivity over the rat d2 receptor , neither compound showing measurable binding to the rat d2 receptor at 100 m . however , both ( )-11 and ( )-13 have significant affinity for the rat d1-like receptors with ki values of 2.2 and 1.8 m , respectively . because ( )-11 showed high affinity for the rat d3 receptor , we resolved the stereoisomers , ( 1s,2r)-11 and ( 1r,2s)-11 ) . ( 1r,2s)-11 has a ki value of 2.7 nm to the rat d3 receptor and is > 100 times more potent than ( 1s,2r)-11 ( ki = 457 nm ) . furthermore , ( 1r,2s)-11 has no appreciable binding to the rat d2 receptor at concentrations as high as 300 m and consequently has > 100,000-fold selectivity for the rat d3 receptor over the rat d2 receptor . ( 1r,2s)-11 shows weak binding affinity to the d1-like receptors , with a ki value of 25.8 m , and has > 9000-fold selectivity for the rat d3 receptor over the rat d1-like receptors . we next assessed the binding affinities of ( )-11 , ( )-12 , ( )-13 , and ( 1r,2s)-11 to the human d2 and d3 receptors using transfected cell lines and included several previously reported d2/d3 antagonists as controls . the data are provided in table 2 . racemic compounds ( )-11 , ( )-12 , ( )-13 , and the pure enantiomer ( 1r,2s)-11 have ki values to the human d3 receptor of 2.61 , 22.1 , 37.9 , and 2.80 nm , respectively . these values are similar to their respective ki values of 4.6 , 19 , 26 , and 2.7 nm to the rat d3 receptor . compounds ( )-11 , ( )-12 , ( )-13 , and ( 1r,2s)-11 have ki values , respectively , of 667 , 923 , 1220 , and 623 nm , to the human d2 receptor . thus , with the exception of ( )-12 , these compounds have higher binding affinities to the human d2 receptor than to the rat d2 receptor . the selectivities of ( )-11 and ( )-13 and the pure enantiomer ( 1r,2s)-11 for the human d3 receptor over the human d2 receptor are 256- , 32- , and 223-fold , respectively . these are lower than the selectivities observed for these compounds for the rat d3 receptor over the rat d2 receptor . the known d3 antagonists n - methylspiperone , eticlopride , raclopride , and butaclamol , all bind to the human d3 receptor with high affinities but show no selectivity between the human d2 and d3 receptors ( table 2 ) . in comparison , pg619 and pg648 , two previously reported selective d3 antagonists , bind to the human d3 receptor with ki values of 6.70 and 1.88 nm , respectively , displaying selectivities of 163- and 397-fold respectively , for the human d3 receptor over the human d2 receptor . to assess the functional activity of ( 1r,2s)-11 at the d3 receptor , we tested it in a discoverx d3 functional assay using the u2os cell line transfected with human dopamine d3 receptor . in this assay , a d3 agonist , such as pramipexole , stimulates -arrestin binding to the d3 receptor , while a d3 antagonist , such as 6 ( bp 897 ) , blocks the association of -arrestin induced by a d3 agonist . assessed in this manner , pramipexole has an agonist activity with an ec50 value of 3.7 0.65 nm . ( 1r,2s)-11 has no agonist activity ( ec50 > 100 m ) , but it dose - dependently inhibits the binding of -arrestin to the d3 receptor induced by 100 nm of pramipexole and has an ic50 value of 327 126 nm ( table 3 ) . in comparison , 6 shows no agonist activity but has potent antagonist activity , with an ic50 value of 13 1.7 nm ( table 3 ) . hence , ( 1r,2s)-11 is a potent d3 antagonist , albeit less potent than 6 . we performed a schild regression analysis for ( 1r,2s)-11 in the human d3 functional assay ( figure 4 ) . in these experiments , d3 activity , measured on the basis of -arrestin binding to the receptor , was stimulated by pramipexole in the presence of three concentrations of ( 1r,2s)-11 . the data were analyzed according to the methods of kenakin . in the schild plot ( the inset in figure 4 ) , it can be seen that increasing concentrations of ( 1r,2s)-11 shift the pramipexole dose response curve to the right , consistent with antagonist activity , with a kb value of 20 nm . the slope of the schild plot was 0.83 , close to unity , indicating that ( 1r,2s)-11 is a competitive d3 antagonist . data are the mean sem of 36 independent determinations ( 2 determinations if inactive ) . schild analysis of ( 1r,2s)-11 at the human d3 receptor in the discoverx pathhunter express -arrestin assay . schild transformation ( insert ) indicated a pa2 of 7.96 , corresponding to a kb value of 20 nm . enantiomerically pure ( 1r,2s)-2-phenylcyclopropanamine and ( 1s,2r)-2-phenylcyclo - propanamine were obtained by resolution of commercially available ( )-trans-2-phenylcyclopropylamine ( 8) according to the reported method . reductive amination of ( 1r,2s)-8 and ( 1s,2r)-8 using propionaldehyde and nabh4 gave ( 1r,2s)-9 and ( 1s,2r)-9 in good yield . reductive amination of ( 1r,2s)-9 and ( 1s,2r)-9 with n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naphthamide using sodium triacetoxyborohydride as a reductant gave ( 1r,2s)-10 and ( 1s,2r)-10 reductive amination of the appropriate commercially available racemic chloro - substituted trans-2-phenylcyclopropanamine ( )-1416 afforded ( )-1719 . the final compounds ( )-1113 were prepared by reductive amination of ( )-1719 with n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naphthamide . reagents and conditions : ( a ) propionaldehyde , nabh4 , meoh , rt ; ( b ) n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naphthamide , nabh(oac)3 , hoac , dcm , rt , 4 h. the synthesis of ( 1s,2r)-11 is shown in scheme 2 . the optically pure key intermediate ( 1s,2s)-2-(4-chlorophenyl)cyclopropane - carboxylic acid 24 was prepared according to a reported method . treatment of the acid chloride 20 with sodium ( 1r)-(+)-2,10-camphorsultam afforded the enoyl sultams 21 in excellent yield ( > 93% ) . reaction of 21 with diazomethane in the presence of a catalytic amount of palladium acetate gave a cyclopropanated diastereomeric mixture of ( 1r)-(+)-2,10-camphorsultam-(1s,2s)-2-phenylcyclopropane - carboxamide 22 and ( 1r)-(+)-2,10-camphorsultam-(1r,2r)-2-phenyl - cyclopropane - carboxamide 23 in a ratio of 7:1 . treatment of 22 with titanium isopropoxide in benzyl alcohol , followed by lithium hydroxide hydrolysis and acidification , afforded the cyclopropanecarboxylic acid ( 1s,2s)-24 . boc - protected ( 1s,2r)-2-phenylcyclopropylamine 25 was obtained from the carboxylic acid using a curtius rearrangement of the corresponding acyl azide followed by addition of t - butanol to the isocyanate intermediate . removal of the boc group , followed by reductive amination , gave ( 1s,2r)-17 . the final compound ( 1s,2r)-11 was obtained by reductive - amination of ( 1s,2r)-17 with n-(cis-3-hydroxy-3-(2-oxoethyl)cyclo - butyl)-2-naphthamide . ( 1r,2s)-11 was made by a method similar to that used for ( 1s,2r)-11 , except that ( 1s)-()-2,10-camphorsultam was used as a chiral auxiliary . reagents and conditions : ( a ) ( 1r)-(+)-2,10-camphorsultam , nah , thf , 0 c , 30 min , then rt overnight ; ( b ) ch2n2 , pd(oac)2 , dcm , rt , 10 h ; ( c ) ( i)ti(iopr)4 , bzoh , 150 c , 30 min , ( ii ) 2 m lioh , meoh , rt , 2 h , ( iii ) 4 m hci ; ( d ) ( i ) ethyl chloroformate . et3n , acetone , 0 c , 2 h , ( ii ) nan3,1 h , ( iii ) 90 c , toluene , 3 h , ( iv ) butoh , reflux , 16 h. ( e ) tfa , dcm , rt , 12 h ; ( f ) propionaldehyde , nabh4 , meoh , rt ; ( g ) n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naprittiamlde , nabh(oac)3 , hoac , dcm , rt , 4 h. starting from tranylcypromine , we have designed and synthesized a class of potent and selective dopamine d3 receptor ligands . the best compound , ( 1r,2s)-11 , had a ki value of 2.7 nm to the rat d3 receptor and displayed a selectivity of > 100000-fold over the rat d2 receptor and > 9000-fold over the rat d1-like receptors . ( 1r,2s)-11 binds to the human d3 receptor with a ki value of 2.8 nm and displays 223-fold selectivity over the human d2 receptor . functional data and schild analysis showed that ( 1r,2s)-11 is a potent and competitive antagonist at the human d3 receptor . ( 1r,2s)-11 ( cj-1882 ) is being further evaluated for its pharmacokinetics , brain bioavailability , and therapeutic potential for the treatment of drug abuse . reactions were monitored by tlc carried out on 250 m silica gel plates 60f-254 ( e. merck ) using uv light as visualizing agent . silica gel 60 , particle size 1540 m ( e. merck ) , was used for flash column chromatography . nmr spectra were recorded on a bruker avance 300 spectrometer ( 300 mhz ) . chemical shifts ( ) are reported as values ( ppm ) downfield relative to tms as an internal standard , with multiplicities reported in the standard manner . all final compounds have purities > 95% , as determined by hplc ( uv detection at 254 nm ) . propionaldehyde ( 82 mg , 1.41 mmol ) was added to a solution of ( 1s,2r)-2-phenylcyclopropanamine ( 188 mg , 1.41 mmol ) in meoh ( 10 ml ) , and the reaction mixture was stirred at room temperature for 2 h. sodium borohydride ( 79 mg , 2.12 mmol ) was then added , and the mixture was stirred at room temperature for 1 h. the reaction was quenched with h2o ( 30 ml ) and extracted with ethyl acetate ( 40 ml ) . the residue was chromatographed ( hexane : etoac = 50:50 ) to give ( 1s,2r)-9 ( 178 mg , 72% yield ) as a colorless oil . h nmr ( cd3od , 300 mhz ) 7.377.12 ( m , 5h ) , 3.203.08 ( m , 2h ) , 2.972.90 ( m , 1h ) , 2.562.45 ( m , 1h ) , 1.821.70 ( m , 2h ) , 1.571.45 ( m , 1h ) , 1.36 ( dd , j = 6.7 , 14.4 hz , 1h ) , 1.04 ( t , j = 7.4 hz ) . c nmr ( cd3od , 75 mhz ) 139.42 , 129.68 , 127.91 , 127.56 , 50.88 , 39.04 , 22.32 , 20.55 , 13.35 , 11.19 . propionaldehyde ( 82 mg , 1.41 mmol ) was added to a solution of ( 1r,2s)-2-phenylcyclopropanamine ( 188 mg , 1.41 mmol ) in meoh ( 10 ml ) , and the reaction mixture was stirred at room temperature for 2 h. sodium borohydride ( 79 mg , 2.12 mmol ) was then added , and the mixture was stirred at room temperature for 1 h. the reaction was quenched with h2o ( 30 ml ) and extracted with etoac ( 40 ml ) . the residue was chromatographed ( hexane : etoac = 50:50 ) to give ( 1r,2s)-9 ( 171 mg , 69% yield ) as a colorless oil . h nmr ( cd3od , 300 mhz ) 7.377.12 ( m , 5h ) , 3.203.08 ( m , 2h ) , 2.972.90 ( m , 1h ) , 2.562.45 ( m , 1h ) , 1.821.70 ( m , 2h ) , 1.571.45 ( m , 1h ) , 1.36 ( dd , j = 6.7 , 14.4 hz , 1h ) , 1.04 ( t , j = 7.4 hz ) . c nmr ( cd3od , 75 mhz ) 139.42 , 129.68 , 127.91 , 127.56 , 50.88 , 39.04 , 22.32 , 20.55 , 13.35 , 11.19 . n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naphthamide ( 80 mg , 0.28 mmol ) , sodium triacetoxyborohydride ( 90 mg , 0.43 mmol ) , and acoh ( 26 mg , 0.43 mmol ) were added to a solution of ( 1s,2r)-9 ( 50 mg , 0.28 mmol ) in ch2cl2 ( 20 ml ) . the mixture was stirred at room temperature for 4 h and then quenched by addition of h2o ( 30 ml ) . the organic solvent was removed under vacuum , and the residue was chromatographed ( meoh : etoac = 10:90 ) to give ( 1s,2r)-10 ( 43 mg , 34% yield ) as a colorless oil . h nmr ( cd3od , 300 mhz ) 8.39 ( s , 1h ) , 8.007.80 ( m , 4h ) , 7.607.50 ( m , 2h ) , 7.407.20 ( m , 5h ) , 4.254.00 ( m , 1h ) , 3.603.25 ( m , 4h ) , 3.203.02 ( m , 1h ) , 2.752.60 ( m , 3h ) , 2.402.09 ( m , 4h ) , 1.901.65 ( m , 3h ) , 1.53 ( dd , j = 7.1 , 14.4 hz , 1h ) , 1.05 ( t , j = 7.4 hz , 3h ) . c nmr ( cd3od , 75 mhz ) 169.90 , 164.83 , 138.89 , 136.25 , 133.99 , 132.65 , 129.99 , 129.91 , 129.31 , 128.85 , 128.76 , 128.21 , 127.86 , 127.18 , 124.91 , 68.81 , 58.31 , 53.29 , 46.48 , 44.19 , 44.09 , 38.24 , 23.66 , 18.84 , 14.49 , 11.23 . n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naphthamide ( 80 mg , 0.28 mmol ) , sodium triacetoxyborohydride ( 90 mg , 0.43 mmol ) , and acoh ( 26 mg , 0.43 mmol ) were added to a solution of ( 1r,2s)-9 ( 50 mg , 0.28 mmol ) in ch2cl2 ( 20 ml ) , and the mixture was stirred at room temperature for 4 h. the reaction was quenched by addition of h2o ( 30 ml ) , and the mixture was extracted with ch2cl2 ( 30 ml 3 ) . the organic solvent was removed under vacuum , and the residue was chromatographed ( meoh : etoac = 10:90 ) to give ( 1r,2s)-10 ( 36 mg , 29% yield ) as a colorless oil . h nmr ( cd3od , 300 mhz ) 8.39 ( s , 1h ) , 8.007.80 ( m , 4h ) , 7.607.50 ( m , 2h ) , 7.407.20 ( m , 5h ) , 4.254.00 ( m , 1h ) , 3.603.25 ( m , 4h ) , 3.203.02 ( m , 1h ) , 2.752.60 ( m , 3h ) , 2.402.09 ( m , 4h ) , 1.901.65 ( m , 3h ) , 1.53 ( dd , j = 7.1 , 14.4 hz , 1h ) , 1.05 ( t , j = 7.4 hz , 3h ) . c nmr ( cd3od , 75 mhz ) 169.90 , 164.83 , 138.89 , 136.25 , 133.99 , 132.65 , 129.99 , 129.91 , 129.31 , 128.85 , 128.76 , 128.21 , 127.86 , 127.18 , 124.91 , 68.81 , 58.31 , 53.29 , 46.48 , 44.19 , 44.09 , 38.24 , 23.66 , 18.84 , 14.49 , 11.23 . n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naphthamide ( 82 mg , 0.29 mmol ) , sodium triacetoxyborohydride ( 91 mg , 0.43 mmol ) , and acoh ( 26 mg , 0.43 mmol ) were added to a solution of ( )-18 ( 60 mg , 0.29 mmol ) in ch2cl2 ( 20 ml ) , and the mixture was stirred at room temperature for 4 h. the reaction was quenched by addition of h2o ( 30 ml ) , and the mixture was extracted with ch2cl2 ( 30 ml 3 ) . the organic solvent was removed under vacuum , and the residue was chromatographed ( meoh : etoac = 10:90 ) to give ( )-12 ( 49 mg , 36% yield ) as a colorless oil . h nmr ( cd3od , 300 mhz ) 8.39 ( s , 1h ) , 8.007.85 ( m , 4h ) , 7.707.52 ( m , 2h ) , 7.47 ( dd , j = 1.6 , 7.4 hz , 1h ) , 7.407.20 ( m , 2h ) , 7.09 ( dd , j = 2.0 , 7.4 hz , 1h ) , 4.254.02 ( m , 1h ) , 3.703.30 ( m , 4h ) , 3.303.20 ( m , 1h ) , 2.752.60 ( m , 2h ) , 2.402.10 ( m , 4h ) , 2.001.50 ( m , 4h ) , 1.07 ( t , j = 7.3 hz , 3h ) . c nmr ( cd3od , 75 mhz ) 170.16 , 136.48 , 136.40 , 135.76 , 134.23 , 132.87 , 130.91 , 130.19 , 129.95 , 129.52 , 129.05 , 128.97 , 128.91 , 128.06 , 127.59 , 125.09 , 69.09 , 58.35 , 53.41 , 46.50 , 44.48 , 44.29 , 38.48 , 34.56 , 21.52 , 18.85 , 14.44 , 11.42 . ( )-11 was prepared in a manner similar to that used for ( )-12 in 35% yield . h nmr ( cdcl3 , 300 mhz ) 8.28 ( s , 1h ) , 7.907.75 ( m , 4h ) , 7.607.50 ( m , 2h ) , 7.23 ( d , j = 8.5 hz , 2h ) , 6.94 ( d , j = 8.5 hz , 2h ) , 6.53 ( d , j = 7.7 hz , 1h ) , 4.404.20 ( m , 1h ) , 3.002.40 ( m , 6h ) , 2.251.70 ( m , 6h ) , 1.601.50 ( m , 2h ) , 1.251.00 ( m , 2h ) , 0.89 ( t , j = 7.3 hz , 3h ) . c nmr ( cdcl3 , 75 mhz ) 167.21 , 140.12 , 134.93 , 132.82 , 131.83 , 131.72 , 129.14 , 128.71 , 128.67 , 127.95 , 127.84 , 127.58 , 127.25 , 126.96 , 123.75 , 71.19 , 57.69 , 53.27 , 48.76 , 44.73 , 44.57 , 37.51 , 34.44 , 24.94 , 20.15 , 17.02 , 12.23 . ( )-13 was prepared in a manner similar to that used for ( )-12 in 32% yield . h nmr ( cd3od , 300 mhz ) 8.39 ( s , 1h ) , 8.007.80 ( m , 4h ) , 7.707.50 ( m , 2h ) , 7.407.10 ( m , 4h ) , 4.254.10 ( m , 1h ) , 3.603.10 ( m , 5h ) , 2.752.65 ( m , 3h ) , 2.352.10 ( m , 4h ) , 1.901.50 ( m , 4h ) , 1.05 ( t , j = 7.3 hz , 3h ) . c nmr ( cd3od , 75 mhz ) 170.14 , 141.61 , 136.45 , 136.01 , 134.20 , 132.86 , 131.60 , 130.18 , 129.50 , 129.04 , 128.95 , 128.51 , 128.04 , 127.45 , 126.01 , 125.09 , 69.05 , 58.49 , 53.47 , 46.64 , 44.42 , 44.27 , 38.47 , 34.50 , 23.42 , 19.02 , 14.99 , 11.41 . n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naphthamide ( 191 mg , 0.67 mmol ) , sodium triacetoxyborohydride ( 212 mg , 1.01 mmol ) , and acoh ( 60 mg , 1.01 mmol ) were added to a solution of ( 1s,2r)-17 ( 140 mg , 0.67 mmol ) in ch2cl2 ( 20 ml ) , and the mixture was stirred at room temperature for 4 h. the reaction was quenched by addition of h2o ( 30 ml ) , and the mixture was extracted with ch2cl2 ( 30 ml 3 ) . the organic solvent was removed under vacuum , and the residue was chromatographed ( meoh : etoac = 10:90 ) to give ( 1s,2r)-11 ( 79 mg , 25% yield ) as a colorless oil . h nmr ( cdcl3 , 300 mhz ) 8.28 ( s , 1h ) , 7.907.75 ( m , 4h ) , 7.607.50 ( m , 2h ) , 7.23 ( d , j = 8.5 hz , 2h ) , 6.94 ( d , j = 8.5 hz , 2h ) , 6.53 ( d , j = 7.7 hz , 1h ) , 4.404.20 ( m , 1h ) , 3.002.40 ( m , 6h ) , 2.251.70 ( m , 6h ) , 1.601.50 ( m , 2h ) , 1.251.00 ( m , 2h ) , 0.89 ( t , j = 7.3 hz , 3h ) . c nmr ( cdcl3 , 75 mhz ) 167.21 , 140.12 , 134.93 , 132.82 , 131.83 , 131.72 , 129.14 , 128.71 , 128.67 , 127.95 , 127.84 , 127.58 , 127.25 , 126.96 , 123.75 , 71.19 , 57.69 , 53.27 , 48.76 , 44.73 , 44.57 , 37.51 , 34.44 , 24.94 , 20.15 , 17.02 , 12.23 . ( 1r,2s)-11 was prepared in 35% yield in a manner similar to that used for ( 1s,2r)-11 , except that ( 1s)-()-2,10-camphorsultam was used as a chiral auxiliary . h nmr ( cdcl3 , 300 mhz ) 8.28 ( s , 1h ) , 7.907.75 ( m , 4h ) , 7.607.50 ( m , 2h ) , 7.23 ( d , j = 8.5 hz , 2h ) , 6.94 ( d , j = 8.5 hz , 2h ) , 6.53 ( d , j = 7.7 hz , 1h ) , 4.404.20 ( m , 1h ) , 3.002.40 ( m , 6h ) , 2.251.70 ( m , 6h ) , 1.601.50 ( m , 2h ) , 1.251.00 ( m , 2h ) , 0.89 ( t , j = 7.3 hz , 3h ) . c nmr ( cdcl3 , 75 mhz ) 167.21 , 140.12 , 134.93 , 132.82 , 131.83 , 131.72 , 129.14 , 128.71 , 128.67 , 127.95 , 127.84 , 127.58 , 127.25 , 126.96 , 123.75 , 71.19 , 57.69 , 53.27 , 48.76 , 44.73 , 44.57 , 37.51 , 34.44 , 24.94 , 20.15 , 17.02 , 12.23 . the binding affinities of all the synthetic compounds were determined at the d3 , d2 , and d1-like receptors in membranes prepared from the brains of adult , male sprague dawley rats ( pel - freez , rogers , ar ) . the [ h]r-(+)-7-oh - dpat binding assay for the rat d3 dopamine receptors was performed as described . a rat ventral striatum ( nucleus accumbens and olfactory tubercles ) was prepared in assay buffer ( 50 mm tris , 1 mm edta ; ph 7.4 at 23 c ) to yield a final concentration of 10 mg original wet weight ( oww)/ml . membranes were incubated with [ h]r-(+)-7-oh - dpat ( 0.15 nm , sa = 163 ci / mmol ; ge healthcare ) and different concentrations of the test compounds ( 10 to 10 m ) . assay tubes were incubated at 23 c for 90 min . the reaction was terminated by rapid vacuum filtration . ki values were calculated using kd = 0.15 nm for [ h]7-oh - dpat and are expressed as the mean sem of 35 independent determinations . [ h]spiperone binding assays for rat d2-like receptors were performed as described for [ h]r-(+)-7-oh - dpat with the following modifications . assays were performed using membranes prepared from rat caudate - putamen , which expresses d2 receptors in high density but with very low levels of d3 receptors , and the final membrane homogenate concentration was 1.5 mg oww / ml . the assay buffer was 50 mm tris - hcl , 5 mm kcl , 2 mm mgcl2 , and 2 mm cacl2 , ph 7.4 at 23 c ; the concentration of [ h]spiperone ( 6096 ci / mmol ; ge healthcare , perkinelmer , or american radiolabeled chemicals ) was 0.2 nm , and the incubation time was 90 min at 23 c . nonspecific binding was defined in the presence of 1 m ( + ) -butaclamol ( sigma - aldrich ) . ki values were calculated using the experimentally determined kd value for [ h]spiperone of 0.4 nm . [ h ] sch 23390 binding assay for rat d1-like dopamine receptors was performed as described for [ h]spiperone binding except the concentration of [ h]sch 23390 ( 60 ci / mmol ; american radiolabeled chemicals ) was 0.3 nm . ki values were calculated using the kd value for [ h]sch 23390 of 0.3 nm . hek293 cells stably expressing human dopamine d2 and d3 receptors were grown in a 1:1 mixture of dmem and ham s f12 culture media , supplemented with 20 mm hepes , 2 mm l - glutamine , 0.1 mm nonessential amino acids , 1 antibiotic / antimycotic , 10% heat - inactivated fetal bovine serum , and 200 g / ml hygromycin ( life technologies , grand island , ny ) and kept in an incubator at 37 c and 5% co2 . upon reaching 8090% confluence , cells were harvested using premixed earle s balanced salt solution ( ebss ) with 5 m edta ( life technologies ) and centrifuged at 3000 rpm for 10 min at 21 c . the supernatant was removed , and the pellet was resuspended in 10 ml of hypotonic lysis buffer ( 5 mm mgcl26h2o , 5 mm tris , ph 7.4 at 4 c ) and centrifuged at 20000 rpm for 30 min at 4 c . / l earle s balanced salts without phenol red ( us biological , salem , ma ) , 2.2 g / l sodium bicarbonate , ph to 7.4 . a bradford protein assay ( bio - rad , hercules , ca ) was used to determine the protein concentration , and membranes were diluted to 500 g / ml and stored in a 80 c freezer for later use . immediately prior to testing , all test compounds were freshly dissolved in 30% dmso and 70% h2o to a stock concentration of 100 m . to assist the solubilization of free - base compounds , 10 l of glacial acetic acid was added along with the dmso . each test compound was then diluted into 13 half - log serial dilutions using 30% dmso vehicle ; final test concentrations ranged from 10 m to 10 pm . previously frozen membranes were diluted in fresh ebss to a 100 g / ml stock for binding . radioligand competition experiments were conducted in glass tubes containing 300 l of fresh ebss buffer with 0.2 mm sodium metabisulfite , 50 l of diluted test compound , 100 l of membranes ( 10 g total protein ) , and 50 l of [ h]n - methylspiperone ( 0.4 nm final concentration ; perkinelmer ) . nonspecific binding was determined using 10 m butaclamol ( sigma - aldrich , st . louis , mo ) , and total binding was determined with 30% dmso vehicle . all compound dilutions were tested in triplicate and the reaction incubated for 1 h at room temperature . the reaction was terminated by filtration through whatman gf / b filters , presoaked for 1 h in 0.5% polyethylenimine , using a brandel r48 filtering manifold ( brandel instruments , gaithersburg , md ) . the filters were washed 3 times with 3 ml of ice - cold ebss buffer and transferred to scintillation vials . then 3 ml of cytoscint liquid scintillation cocktail ( mp biomedicals , solon , oh ) was added and vials were counted using a perkinelmer tri - carb 2910 tr liquid scintillation counter ( waltham , ma ) . ic50 values for each compound were determined from dose response curves , and ki values were calculated using the cheng prusoff equation . these analyses were performed using graphpad prism version 5.00 for windows ( graphpad software , san diego , ca ) . reported ki values were determined from least three independent experiments . functional activity at the d3 receptor was determined using a pathhunter express -arrestin assay kit ( discoverx , fremont , ca ) for human d3 receptors transfected in u20s cells . compounds were screened for agonist and antagonist activity according to the manufacturer s instructions . data were analyzed using sigmaplot 10 and are presented as the mean sem of 46 independent determinations . for the schild analysis , d3 activity was stimulated by pramipexole in the presence of 3 concentrations of ( 1r,2s)-11 and analyzed according to the methods of kenakin .

we report a class of potent and selective dopamine d3 receptor antagonists based upon tranylcypromine . although tranylcypromine has a low affinity for the rat d3 receptor ( ki = 12.8 m ) , our efforts have yielded ( 1r,2s)-11 ( cj-1882 ) , which has ki values of 2.7 and 2.8 nm at the rat and human dopamine d3 receptors , respectively , and displays respective selectivities of > 10000-fold and 223-fold over the rat and human d2 receptors . evaluation in a -arrestin functional assay showed that ( 1r,2s)-11 is a potent and competitive antagonist at the human d3 receptor .

Introduction Results and Discussion Summary Experimental Section

the dopamine-3 ( d3 ) receptor subtype has been identified as an important target for agents currently in clinical use for the treatment of a variety of neurological diseases , including schizophrenia , parkinson s disease , and depression . however , all of the clinically approved drugs targeting the d3 receptor have a very limited selectivity over d2 receptors and other off - targets . considerable effort has been devoted in the past decade to the design of potent and selective d3 ligands . although it was initially challenging to design highly selective d3 ligands , due to the high degree of sequence homology between the d2 and d3 receptors , recent sar studies have demonstrated the feasibility of such a design . for example , upon the basis of pramipexole ( 1 ) , a potent d3 agonist with only modest selectivity over the d2 receptor , we designed and prepared compounds cj-1638 ( 2 ) and cj-1639 ( 3 ) , which are potent and selective d3 agonists ( figure 1 ) . compounds 2 and 3 bind to the d3 receptor , with ki values < 1 nm and display > 1000-fold selectivity over both the d1 and d2 receptors . not only have highly d3 receptor selective ligands been discovered , but the roles of the orthosteric site and a secondary binding pocket have further defined the drug protein interactions responsible for affinity , selectivity , and efficacy . potent and selective d3 antagonists may have a therapeutic potential for the treatment of drug addictions and relate disorders . herein we report the design and sar study of a new class of d3 antagonists . analysis of several classes of known d3 antagonists shows that their structures can be divided into three regions as shown in figure 2 : the headgroup ( in blue ) , the linker ( in black ) , and the tail ( in red ) . we first selected a new head group with these features for the design and development of a new class of d3 antagonists . chemical structures of previous dopamine d3 receptor antagonists . our data showed that tranylcypromine has a weak affinity for the d3 receptor with a ki value = 12.8 m and displays 4-fold selectivity over the d2 receptor ( table 1 ) . although tranylcypromine is a weak d3 ligand and has a very limited selectivity over the d2 receptor , it was used as a starting point for our sar study , which has ultimately yielded a class of potent and selective d3 antagonists . our previous study showed that the propyl substituent on the amine group in pramipexole enhances the binding affinity to the d3 receptors by at least 1 order of magnitude . binding data showed that both ( 1s,2r)-9 and ( 1r,2s)-9 bind to the rat d3 receptor with ki values of 1.2 m and display approximately 80-fold selectivity over the d2 receptor ( table 1 ) . thus , addition of a propyl group to the primary amine in tranylcypromine indeed improves the binding affinity for the d3 receptor , as well as the selectivity over the d2 receptor . our previous study showed that introduction of appropriate linker and tail groups in compounds 2 and 3 onto the amine group in pramipexole significantly enhanced its selectivity for the rat d3 receptor over that for the d2 receptor . compounds ( 1s,2r)-10 and ( 1r,2s)-10 have ki values of 108 and 44 nm to the rat d3 receptor , respectively , representing a 1020-fold improvement over ( 1s,2r)-9 and ( 1r,2s)-9 . however , in contrast to the marked improved selectivity of compound 2 over pramipexole for the d3 receptor over the d2 receptor observed in our previous study , both ( 1s,2r)-10 and ( 1r,2s)-10 only have modest 2030-fold selectivity over the rat d2 receptor . because ( 1s,2r)-10 and ( 1r,2s)-10 do not differ markedly in their binding affinity to the rat d3 receptor or their selectivity over the rat d2 receptor , we first synthesized and evaluated the racemic forms of 1113 . ( )-12 , and ( )-13 have ki values of 4.6 , 19 , and 28 nm , respectively , to the rat d3 receptor . ( )-11 and ( )-13 also display high ( > 10000 times ) selectivity over the rat d2 receptor , neither compound showing measurable binding to the rat d2 receptor at 100 m . however , both ( )-11 and ( )-13 have significant affinity for the rat d1-like receptors with ki values of 2.2 and 1.8 m , respectively . because ( )-11 showed high affinity for the rat d3 receptor , we resolved the stereoisomers , ( 1s,2r)-11 and ( 1r,2s)-11 ) . ( 1r,2s)-11 has a ki value of 2.7 nm to the rat d3 receptor and is > 100 times more potent than ( 1s,2r)-11 ( ki = 457 nm ) . furthermore , ( 1r,2s)-11 has no appreciable binding to the rat d2 receptor at concentrations as high as 300 m and consequently has > 100,000-fold selectivity for the rat d3 receptor over the rat d2 receptor . ( 1r,2s)-11 shows weak binding affinity to the d1-like receptors , with a ki value of 25.8 m , and has > 9000-fold selectivity for the rat d3 receptor over the rat d1-like receptors . we next assessed the binding affinities of ( )-11 , ( )-12 , ( )-13 , and ( 1r,2s)-11 to the human d2 and d3 receptors using transfected cell lines and included several previously reported d2/d3 antagonists as controls . racemic compounds ( )-11 , ( )-12 , ( )-13 , and the pure enantiomer ( 1r,2s)-11 have ki values to the human d3 receptor of 2.61 , 22.1 , 37.9 , and 2.80 nm , respectively . these values are similar to their respective ki values of 4.6 , 19 , 26 , and 2.7 nm to the rat d3 receptor . compounds ( )-11 , ( )-12 , ( )-13 , and ( 1r,2s)-11 have ki values , respectively , of 667 , 923 , 1220 , and 623 nm , to the human d2 receptor . thus , with the exception of ( )-12 , these compounds have higher binding affinities to the human d2 receptor than to the rat d2 receptor . the selectivities of ( )-11 and ( )-13 and the pure enantiomer ( 1r,2s)-11 for the human d3 receptor over the human d2 receptor are 256- , 32- , and 223-fold , respectively . these are lower than the selectivities observed for these compounds for the rat d3 receptor over the rat d2 receptor . the known d3 antagonists n - methylspiperone , eticlopride , raclopride , and butaclamol , all bind to the human d3 receptor with high affinities but show no selectivity between the human d2 and d3 receptors ( table 2 ) . in comparison , pg619 and pg648 , two previously reported selective d3 antagonists , bind to the human d3 receptor with ki values of 6.70 and 1.88 nm , respectively , displaying selectivities of 163- and 397-fold respectively , for the human d3 receptor over the human d2 receptor . to assess the functional activity of ( 1r,2s)-11 at the d3 receptor , we tested it in a discoverx d3 functional assay using the u2os cell line transfected with human dopamine d3 receptor . in this assay , a d3 agonist , such as pramipexole , stimulates -arrestin binding to the d3 receptor , while a d3 antagonist , such as 6 ( bp 897 ) , blocks the association of -arrestin induced by a d3 agonist . ( 1r,2s)-11 has no agonist activity ( ec50 > 100 m ) , but it dose - dependently inhibits the binding of -arrestin to the d3 receptor induced by 100 nm of pramipexole and has an ic50 value of 327 126 nm ( table 3 ) . hence , ( 1r,2s)-11 is a potent d3 antagonist , albeit less potent than 6 . we performed a schild regression analysis for ( 1r,2s)-11 in the human d3 functional assay ( figure 4 ) . in the schild plot ( the inset in figure 4 ) , it can be seen that increasing concentrations of ( 1r,2s)-11 shift the pramipexole dose response curve to the right , consistent with antagonist activity , with a kb value of 20 nm . the slope of the schild plot was 0.83 , close to unity , indicating that ( 1r,2s)-11 is a competitive d3 antagonist . schild analysis of ( 1r,2s)-11 at the human d3 receptor in the discoverx pathhunter express -arrestin assay . ( 1r,2s)-11 was made by a method similar to that used for ( 1s,2r)-11 , except that ( 1s)-()-2,10-camphorsultam was used as a chiral auxiliary . et3n , acetone , 0 c , 2 h , ( ii ) nan3,1 h , ( iii ) 90 c , toluene , 3 h , ( iv ) butoh , reflux , 16 h. ( e ) tfa , dcm , rt , 12 h ; ( f ) propionaldehyde , nabh4 , meoh , rt ; ( g ) n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naprittiamlde , nabh(oac)3 , hoac , dcm , rt , 4 h. starting from tranylcypromine , we have designed and synthesized a class of potent and selective dopamine d3 receptor ligands . the best compound , ( 1r,2s)-11 , had a ki value of 2.7 nm to the rat d3 receptor and displayed a selectivity of > 100000-fold over the rat d2 receptor and > 9000-fold over the rat d1-like receptors . ( 1r,2s)-11 binds to the human d3 receptor with a ki value of 2.8 nm and displays 223-fold selectivity over the human d2 receptor . functional data and schild analysis showed that ( 1r,2s)-11 is a potent and competitive antagonist at the human d3 receptor . ( 1r,2s)-11 ( cj-1882 ) is being further evaluated for its pharmacokinetics , brain bioavailability , and therapeutic potential for the treatment of drug abuse . propionaldehyde ( 82 mg , 1.41 mmol ) was added to a solution of ( 1s,2r)-2-phenylcyclopropanamine ( 188 mg , 1.41 mmol ) in meoh ( 10 ml ) , and the reaction mixture was stirred at room temperature for 2 h. sodium borohydride ( 79 mg , 2.12 mmol ) was then added , and the mixture was stirred at room temperature for 1 h. the reaction was quenched with h2o ( 30 ml ) and extracted with ethyl acetate ( 40 ml ) . propionaldehyde ( 82 mg , 1.41 mmol ) was added to a solution of ( 1r,2s)-2-phenylcyclopropanamine ( 188 mg , 1.41 mmol ) in meoh ( 10 ml ) , and the reaction mixture was stirred at room temperature for 2 h. sodium borohydride ( 79 mg , 2.12 mmol ) was then added , and the mixture was stirred at room temperature for 1 h. the reaction was quenched with h2o ( 30 ml ) and extracted with etoac ( 40 ml ) . n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naphthamide ( 80 mg , 0.28 mmol ) , sodium triacetoxyborohydride ( 90 mg , 0.43 mmol ) , and acoh ( 26 mg , 0.43 mmol ) were added to a solution of ( 1s,2r)-9 ( 50 mg , 0.28 mmol ) in ch2cl2 ( 20 ml ) . n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naphthamide ( 80 mg , 0.28 mmol ) , sodium triacetoxyborohydride ( 90 mg , 0.43 mmol ) , and acoh ( 26 mg , 0.43 mmol ) were added to a solution of ( 1r,2s)-9 ( 50 mg , 0.28 mmol ) in ch2cl2 ( 20 ml ) , and the mixture was stirred at room temperature for 4 h. the reaction was quenched by addition of h2o ( 30 ml ) , and the mixture was extracted with ch2cl2 ( 30 ml 3 ) . n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naphthamide ( 82 mg , 0.29 mmol ) , sodium triacetoxyborohydride ( 91 mg , 0.43 mmol ) , and acoh ( 26 mg , 0.43 mmol ) were added to a solution of ( )-18 ( 60 mg , 0.29 mmol ) in ch2cl2 ( 20 ml ) , and the mixture was stirred at room temperature for 4 h. the reaction was quenched by addition of h2o ( 30 ml ) , and the mixture was extracted with ch2cl2 ( 30 ml 3 ) . n-(cis-3-hydroxy-3-(2-oxoethyl)cyclobutyl)-2-naphthamide ( 191 mg , 0.67 mmol ) , sodium triacetoxyborohydride ( 212 mg , 1.01 mmol ) , and acoh ( 60 mg , 1.01 mmol ) were added to a solution of ( 1s,2r)-17 ( 140 mg , 0.67 mmol ) in ch2cl2 ( 20 ml ) , and the mixture was stirred at room temperature for 4 h. the reaction was quenched by addition of h2o ( 30 ml ) , and the mixture was extracted with ch2cl2 ( 30 ml 3 ) . ( 1r,2s)-11 was prepared in 35% yield in a manner similar to that used for ( 1s,2r)-11 , except that ( 1s)-()-2,10-camphorsultam was used as a chiral auxiliary . the binding affinities of all the synthetic compounds were determined at the d3 , d2 , and d1-like receptors in membranes prepared from the brains of adult , male sprague dawley rats ( pel - freez , rogers , ar ) . the [ h]r-(+)-7-oh - dpat binding assay for the rat d3 dopamine receptors was performed as described . assays were performed using membranes prepared from rat caudate - putamen , which expresses d2 receptors in high density but with very low levels of d3 receptors , and the final membrane homogenate concentration was 1.5 mg oww / ml . the assay buffer was 50 mm tris - hcl , 5 mm kcl , 2 mm mgcl2 , and 2 mm cacl2 , ph 7.4 at 23 c ; the concentration of [ h]spiperone ( 6096 ci / mmol ; ge healthcare , perkinelmer , or american radiolabeled chemicals ) was 0.2 nm , and the incubation time was 90 min at 23 c . hek293 cells stably expressing human dopamine d2 and d3 receptors were grown in a 1:1 mixture of dmem and ham s f12 culture media , supplemented with 20 mm hepes , 2 mm l - glutamine , 0.1 mm nonessential amino acids , 1 antibiotic / antimycotic , 10% heat - inactivated fetal bovine serum , and 200 g / ml hygromycin ( life technologies , grand island , ny ) and kept in an incubator at 37 c and 5% co2 . a bradford protein assay ( bio - rad , hercules , ca ) was used to determine the protein concentration , and membranes were diluted to 500 g / ml and stored in a 80 c freezer for later use . radioligand competition experiments were conducted in glass tubes containing 300 l of fresh ebss buffer with 0.2 mm sodium metabisulfite , 50 l of diluted test compound , 100 l of membranes ( 10 g total protein ) , and 50 l of [ h]n - methylspiperone ( 0.4 nm final concentration ; perkinelmer ) . louis , mo ) , and total binding was determined with 30% dmso vehicle . ic50 values for each compound were determined from dose response curves , and ki values were calculated using the cheng prusoff equation . functional activity at the d3 receptor was determined using a pathhunter express -arrestin assay kit ( discoverx , fremont , ca ) for human d3 receptors transfected in u20s cells . for the schild analysis , d3 activity was stimulated by pramipexole in the presence of 3 concentrations of ( 1r,2s)-11 and analyzed according to the methods of kenakin .

the primary objective of this initial study was to evaluate f - sodium fluoride ( f - naf ) pet / ct scanning for helping to correctly manage patients after spinal fusion surgery ( arthrodesis ) . spinal fusion is a common procedure performed to treat a variety of spine conditions including degenerative disease , spondylolisthesis , and vertebral deformities [ 1 , 2 ] . after spinal fusion , suboptimal outcomes may be caused by infection , hardware loosening , non - union of fused vertebrae , and/or incomplete growth of bone grafts . patients who have recurrent symptoms caused by these complications after spinal fusion surgery undergo standard evaluation by clinical examination and conventional imaging . however , whether a patient requires surgical intervention is difficult to ascertain because ct and/or mri will often show extensive and nonspecific postoperative changes [ 3 , 4 ] . the physiology of f - naf is similar to that of tc - mdp used in traditional bone scanning . however , f - naf pet / ct is significantly faster ( both uptake and acquisition ) , provides superior spatial resolution , and is widely available in the us and europe [ 57 ] . this was a pilot investigation of the ability of f - naf pet / ct to identify spinal sites requiring surgical revision after fusion surgery and therefore help orthopedists stratify patients into surgical or conservative management . validation of the pet / ct results was based on findings on surgery or clinical follow - up of at least 15 months . this study was conducted as a clinical trial and was fully compliant with the approval from the ethics committees at our respective institutions . this trial was a single - cohort prospective study of consecutive patients enrolled at two institutions between april 2009 and august 2011 . the inclusion criteria consisted of identifying patients who had recurrent symptoms after prior spinal fusion surgery . additionally , these patients had had a clinical evaluation , physical examination and ct scan that had failed to identify a specific contributing source of the symptoms , and the course of management remained uncertain . however , if a patient had undergone these examinations , then the inclusion criteria stipulated that these also did not adequately elucidate the course of management . pediatric patients , pregnant women and patients unfit for additional surgery were excluded . included in the study were 22 patients ( 16 women , 6 men ; age range 3680 years ) with recurrent back pain after spinal surgery and an equivocal physical examination and conventional imaging . the patients underwent f - naf pet / ct at me de deus hospital in porto alegre , brazil ( 18 patients ) and stanford university hospital ( 4 patients ) . all f - naf pet / ct scanning was performed at least 4 months after the most recent surgery ( range 496 months after surgery , median 21 months ; table 1 ) and all scanning was performed within 4 months of presentation with recurrent symptoms.table 1key findings in all patientspatienttime since surgery ( months)imaging resultsclinical follow - uppain scorestatistical categorynaf pet findingssuvmaxfusion ctfindings on surgical explorationconservative managementprior to treatmentat 15-month follow - up18abnormal activity around cage and hardware at l4-l513.4subchondral sclerosiscage failure and screw loosening41true positive212increased activity at bone graft18.1linear radiolucency at graftscrew loosening and bone graft fracture42true positivefocus at l4 right screw20.3radiolucency around screw true positive360focus at l3-l4 screw12.1radiolucency around screwl3-l4 screw loosening40true positive412intensely increased activity at left l4 bone graft18.5sclerotic changes in bone graftparavertebral bone graft fracture41true positive536normal6.2 ( at cervical screws)no obvious abnormalities requiring surgeryregional anesthetic block and clinical surveillance ; long - term follow - up without pain40true negative648normal5.8 ( at upper thoracic screws)no obvious abnormalities requiring surgeryregional anesthetic block and clinical surveillance ; long - term follow - up without pain with no intervention40true negative726abnormal activity at bone graft right l5-s122.2heterogeneous bone sclerosispseudoarthrosis ( abnormal mobility ) in bone graft on right42true positive88abnormal activity at bone graft l512.8likely fracture on the right side of graftparavertebral bone graft fracture and loosening of right screw in s140true positiveasymmetric focus at right s1 screw11.2radiolucency around screwtrue positive912increased uptake around cage at l5-s113.3subtle increased sclerosis at cage tippseudoarthrosis in the bone graft ; fixation cage hardware at l5 loose40true positiveabnormal activity at left l5 bone graft14.2bone reabsorption in region around grafttrue positive1011increased uptake between right paravertebral graft and right iliac bone23.5abnormal bone resorptionpoor graft healing and necrosis at right iliac bone causing an abnormal neojoint40true positive1136mild increased uptake at right l4 screw within normal limits6.9completely normal l4 screwregional anesthetic block and clinical surveillance ; good clinical response in long term41true negative1217mild activity in l2 vertebral body within normal limits5.9completely normal l2 vertebral bodyclinical surveillance ; improved symptoms ; without pain at time of report42true negative134increased uptake in left bone graft l4-l516.3heterogeneous bone at l4-l5bone graft biopsy revealed normal bone4n / afalse positive1472increased activity in bone graft at l5-s121.2arthrosis at facet joints l5-s1pseudoarthrosis at l5-s1 graft site41true positive1517borderline abnormal focus at proximal rod on right side of l3 likely within normal limits6.2mild osteolysis at l3regional anesthetic block with good clinical response ; symptoms resolved without further intervention40true negative1696increased uptake in left l5 screw7.4lucency around l5 screwgraft fracture and distal screw loosening40true positiveintensely increased activity at l5-s1 bone graft13.8possible graft fracturetrue positive1713focal increased uptake in proximal cage hardware19.8mild linear bone resorptionposterior approach showed proximal and distal cage loosening41true positive1823focal increased uptake in middle of hardware at c418.7very mild lucency around hardware at c4micromobility and cage loosening consistent with psuedarthrosis at c441true positive198increase uptake in cage at c5-c613.4very mild lucency zone related to bone graft maturation or cage mobilitycage loosening40true positive2024intense focus right side of cage at l5-s120.7signs of loosening at cagefollow - up examination showed worsening pain ; surgeon s impression was hardware loosening because new ct scan showed right bone graft reabsorption and lucency at screw ; patient averse to surgery41true positiveincreased uptake in s1 screws11.5ct with lucency area at s1 screwstrue positive2119normal7.3 at l4-l5 hardwareno obvious abnormalities requiring surgeryclinical surveillance ; follow - up clinical evaluation showed low back pain related to sacroiliitis unrelated to prior surgery42true negative2272intense focus at right l4 screw / bone graft21.1subtle cortical resorption around screw / graft areavertebral body necrosis at l4-l5 with screw loosening40true positivein patients with more than one abnormal focus each lesion is listed separatelygraded : 0 no pain , 1 mild pain , 2 moderate pain , 3 severe pain , 4 severe and debilitating painstatistical category assigned to each lesion . if a patient had at least one true - positive finding on surgery , then the imaging results were considered true positive in the patient - by - patient analysisdeveloped bladder cancer key findings in all patients in patients with more than one abnormal focus each lesion is listed separately graded : 0 no pain , 1 mild pain , 2 moderate pain , 3 severe pain , 4 severe and debilitating pain statistical category assigned to each lesion . if a patient had at least one true - positive finding on surgery , then the imaging results were considered true positive in the patient - by - patient analysis developed bladder cancer the pet / ct system used at me de deus hospital was a phillips gemini tf scanner with a brilliance 16-slice ct scanner and at stanford university hospital was a ge discovery 600 pet / ct system with a 16-slice ct scanner . the ct part of the pet / ct acquisition was designed to be equivalent to a stand - alone diagnostic ct protocol optimized for bone / spine imaging : 1.252 mm slice thickness , 1 mm increment , 100140 kv , 180230 mas , and matrix size 512 512 pixels . the pet scanning protocol included injection of 222370 mbq ( 610 mci ) f - naf followed by 45 min for tracer uptake . pet scanning consisted of 150250 s per bed position acquisition time . the number of bed positions varied from five to eight depending on the amount of spine to be covered . images were reconstructed using the ordered subset expected maximization algorithm with four to eight iterations . a nuclear medicine physician and a radiologist with musculoskeletal expertise reviewed all pet / ct images together with the primary objective of identifying or ruling out the presence of lesions amenable to surgical intervention . the most important feature for identifying an abnormality requiring surgery was focal and well - circumscribed activity clearly above the background spine that coregistered on the ct image with sites typical for hardware failure or pseudoarthrosis ( such as the ends of fixation rods and cages , screw shafts , bone grafts ) . standardized uptake values ( suv ) were used to compare individual foci against background spine activity , but an absolute threshold level for positivity was not used . clinical history and prior ct imaging pet images not corrected for attenuation were analyzed when needed to identify attenuation artifacts caused by the metallic implants . incidental foci such as osteophytes , benign bone lesions , and mild degenerative changes were noted but were not formally tabulated . results of f - naf pet / ct scanning were then communicated to the referring orthopedist and tabulated in detail for analysis and validation ( table 1 ) . based primarily on f - naf pet / ct imaging , the patient then underwent either surgical exploration ( with possible intervention ) , or conservative management . surgical exploration consisted of the orthopedic surgeon probing and manually testing the exact region for loosening and hardware failure at sites of abnormal tracer activity . further , extracted hardware and bone tissue underwent histopathological analysis for evidence of infection or bone necrosis . conservative management included : regional anesthetic nerve blockade ( to provide palliative short - term relief ) , physical therapy , medication , and bed rest . pet / ct abnormalities were considered true positive if they were confirmed on surgery or if by the 15-month clinical follow - up other adjunctive examinations and data were wholly consistent with a surgically relevant lesion . pet / ct foci were considered false positive if no operable abnormality was found on surgery or if symptoms improved without surgical intervention . negative pet / ct scans were considered true negative if symptoms improved with nonsurgical management and/or were corroborated as stable or resolved on adjunctive imaging examinations such as ct and mri . negative pet / ct scans were considered false negative if surgical intervention was ultimately required . the intensity of the patient s pain was graded from 0 to 4 prior to initiation of treatment and at the 15-month follow - up as follows : 0 no pain , 1 mild pain , 2 moderate pain , 3 severe pain , 4 severe and debilitating pain . a standard 2 2 contingency table was used to calculate the sensitivity and specificity on a patient - by - patient basis . findings on surgery or the 15-month clinical follow - up were utilized as the gold standard . this study was conducted as a clinical trial and was fully compliant with the approval from the ethics committees at our respective institutions . this trial was a single - cohort prospective study of consecutive patients enrolled at two institutions between april 2009 and august 2011 . the inclusion criteria consisted of identifying patients who had recurrent symptoms after prior spinal fusion surgery . additionally , these patients had had a clinical evaluation , physical examination and ct scan that had failed to identify a specific contributing source of the symptoms , and the course of management remained uncertain . however , if a patient had undergone these examinations , then the inclusion criteria stipulated that these also did not adequately elucidate the course of management . pediatric patients , pregnant women and patients unfit for additional surgery were excluded . included in the study were 22 patients ( 16 women , 6 men ; age range 3680 years ) with recurrent back pain after spinal surgery and an equivocal physical examination and conventional imaging . the patients underwent f - naf pet / ct at me de deus hospital in porto alegre , brazil ( 18 patients ) and stanford university hospital ( 4 patients ) . all f - naf pet / ct scanning was performed at least 4 months after the most recent surgery ( range 496 months after surgery , median 21 months ; table 1 ) and all scanning was performed within 4 months of presentation with recurrent symptoms.table 1key findings in all patientspatienttime since surgery ( months)imaging resultsclinical follow - uppain scorestatistical categorynaf pet findingssuvmaxfusion ctfindings on surgical explorationconservative managementprior to treatmentat 15-month follow - up18abnormal activity around cage and hardware at l4-l513.4subchondral sclerosiscage failure and screw loosening41true positive212increased activity at bone graft18.1linear radiolucency at graftscrew loosening and bone graft fracture42true positivefocus at l4 right screw20.3radiolucency around screw true positive360focus at l3-l4 screw12.1radiolucency around screwl3-l4 screw loosening40true positive412intensely increased activity at left l4 bone graft18.5sclerotic changes in bone graftparavertebral bone graft fracture41true positive536normal6.2 ( at cervical screws)no obvious abnormalities requiring surgeryregional anesthetic block and clinical surveillance ; long - term follow - up without pain40true negative648normal5.8 ( at upper thoracic screws)no obvious abnormalities requiring surgeryregional anesthetic block and clinical surveillance ; long - term follow - up without pain with no intervention40true negative726abnormal activity at bone graft right l5-s122.2heterogeneous bone sclerosispseudoarthrosis ( abnormal mobility ) in bone graft on right42true positive88abnormal activity at bone graft l512.8likely fracture on the right side of graftparavertebral bone graft fracture and loosening of right screw in s140true positiveasymmetric focus at right s1 screw11.2radiolucency around screwtrue positive912increased uptake around cage at l5-s113.3subtle increased sclerosis at cage tippseudoarthrosis in the bone graft ; fixation cage hardware at l5 loose40true positiveabnormal activity at left l5 bone graft14.2bone reabsorption in region around grafttrue positive1011increased uptake between right paravertebral graft and right iliac bone23.5abnormal bone resorptionpoor graft healing and necrosis at right iliac bone causing an abnormal neojoint40true positive1136mild increased uptake at right l4 screw within normal limits6.9completely normal l4 screwregional anesthetic block and clinical surveillance ; good clinical response in long term41true negative1217mild activity in l2 vertebral body within normal limits5.9completely normal l2 vertebral bodyclinical surveillance ; improved symptoms ; without pain at time of report42true negative134increased uptake in left bone graft l4-l516.3heterogeneous bone at l4-l5bone graft biopsy revealed normal bone4n / afalse positive1472increased activity in bone graft at l5-s121.2arthrosis at facet joints l5-s1pseudoarthrosis at l5-s1 graft site41true positive1517borderline abnormal focus at proximal rod on right side of l3 likely within normal limits6.2mild osteolysis at l3regional anesthetic block with good clinical response ; symptoms resolved without further intervention40true negative1696increased uptake in left l5 screw7.4lucency around l5 screwgraft fracture and distal screw loosening40true positiveintensely increased activity at l5-s1 bone graft13.8possible graft fracturetrue positive1713focal increased uptake in proximal cage hardware19.8mild linear bone resorptionposterior approach showed proximal and distal cage loosening41true positive1823focal increased uptake in middle of hardware at c418.7very mild lucency around hardware at c4micromobility and cage loosening consistent with psuedarthrosis at c441true positive198increase uptake in cage at c5-c613.4very mild lucency zone related to bone graft maturation or cage mobilitycage loosening40true positive2024intense focus right side of cage at l5-s120.7signs of loosening at cagefollow - up examination showed worsening pain ; surgeon s impression was hardware loosening because new ct scan showed right bone graft reabsorption and lucency at screw ; patient averse to surgery41true positiveincreased uptake in s1 screws11.5ct with lucency area at s1 screwstrue positive2119normal7.3 at l4-l5 hardwareno obvious abnormalities requiring surgeryclinical surveillance ; follow - up clinical evaluation showed low back pain related to sacroiliitis unrelated to prior surgery42true negative2272intense focus at right l4 screw / bone graft21.1subtle cortical resorption around screw / graft areavertebral body necrosis at l4-l5 with screw loosening40true positivein patients with more than one abnormal focus each lesion is listed separatelygraded : 0 no pain , 1 mild pain , 2 moderate pain , 3 severe pain , 4 severe and debilitating painstatistical category assigned to each lesion . if a patient had at least one true - positive finding on surgery , then the imaging results were considered true positive in the patient - by - patient analysisdeveloped bladder cancer key findings in all patients in patients with more than one abnormal focus each lesion is listed separately graded : 0 no pain , 1 mild pain , 2 moderate pain , 3 severe pain , 4 severe and debilitating pain statistical category assigned to each lesion . if a patient had at least one true - positive finding on surgery , then the imaging results were considered true positive in the patient - by - patient analysis developed bladder cancer the pet / ct system used at me de deus hospital was a phillips gemini tf scanner with a brilliance 16-slice ct scanner and at stanford university hospital was a ge discovery 600 pet / ct system with a 16-slice ct scanner . the ct part of the pet / ct acquisition was designed to be equivalent to a stand - alone diagnostic ct protocol optimized for bone / spine imaging : 1.252 mm slice thickness , 1 mm increment , 100140 kv , 180230 mas , and matrix size 512 512 pixels . the pet scanning protocol included injection of 222370 mbq ( 610 mci ) f - naf followed by 45 min for tracer uptake . pet scanning consisted of 150250 s per bed position acquisition time . the number of bed positions varied from five to eight depending on the amount of spine to be covered . images were reconstructed using the ordered subset expected maximization algorithm with four to eight iterations . a nuclear medicine physician and a radiologist with musculoskeletal expertise reviewed all pet / ct images together with the primary objective of identifying or ruling out the presence of lesions amenable to surgical intervention . the most important feature for identifying an abnormality requiring surgery was focal and well - circumscribed activity clearly above the background spine that coregistered on the ct image with sites typical for hardware failure or pseudoarthrosis ( such as the ends of fixation rods and cages , screw shafts , bone grafts ) . foci without any significant ct abnormality were downgraded in suspicion . standardized uptake values ( suv ) were used to compare individual foci against background spine activity , but an absolute threshold level for positivity was not used . clinical history and prior ct imaging pet images not corrected for attenuation were analyzed when needed to identify attenuation artifacts caused by the metallic implants . incidental foci such as osteophytes , benign bone lesions , and mild degenerative changes were noted but were not formally tabulated . results of f - naf pet / ct scanning were then communicated to the referring orthopedist and tabulated in detail for analysis and validation ( table 1 ) . based primarily on f - naf pet / ct imaging , the patient then underwent either surgical exploration ( with possible intervention ) , or conservative management . surgical exploration consisted of the orthopedic surgeon probing and manually testing the exact region for loosening and hardware failure at sites of abnormal tracer activity . further , extracted hardware and bone tissue underwent histopathological analysis for evidence of infection or bone necrosis . conservative management included : regional anesthetic nerve blockade ( to provide palliative short - term relief ) , physical therapy , medication , and bed rest . pet / ct abnormalities were considered true positive if they were confirmed on surgery or if by the 15-month clinical follow - up other adjunctive examinations and data were wholly consistent with a surgically relevant lesion . pet / ct foci were considered false positive if no operable abnormality was found on surgery or if symptoms improved without surgical intervention . negative pet / ct scans were considered true negative if symptoms improved with nonsurgical management and/or were corroborated as stable or resolved on adjunctive imaging examinations such as ct and mri . negative pet / ct scans were considered false negative if surgical intervention was ultimately required . the intensity of the patient s pain was graded from 0 to 4 prior to initiation of treatment and at the 15-month follow - up as follows : 0 no pain , 1 mild pain , 2 moderate pain , 3 severe pain , 4 severe and debilitating pain . a standard 2 2 contingency table was used to calculate the sensitivity and specificity on a patient - by - patient basis . findings on surgery or the 15-month clinical follow - up were utilized as the gold standard . in total , 116 patients were evaluated for recurrent pain during the study period . of these , standard clinical evaluation and imaging produced an adequate management plan in 64 patients ( 55 % ) while 52 patients ( 45 % ) did not have conclusive results and met the inclusion criteria for f - naf pet / ct . overall , 22 patients were enrolled and consented to the study while the remaining 30 did not consent to imaging for a variety of reasons including fear of extra radiation , schedule conflict , and/or additional time commitment . some patients had very long - lasting relief from their original surgery while others presented soon after surgery with new or recurrent symptoms . also not unexpectedly , the symptomatology may have differed at the initial spinal fusion and at the time of the follow - up pet / ct examination including the exact location and/or the descriptive quality of the complaint . all patients had prior multilevel hardware placement exhibiting varying degrees of abnormal anatomical findings where postsurgical healing versus pathological findings could not be clearly differentiated on ct alone . the interpreting physicians found that 16 of the 22 patients had an f - naf pet / ct scan that had at least one abnormality amenable to surgical intervention ( figs . 1 and 2 ) . the abnormal foci were located at the following sites : screws ( six ) , cages / rods / fixation hardware ( six ) , and bone grafts ( nine ) ( table 1 ) . the maximum suv ( suvmax ) of these foci ranged from 7.4 to 23.5 ( suvmax of all foci : average 15.9 , median 14.2 ) . the background suvmax was recorded from the closest comparable vertebral structure without a lesion and ranged from 4.4 to 5.9 ( average 5.2 , median 5.4).fig . 1pet / ct images in patient 16 a intense and asymmetric f - naf activity is apparent in the left l5 screw ( yellow arrow , suv 7.4 ) . relatively normal tracer activity is apparent in the other hardware ( blue arrows , suv 5.25.5 ) . b zoomed ct image of the left l5 screw demonstrates possible radiolucency around the screw ( yellow arrows)fig . a anterior view 3-d fusion pet / ct maximal intensity projection image demonstrates intensely abnormal activity at the right side of l5-s1 ( yellow arrow ) that was confirmed to be bone graft instability requiring surgical revision . b pet , pet / ct fusion , and ct images show markedly asymmetric activity ( yellow arrows ) at the right l5-s1 lamina and facet which is the site of a prior bone graft . relatively normal activity is apparent within the bone graft located on the contralateral side ( blue arrows ) . increased sclerosis is apparent on the ct image ( white arrow ) at the right l5-s1 graft compared to the left that was initially described as postoperative changes ( and difficult to differentiate from graft failure ) on the initial ct scan pet / ct images in patient 16 a intense and asymmetric f - naf activity is apparent in the left l5 screw ( yellow arrow , suv 7.4 ) . relatively normal tracer activity is apparent in the other hardware ( blue arrows , suv 5.25.5 ) . b zoomed ct image of the left l5 screw demonstrates possible radiolucency around the screw ( yellow arrows ) pet / ct images in patient 7 . a anterior view 3-d fusion pet / ct maximal intensity projection image demonstrates intensely abnormal activity at the right side of l5-s1 ( yellow arrow ) that was confirmed to be bone graft instability requiring surgical revision . b pet , pet / ct fusion , and ct images show markedly asymmetric activity ( yellow arrows ) at the right l5-s1 lamina and facet which is the site of a prior bone graft . relatively normal activity is apparent within the bone graft located on the contralateral side ( blue arrows ) . increased sclerosis is apparent on the ct image ( white arrow ) at the right l5-s1 graft compared to the left that was initially described as postoperative changes ( and difficult to differentiate from graft failure ) on the initial ct scan of the 16 patients with abnormal pet / ct results , 15 went on to surgical exploration . the orthopedist found abnormalities in 14 of 15 of these patients that correlated precisely with the pet / ct findings and led to repair of the hardware or spine . in one patient ( patient 13 , table 1 ) a biopsy of a bone graft revealed normal bone growth and the pet / ct result was therefore false positive ( table 1 ) . one patient ( patient 20 , table 1 ) with pet / ct abnormalities was not receptive to surgical intervention and was therefore followed clinically rather than subjected to surgery ( table 1 ) . a repeat ct scan at 15 months revealed a graft fracture and hardware loosening and the pet / ct result was therefore true positive . of the 22 patients , 6 did not have any foci on f - naf pet / ct that appeared amenable to surgery and conservative treatment was recommended . in these normal - appearing patients , the suvmax was recorded from skeletal sites that had undergone surgery , and the values ranged from 5.8 to 7.3 ( suvmax : average 6.4 , median 6.2 ) . the background suvmax values obtained from comparable spinal structures external to the surgical sites ranged from 5.2 to 6.6 ( average 5.6 , median 5.4 ) . in all six patients , pain had improved without surgery at the 15-month follow - up and the imaging results were scored as true negative ( table 1 ) . on a patient - by - patient basis , the sensitivity and specificity of the ability of f - naf pet / ct to identify the presence of an abnormality requiring surgical intervention were 100 % ( 95 % ci 78.2100 % ) and 85.7 % ( 95 % ci 42.199.6 % ) , respectively . on a lesion - by - lesion basis , 20 of the 21 abnormal foci ( in 16 subjects ) identified by f - naf pet / ct were confirmed as true positive while 1 of 20 was false positive . f - naf pet / ct imaging appeared to provide accurate adjunctive information to the standard work - up . specifically , 14 of 15 patients who underwent surgical exploration based on pet / ct results were confirmed to have lesions requiring surgical intervention by either histopathology or direct manual probing . overall , 15 of 16 patients were correctly identified by pet / ct as having a lesion requiring surgery after initial evaluation was equivocal regarding the need for surgery . conversely , all of the six patients with pet / ct results indicating that surgery was not required were found after 15 months of follow - up to have improved ( by clinical assessment and reported pain ) . it should be noted that the timing of postoperative inflammation and its effect on f - naf pet / ct can not be deduced from this project . the single false - positive result in this study occurred in a patient ( patient 13 , table 1 ) who underwent scanning just 4 months after spinal fusion . nevertheless , most of the postoperative spine in all patients showed normal f - naf activity compared to background vertebrae despite extensive hardware placement , suggesting that when given enough time , the tracer has relatively little nonspecific uptake at sites of prior surgery ( figs . 1 , 2 and 3).fig . 3comparison of patient 5 and patient 17 . a patient 5 : oblique 3-d pet / ct fusion maximal intensity projection image , axial noncontrast ct image , and axial coregistered pet image show metallic spinal fixation hardware in the cervical neck ( yellow arrows ) . b patient 17 : oblique 3-d pet / ct fusion maximal intensity projection image , axial noncontrast ct , and axial coregistered pet image show metallic hardware in the cervical neck . an abnormally intense focus is apparent in the proximal spinal fixation cage ( red arrows ) , and very subtle radiolucency around the metallic fixation cage ( blue arrows ) is apparent on the ct image . the patient was referred for surgical exploration and hardware loosening and abnormal spinal mobility was confirmed in this region comparison of patient 5 and patient 17 . a patient 5 : oblique 3-d pet / ct fusion maximal intensity projection image , axial noncontrast ct image , and axial coregistered pet image show metallic spinal fixation hardware in the cervical neck ( yellow arrows ) . b patient 17 : oblique 3-d pet / ct fusion maximal intensity projection image , axial noncontrast ct , and axial coregistered pet image show metallic hardware in the cervical neck . an abnormally intense focus is apparent in the proximal spinal fixation cage ( red arrows ) , and very subtle radiolucency around the metallic fixation cage ( blue arrows ) is apparent on the ct image . the patient was referred for surgical exploration and hardware loosening and abnormal spinal mobility was confirmed in this region our results substantiate and expand on those of a recent study by fischer et al . that showed the potential of f - naf pet / ct for characterizing orthopedic hardware incorporation . however , this prior study lacked outcome data and abnormalities found on f - naf pet / ct were described without follow - up information to validate the imaging results . the literature on the use of f - naf pet / ct for evaluating the postoperative spine is otherwise very scant . the use of spect tracers such as tc - mdp has been described more extensively , although very few studies included integrated spect / ct which would be more comparable to pet / ct . a contemporary study by damgaard et al . using tc - mdp spect / ct suggested possible utility for detecting lack of fusion of metallic implants , but this study suffered from the smallness of the cohort which comprised only nine subjects and a retrospective design . a review by scharf in 2010 also noted the potential for integrated spect / ct in a variety of conditions including evaluation of spinal fusion . however , this was not a formal scientific evaluation of data but merely a descriptive report of two case studies . studies prior to 2000 investigated the utility of tc - mdp spect ( without integrated ct ) more comprehensively [ 11 , 12 ] . a 1987 study by slizofski et al . prospectively evaluated 15 symptomatic patients following lumbar fusion and found spect to have a sensitivity and specificity of 78 % and 83 % , respectively , for identifying offending osseous sites of recurrent pain . a larger study by gates and mcdonald showed similar results in 63 patients following lumbar surgery . while a statistical analysis was not presented , the authors concluded that spect imaging is particularly useful for excluding bony causes of recurrent back pain while positive lesions are helpful for identifying causative abnormalities such as facet arthropathy and pseudoarthrosis . in both studies ( slizofski et al . and gates and mcdonald ) , validation of spect results utilized findings at surgery as well as other imaging . however , the added value of spect versus existing conventional imaging , such as ct , plain radiography or mri , was not directly addressed , and it is not clear whether spect would aid treatment decisions beyond conventional work - up . there are no studies that have directly compared f - naf pet / ct with tc - mdp spect or spect / ct in the same cohort of patients although , as stated in the introduction , f - naf pet / ct is superior to tc - mdp spect / ct in terms of speed and image quality . the pilot cohort investigated in our project was small and our high reported sensitivity and specificity should be viewed with caution . nevertheless , the data and image quality appear promising and a larger clinical evaluation is warranted , including possibly comparing the outcomes of two management approaches in different cohorts , ct and one / ct and one without . in this prospective pilot investigation of 22 patients , f - naf pet / ct imaging demonstrated potential for aiding management of patients with recurrent pain after spinal fusion surgery by helping to correctly identify those requiring surgical intervention . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

purposea pilot study was performed in patients with recurrent back pain after spinal fusion surgery to evaluate the ability of 18f - naf pet / ct imaging to correctly identify those requiring surgical intervention and to locate a site amenable to surgical intervention.methodsin this prospective study 22 patients with recurrent back pain after spinal surgery and with equivocal findings on physical examination and ct were enrolled for evaluation with 18f - naf pet / ct . all pet / ct images were prospectively reviewed with the primary objective of identifying or ruling out the presence of lesions amenable to surgical intervention . the pet / ct results were then validated during surgical exploration or clinical follow - up of at least 15 months.resultsabnormal 18f - naf foci were found in 16 of the 22 patients , and surgical intervention was recommended . these foci were located at various sites : screws , cages , rods , fixation hardware , and bone grafts . in 6 of the 22 patients no foci requiring surgical intervention were found . validation of the results by surgery ( 15 patients ) or on clinical follow - up ( 7 patients ) showed that 18f - naf pet / ct correctly predicted the presence of an abnormality requiring surgical intervention in 15 of 16 patients and was falsely positive in 1 of 16.conclusionin this initial investigation , 18f - naf pet / ct imaging showed potential utility for evaluation of recurrent symptoms after spinal fusion surgery by identifying those patients requiring surgical management .

Introduction Materials and methods Patients Scanning Interpretation Clinical management and outcome measurement Statistics Results Discussion Conclusion Conflicts of interest Open Access

the primary objective of this initial study was to evaluate f - sodium fluoride ( f - naf ) pet / ct scanning for helping to correctly manage patients after spinal fusion surgery ( arthrodesis ) . however , f - naf pet / ct is significantly faster ( both uptake and acquisition ) , provides superior spatial resolution , and is widely available in the us and europe [ 57 ] . this was a pilot investigation of the ability of f - naf pet / ct to identify spinal sites requiring surgical revision after fusion surgery and therefore help orthopedists stratify patients into surgical or conservative management . validation of the pet / ct results was based on findings on surgery or clinical follow - up of at least 15 months . the inclusion criteria consisted of identifying patients who had recurrent symptoms after prior spinal fusion surgery . additionally , these patients had had a clinical evaluation , physical examination and ct scan that had failed to identify a specific contributing source of the symptoms , and the course of management remained uncertain . included in the study were 22 patients ( 16 women , 6 men ; age range 3680 years ) with recurrent back pain after spinal surgery and an equivocal physical examination and conventional imaging . all f - naf pet / ct scanning was performed at least 4 months after the most recent surgery ( range 496 months after surgery , median 21 months ; table 1 ) and all scanning was performed within 4 months of presentation with recurrent symptoms.table 1key findings in all patientspatienttime since surgery ( months)imaging resultsclinical follow - uppain scorestatistical categorynaf pet findingssuvmaxfusion ctfindings on surgical explorationconservative managementprior to treatmentat 15-month follow - up18abnormal activity around cage and hardware at l4-l513.4subchondral sclerosiscage failure and screw loosening41true positive212increased activity at bone graft18.1linear radiolucency at graftscrew loosening and bone graft fracture42true positivefocus at l4 right screw20.3radiolucency around screw true positive360focus at l3-l4 screw12.1radiolucency around screwl3-l4 screw loosening40true positive412intensely increased activity at left l4 bone graft18.5sclerotic changes in bone graftparavertebral bone graft fracture41true positive536normal6.2 ( at cervical screws)no obvious abnormalities requiring surgeryregional anesthetic block and clinical surveillance ; long - term follow - up without pain40true negative648normal5.8 ( at upper thoracic screws)no obvious abnormalities requiring surgeryregional anesthetic block and clinical surveillance ; long - term follow - up without pain with no intervention40true negative726abnormal activity at bone graft right l5-s122.2heterogeneous bone sclerosispseudoarthrosis ( abnormal mobility ) in bone graft on right42true positive88abnormal activity at bone graft l512.8likely fracture on the right side of graftparavertebral bone graft fracture and loosening of right screw in s140true positiveasymmetric focus at right s1 screw11.2radiolucency around screwtrue positive912increased uptake around cage at l5-s113.3subtle increased sclerosis at cage tippseudoarthrosis in the bone graft ; fixation cage hardware at l5 loose40true positiveabnormal activity at left l5 bone graft14.2bone reabsorption in region around grafttrue positive1011increased uptake between right paravertebral graft and right iliac bone23.5abnormal bone resorptionpoor graft healing and necrosis at right iliac bone causing an abnormal neojoint40true positive1136mild increased uptake at right l4 screw within normal limits6.9completely normal l4 screwregional anesthetic block and clinical surveillance ; good clinical response in long term41true negative1217mild activity in l2 vertebral body within normal limits5.9completely normal l2 vertebral bodyclinical surveillance ; improved symptoms ; without pain at time of report42true negative134increased uptake in left bone graft l4-l516.3heterogeneous bone at l4-l5bone graft biopsy revealed normal bone4n / afalse positive1472increased activity in bone graft at l5-s121.2arthrosis at facet joints l5-s1pseudoarthrosis at l5-s1 graft site41true positive1517borderline abnormal focus at proximal rod on right side of l3 likely within normal limits6.2mild osteolysis at l3regional anesthetic block with good clinical response ; symptoms resolved without further intervention40true negative1696increased uptake in left l5 screw7.4lucency around l5 screwgraft fracture and distal screw loosening40true positiveintensely increased activity at l5-s1 bone graft13.8possible graft fracturetrue positive1713focal increased uptake in proximal cage hardware19.8mild linear bone resorptionposterior approach showed proximal and distal cage loosening41true positive1823focal increased uptake in middle of hardware at c418.7very mild lucency around hardware at c4micromobility and cage loosening consistent with psuedarthrosis at c441true positive198increase uptake in cage at c5-c613.4very mild lucency zone related to bone graft maturation or cage mobilitycage loosening40true positive2024intense focus right side of cage at l5-s120.7signs of loosening at cagefollow - up examination showed worsening pain ; surgeon s impression was hardware loosening because new ct scan showed right bone graft reabsorption and lucency at screw ; patient averse to surgery41true positiveincreased uptake in s1 screws11.5ct with lucency area at s1 screwstrue positive2119normal7.3 at l4-l5 hardwareno obvious abnormalities requiring surgeryclinical surveillance ; follow - up clinical evaluation showed low back pain related to sacroiliitis unrelated to prior surgery42true negative2272intense focus at right l4 screw / bone graft21.1subtle cortical resorption around screw / graft areavertebral body necrosis at l4-l5 with screw loosening40true positivein patients with more than one abnormal focus each lesion is listed separatelygraded : 0 no pain , 1 mild pain , 2 moderate pain , 3 severe pain , 4 severe and debilitating painstatistical category assigned to each lesion . if a patient had at least one true - positive finding on surgery , then the imaging results were considered true positive in the patient - by - patient analysisdeveloped bladder cancer key findings in all patients in patients with more than one abnormal focus each lesion is listed separately graded : 0 no pain , 1 mild pain , 2 moderate pain , 3 severe pain , 4 severe and debilitating pain statistical category assigned to each lesion . if a patient had at least one true - positive finding on surgery , then the imaging results were considered true positive in the patient - by - patient analysis developed bladder cancer the pet / ct system used at me de deus hospital was a phillips gemini tf scanner with a brilliance 16-slice ct scanner and at stanford university hospital was a ge discovery 600 pet / ct system with a 16-slice ct scanner . the ct part of the pet / ct acquisition was designed to be equivalent to a stand - alone diagnostic ct protocol optimized for bone / spine imaging : 1.252 mm slice thickness , 1 mm increment , 100140 kv , 180230 mas , and matrix size 512 512 pixels . a nuclear medicine physician and a radiologist with musculoskeletal expertise reviewed all pet / ct images together with the primary objective of identifying or ruling out the presence of lesions amenable to surgical intervention . results of f - naf pet / ct scanning were then communicated to the referring orthopedist and tabulated in detail for analysis and validation ( table 1 ) . based primarily on f - naf pet / ct imaging , the patient then underwent either surgical exploration ( with possible intervention ) , or conservative management . pet / ct abnormalities were considered true positive if they were confirmed on surgery or if by the 15-month clinical follow - up other adjunctive examinations and data were wholly consistent with a surgically relevant lesion . additionally , these patients had had a clinical evaluation , physical examination and ct scan that had failed to identify a specific contributing source of the symptoms , and the course of management remained uncertain . included in the study were 22 patients ( 16 women , 6 men ; age range 3680 years ) with recurrent back pain after spinal surgery and an equivocal physical examination and conventional imaging . the patients underwent f - naf pet / ct at me de deus hospital in porto alegre , brazil ( 18 patients ) and stanford university hospital ( 4 patients ) . all f - naf pet / ct scanning was performed at least 4 months after the most recent surgery ( range 496 months after surgery , median 21 months ; table 1 ) and all scanning was performed within 4 months of presentation with recurrent symptoms.table 1key findings in all patientspatienttime since surgery ( months)imaging resultsclinical follow - uppain scorestatistical categorynaf pet findingssuvmaxfusion ctfindings on surgical explorationconservative managementprior to treatmentat 15-month follow - up18abnormal activity around cage and hardware at l4-l513.4subchondral sclerosiscage failure and screw loosening41true positive212increased activity at bone graft18.1linear radiolucency at graftscrew loosening and bone graft fracture42true positivefocus at l4 right screw20.3radiolucency around screw true positive360focus at l3-l4 screw12.1radiolucency around screwl3-l4 screw loosening40true positive412intensely increased activity at left l4 bone graft18.5sclerotic changes in bone graftparavertebral bone graft fracture41true positive536normal6.2 ( at cervical screws)no obvious abnormalities requiring surgeryregional anesthetic block and clinical surveillance ; long - term follow - up without pain40true negative648normal5.8 ( at upper thoracic screws)no obvious abnormalities requiring surgeryregional anesthetic block and clinical surveillance ; long - term follow - up without pain with no intervention40true negative726abnormal activity at bone graft right l5-s122.2heterogeneous bone sclerosispseudoarthrosis ( abnormal mobility ) in bone graft on right42true positive88abnormal activity at bone graft l512.8likely fracture on the right side of graftparavertebral bone graft fracture and loosening of right screw in s140true positiveasymmetric focus at right s1 screw11.2radiolucency around screwtrue positive912increased uptake around cage at l5-s113.3subtle increased sclerosis at cage tippseudoarthrosis in the bone graft ; fixation cage hardware at l5 loose40true positiveabnormal activity at left l5 bone graft14.2bone reabsorption in region around grafttrue positive1011increased uptake between right paravertebral graft and right iliac bone23.5abnormal bone resorptionpoor graft healing and necrosis at right iliac bone causing an abnormal neojoint40true positive1136mild increased uptake at right l4 screw within normal limits6.9completely normal l4 screwregional anesthetic block and clinical surveillance ; good clinical response in long term41true negative1217mild activity in l2 vertebral body within normal limits5.9completely normal l2 vertebral bodyclinical surveillance ; improved symptoms ; without pain at time of report42true negative134increased uptake in left bone graft l4-l516.3heterogeneous bone at l4-l5bone graft biopsy revealed normal bone4n / afalse positive1472increased activity in bone graft at l5-s121.2arthrosis at facet joints l5-s1pseudoarthrosis at l5-s1 graft site41true positive1517borderline abnormal focus at proximal rod on right side of l3 likely within normal limits6.2mild osteolysis at l3regional anesthetic block with good clinical response ; symptoms resolved without further intervention40true negative1696increased uptake in left l5 screw7.4lucency around l5 screwgraft fracture and distal screw loosening40true positiveintensely increased activity at l5-s1 bone graft13.8possible graft fracturetrue positive1713focal increased uptake in proximal cage hardware19.8mild linear bone resorptionposterior approach showed proximal and distal cage loosening41true positive1823focal increased uptake in middle of hardware at c418.7very mild lucency around hardware at c4micromobility and cage loosening consistent with psuedarthrosis at c441true positive198increase uptake in cage at c5-c613.4very mild lucency zone related to bone graft maturation or cage mobilitycage loosening40true positive2024intense focus right side of cage at l5-s120.7signs of loosening at cagefollow - up examination showed worsening pain ; surgeon s impression was hardware loosening because new ct scan showed right bone graft reabsorption and lucency at screw ; patient averse to surgery41true positiveincreased uptake in s1 screws11.5ct with lucency area at s1 screwstrue positive2119normal7.3 at l4-l5 hardwareno obvious abnormalities requiring surgeryclinical surveillance ; follow - up clinical evaluation showed low back pain related to sacroiliitis unrelated to prior surgery42true negative2272intense focus at right l4 screw / bone graft21.1subtle cortical resorption around screw / graft areavertebral body necrosis at l4-l5 with screw loosening40true positivein patients with more than one abnormal focus each lesion is listed separatelygraded : 0 no pain , 1 mild pain , 2 moderate pain , 3 severe pain , 4 severe and debilitating painstatistical category assigned to each lesion . if a patient had at least one true - positive finding on surgery , then the imaging results were considered true positive in the patient - by - patient analysisdeveloped bladder cancer key findings in all patients in patients with more than one abnormal focus each lesion is listed separately graded : 0 no pain , 1 mild pain , 2 moderate pain , 3 severe pain , 4 severe and debilitating pain statistical category assigned to each lesion . if a patient had at least one true - positive finding on surgery , then the imaging results were considered true positive in the patient - by - patient analysis developed bladder cancer the pet / ct system used at me de deus hospital was a phillips gemini tf scanner with a brilliance 16-slice ct scanner and at stanford university hospital was a ge discovery 600 pet / ct system with a 16-slice ct scanner . the ct part of the pet / ct acquisition was designed to be equivalent to a stand - alone diagnostic ct protocol optimized for bone / spine imaging : 1.252 mm slice thickness , 1 mm increment , 100140 kv , 180230 mas , and matrix size 512 512 pixels . a nuclear medicine physician and a radiologist with musculoskeletal expertise reviewed all pet / ct images together with the primary objective of identifying or ruling out the presence of lesions amenable to surgical intervention . results of f - naf pet / ct scanning were then communicated to the referring orthopedist and tabulated in detail for analysis and validation ( table 1 ) . based primarily on f - naf pet / ct imaging , the patient then underwent either surgical exploration ( with possible intervention ) , or conservative management . pet / ct abnormalities were considered true positive if they were confirmed on surgery or if by the 15-month clinical follow - up other adjunctive examinations and data were wholly consistent with a surgically relevant lesion . also not unexpectedly , the symptomatology may have differed at the initial spinal fusion and at the time of the follow - up pet / ct examination including the exact location and/or the descriptive quality of the complaint . the interpreting physicians found that 16 of the 22 patients had an f - naf pet / ct scan that had at least one abnormality amenable to surgical intervention ( figs . the abnormal foci were located at the following sites : screws ( six ) , cages / rods / fixation hardware ( six ) , and bone grafts ( nine ) ( table 1 ) . b pet , pet / ct fusion , and ct images show markedly asymmetric activity ( yellow arrows ) at the right l5-s1 lamina and facet which is the site of a prior bone graft . b pet , pet / ct fusion , and ct images show markedly asymmetric activity ( yellow arrows ) at the right l5-s1 lamina and facet which is the site of a prior bone graft . increased sclerosis is apparent on the ct image ( white arrow ) at the right l5-s1 graft compared to the left that was initially described as postoperative changes ( and difficult to differentiate from graft failure ) on the initial ct scan of the 16 patients with abnormal pet / ct results , 15 went on to surgical exploration . the orthopedist found abnormalities in 14 of 15 of these patients that correlated precisely with the pet / ct findings and led to repair of the hardware or spine . one patient ( patient 20 , table 1 ) with pet / ct abnormalities was not receptive to surgical intervention and was therefore followed clinically rather than subjected to surgery ( table 1 ) . of the 22 patients , 6 did not have any foci on f - naf pet / ct that appeared amenable to surgery and conservative treatment was recommended . on a patient - by - patient basis , the sensitivity and specificity of the ability of f - naf pet / ct to identify the presence of an abnormality requiring surgical intervention were 100 % ( 95 % ci 78.2100 % ) and 85.7 % ( 95 % ci 42.199.6 % ) , respectively . on a lesion - by - lesion basis , 20 of the 21 abnormal foci ( in 16 subjects ) identified by f - naf pet / ct were confirmed as true positive while 1 of 20 was false positive . f - naf pet / ct imaging appeared to provide accurate adjunctive information to the standard work - up . specifically , 14 of 15 patients who underwent surgical exploration based on pet / ct results were confirmed to have lesions requiring surgical intervention by either histopathology or direct manual probing . overall , 15 of 16 patients were correctly identified by pet / ct as having a lesion requiring surgery after initial evaluation was equivocal regarding the need for surgery . conversely , all of the six patients with pet / ct results indicating that surgery was not required were found after 15 months of follow - up to have improved ( by clinical assessment and reported pain ) . however , this prior study lacked outcome data and abnormalities found on f - naf pet / ct were described without follow - up information to validate the imaging results . in this prospective pilot investigation of 22 patients , f - naf pet / ct imaging demonstrated potential for aiding management of patients with recurrent pain after spinal fusion surgery by helping to correctly identify those requiring surgical intervention .

early progressive mobilization of adult intensive care unit ( icu ) patients has been shown to be safe and feasible and1,2 result in reduced delirium,3 improved functional outcomes,4,5 reduced hospital length of stay,3,6 and reduced mortality in patients with acute respiratory failure.7 in a recent systematic review of physiotherapy in icu , stiller6 suggested that , given the evidence supporting the outcomes for early mobilization , icu physiotherapists should give priority to interventions aimed at early progressive mobilization . however , point - prevalence studies from germany8 and australia and new zealand9 have demonstrated a low incidence of mobilization of patients with an endotracheal tube ( 8% in germany and 0% in australia and new zealand ) . although there are several guidelines available that discuss the implementation of early mobilization in critically ill patients,1013 few papers address the practical challenges faced by clinicians attempting to translate this evidence into practice . our 31-bed mixed tertiary icu had 1,976 admissions in 2015 , with 50% requiring mechanical ventilation . we have been practicing early mobilization and rehabilitation for over a decade , and it is well embedded in our unit culture across the multidisciplinary team ( mdt ) ( physiotherapy , medical , nursing , and support staff14 ) . in this paper , we share the practical tools we have developed to educate and train staff in early mobilization across the spectrum of acuity , including patients with neurological diagnoses . our approach is applicable to general icu patients regardless of whether they are intubated and mechanically ventilated , noninvasively ventilated , or not requiring any ventilatory or airway support . prior to any mobilization episode , a comprehensive assessment and review of safety criteria should occur to minimize risk . this paper is intended to be used in conjunction with published expert consensus statements13,1517 and recommendations and is focused more on the practical implementation of mobilization once a safety assessment has been completed in collaboration with the mdt . we perform a daily assessment of all patients in the icu regarding their suitability for mobilization with the aim of achieving the highest level of mobilization possible each day . to determine the type of mobilization , a stepwise approach is taken ( figure 1 ) . the first two steps are to assess the patient s level of alertness and ability to follow instructions ( eg , using the richmond agitation sedation scale)18 and identify if there are any other safety concerns or barriers to mobilization . the physiotherapist will discuss any safety concerns with senior medical staff to determine whether these factors should preclude mobilization . in particular , the team must also consider whether the patient has sufficient respiratory and cardiovascular reserve to perform the proposed mobilization task and if acceptable limits of organ support can be established to facilitate mobilization ( eg , alteration of ventilator settings or increasing vasoactive infusions ) . if sedation can be weaned or barriers to mobilization overcome ( eg , timing of procedures or tests ) , this is prioritized by the mdt early in the day to facilitate mobilization . if any member of the mdt has concerns about whether mobilization should proceed , this is discussed openly with the icu physiotherapist and senior medical staff . the flowchart in figure 1 also reinforces the need to continuously monitor the patient during mobilization . for each patient , we clearly articulate and agree on target ranges for physiological parameters during mobilization , rather than arbitrary thresholds . it is important to remember that the normal physiological response to exercise includes an increase in heart rate and to allow for this when setting parameters . mobilization may need to temporarily cease due to the patient s physiological response to mobilization ( eg , decrease in spo2 and excessive increase in heart rate ) . however , after a period of monitoring and rest , mobilization may recommence . in the very rare circumstance of an adverse event,14 mobilization ceases immediately and a referral is made for an urgent medical review . ultimately , treatment dosage and intensity should align with the specific goals of treatment for the individual patient , whether this is enhanced respiratory status , maintenance of global function or recovery of strength , endurance or balance deficits , or a combination of these . these goals are collaboratively determined by the physiotherapist , bedside nurse , and patient following assessment . where patients are able to rate their perceived exertion,19 this can be helpful in guiding treatment intensity and has shown to be reliable in mechanically ventilated patients.20 for example , we may encourage a patient to work at a rated perceived exertion of 34 out of 10 while mobilizing around the icu and reassure them that it will feel challenging at this point in their recovery . patients who are awake but must remain on bed rest due to the presence of safety criteria are assessed for their suitability to complete an in - bed exercise program . physiotherapists liaise closely with surgical and medical teams to clarify movement restrictions and duration of bed rest ( eg , following pelvic fracture ) to determine when active mobilization can commence . if the patient is unable to follow commands and actively participate in mobilization ( eg , richmond agitation < 1),18 he or she may be suitable for passive mobilization ( ie , hoist transfer to sit - out - of - bed , figure 2 ) . even patients who are sedated and unresponsive may still benefit from the high sitting position in an appropriate chair to potentially minimize orthostatic intolerance , which is known to occur after just 24 hours of bed rest.21 a portable sling lifter for mobilization is feasible and is a standard practice in our unit . once the decision to actively mobilize a patient is made , the next step requires the physiotherapist to complete a functional assessment , including a sitting balance and strength assessment . if the patient does not have at least 3/5 ( oxford scale ) strength in their lower limbs or safe unsupported sitting balance , the patient commences phase 1 mobilization phase 1 can involve sitting balance retraining ( eg , reaching and returning to midline from the bed or chair ) , strength training including the use of weights or slings , and/or treatment on the tilt table ( figure 3 ) . some patients in the passive mobilization group ( eg , patients with a neurological diagnosis ) may be assessed as suitable for phase 1 mobilization even if they are unable to participate in therapy or their level of consciousness fluctuates . a patient will remain in this phase until they achieve adequate sitting balance and lower limb strength to progress to phase 2 mobilization . if the patient is not able to stand with the assistance of two staff , they progress to the supported weight - bearing phase , which involves the use of a gait harness to facilitate mobilization ( figure 4 ) . a gait harness can be suspended from overhead ceiling tracking or from a sling hoist lifter . for patients with several drains and attachments in the abdomen and thorax , we recommend noncircumferential gait harnesses ( figure 4 ) as these are less likely to result in compression or dislodgement of these items . this sometimes requires the patient to be lifted with a standard sling , and then lowered onto the gait harness either on the edge of the bed or in a chair . we also recommend padding the gait harness with a disposable lining to reduce the need for laundering between treatments . once transferred to a standing position , the patient can be assisted to extend the hips and knees as much as possible and step forward , or practice lateral weight shift . if the patient is able to stand with the assistance of two staff members , they proceed to the active weight - bearing phase ( figure 5 ) . patients may require the use of a gait aid ( eg , a forearm support frame ) , and some patients will require a sling hoist to sit out of bed , with more effective mobilization commencing from the chair . mobilization is progressive both within and between treatments to achieve functional goals ( figure 1 , phase 2 mobilization ) . for some chronically critically ill patients , following discussion and planning with the mdt , it has been possible to complete rehabilitation sessions outside of the bed space , including the balcony area adjacent to our icu and the rehabilitation gym . if rehabilitation is occurring outside the icu location , we ensure an airway management kit is with the patient at all times , as well as nursing and medical escorts as appropriate . for example , we have had a mechanically ventilated patient complete a 30-minute therapy session in the rehabilitation gym , including treadmill training on a portable ventilator . invasive mechanical ventilation , whether through an endotracheal or tracheostomy tube , should not be a barrier for mobilization and has been shown to be safe and feasible.810 considerable planning and preparation is required to ensure safety for both patient and staff . we have developed the plan b mnemonic ( figure 6 ) which is used routinely by staff in our unit before commencing mobilization of a mechanically ventilated patient . preparation and planning are essential , as mobilization of an icu patient requires coordination of the patient , several staff , and equipment . timing of the mobilization episode needs to be negotiated with the bedside nurse and coordinated with other care needs ( eg , procedures and scans ) . in our experience , 3060 minutes is a reasonable time frame to allocate for mobilization of a ventilated patient , including preparation time . mobilization should be timed to coincide with medication peak effectiveness where applicable ( eg , analgesia and bronchodilators ) . equipment requirements may include portable monitoring , overhead ceiling - mounted tracking or portable sling lifters , gait harnesses , tilt table , gait aid ( eg , forearm support frame ) , a suitable recliner chair , and linen . if mobilizing away from the bed space , other equipment may include portable oxygen , portable suction , and either a portable ventilator or resuscitator bag . when using a portable ventilator , a spare oxygen cylinder should accompany the patient to guarantee adequate oxygen supply . the safe weight limit of all equipment should be checked with the patient s body weight . when using gait harnesses , patency of all lines and attachments should be checked prior to proceeding with and at multiple time points during mobilization . preparation of the awake ventilated patient should include provision of a clear explanation of the plan , process , goals , and rationale for mobilization , answering questions , and providing reassurance as required . a final review of the physical environment ( eg , the bed space ) completes the preparation phase . this must include a consideration of lines and attachments to ensure that there is adequate space to perform mobilization safely . if lines are to be removed imminently , consider delaying mobilization until after this has occurred . often , it is possible to rationalize attachments to reduce the number of staff required for safe mobilization . it is highly recommended to remove any unnecessary equipment and clutter out of the area . throughout the mobilization episode , it is imperative that there is a clear leader . in our unit , this is usually the physiotherapist , coordinating between the patient , the bedside nurse , support staff , and sometimes , family members as well . members of the mobilization team must have a clear understanding of their role ( eg , airway , attachments , patient , and frame ) with clear communication throughout the treatment . however , while the physiotherapist may typically lead the mobilization , the person responsible for the airway reserves the right to revert to a leadership role at any point , as airway safety must be the first priority . in this case , in our unit , the physiotherapist would temporarily transfer leadership to the bedside nurse who is usually responsible for the airway . these leadership transfers are articulated clearly so that the whole team is aware of the change to ensure patient safety . emergency equipment including emergency airway and ventilation equipment should be checked as part of routine nursing practices . discussion with the icu medical team prior to mobilization is recommended for any patient with an identified difficult airway lehane score at the time of intubation ) or if there is any concern regarding airway stability . one staff member ( usually the bedside nurse ) is dedicated to the role of airway and is responsible for supporting the airway to prevent tube migration or displacement . for patients with a high sputum load , airway clearance is recommended prior to any mobilization . if mobilizing a patient with a high sputum load away from the bed space , portable suction must be available . the number of staff required to safely mobilize a critically ill patient is risk - assessed on the basis of the patient s functional assessment . the minimum number of staff required to mobilize a ventilated patient in our icu is two ( one bedside nurse for the airway and one physiotherapist for the patient ) , but this is only possible where the patient requires minimal physical support . more commonly , we require between three and five staff members to mobilize a ventilated patient and safely manage all the lines and attachments . it is essential that the mdt members are trained in safe manual handling and have a clear understanding of each other s roles . before mobilization , the leader should clearly articulate the backup plan , which may occur if the patient is unable to complete the planned mobilization episode . the patient should also be aware of the backup plan , so that they are aware of the possible change in direction midtreatment . examples of this include having the bed nearby , a chair , commode , or wheelchair to follow the patient if mobilizing away from the bed space or a gait harness to prevent a fall in a high - risk patient . sometimes the backup plan is simply to sit the patient back in the chair if the patient has insufficient standing ability to proceed . patients who are awake but must remain on bed rest due to the presence of safety criteria are assessed for their suitability to complete an in - bed exercise program . physiotherapists liaise closely with surgical and medical teams to clarify movement restrictions and duration of bed rest ( eg , following pelvic fracture ) to determine when active mobilization can commence . if the patient is unable to follow commands and actively participate in mobilization ( eg , richmond agitation sedation score < 1),18 he or she may be suitable for passive mobilization ( ie , hoist transfer to sit - out - of - bed , figure 2 ) . even patients who are sedated and unresponsive may still benefit from the high sitting position in an appropriate chair to potentially minimize orthostatic intolerance , which is known to occur after just 24 hours of bed rest.21 a portable sling lifter for mobilization is feasible and is a standard practice in our unit . once the decision to actively mobilize a patient is made , the next step requires the physiotherapist to complete a functional assessment , including a sitting balance and strength assessment . if the patient does not have at least 3/5 ( oxford scale ) strength in their lower limbs or safe unsupported sitting balance , the patient commences phase 1 mobilization phase 1 can involve sitting balance retraining ( eg , reaching and returning to midline from the bed or chair ) , strength training including the use of weights or slings , and/or treatment on the tilt table ( figure 3 ) . some patients in the passive mobilization group ( eg , patients with a neurological diagnosis ) may be assessed as suitable for phase 1 mobilization even if they are unable to participate in therapy or their level of consciousness fluctuates . a patient will remain in this phase until they achieve adequate sitting balance and lower limb strength to progress to phase 2 mobilization . if the patient is not able to stand with the assistance of two staff , they progress to the supported weight - bearing phase , which involves the use of a gait harness to facilitate mobilization ( figure 4 ) . a gait harness can be suspended from overhead ceiling tracking or from a sling hoist lifter . for patients with several drains and attachments in the abdomen and thorax , we recommend noncircumferential gait harnesses ( figure 4 ) as these are less likely to result in compression or dislodgement of these items . this sometimes requires the patient to be lifted with a standard sling , and then lowered onto the gait harness either on the edge of the bed or in a chair . we also recommend padding the gait harness with a disposable lining to reduce the need for laundering between treatments . once transferred to a standing position , the patient can be assisted to extend the hips and knees as much as possible and step forward , or practice lateral weight shift . if the patient is able to stand with the assistance of two staff members , they proceed to the active weight - bearing phase ( figure 5 ) . patients may require the use of a gait aid ( eg , a forearm support frame ) , and some patients will require a sling hoist to sit out of bed , with more effective mobilization commencing from the chair . mobilization is progressive both within and between treatments to achieve functional goals ( figure 1 , phase 2 mobilization ) . for some chronically critically ill patients , following discussion and planning with the mdt , it has been possible to complete rehabilitation sessions outside of the bed space , including the balcony area adjacent to our icu and the rehabilitation gym . if rehabilitation is occurring outside the icu location , we ensure an airway management kit is with the patient at all times , as well as nursing and medical escorts as appropriate . for example , we have had a mechanically ventilated patient complete a 30-minute therapy session in the rehabilitation gym , including treadmill training on a portable ventilator . invasive mechanical ventilation , whether through an endotracheal or tracheostomy tube , should not be a barrier for mobilization and has been shown to be safe and feasible.810 considerable planning and preparation is required to ensure safety for both patient and staff . we have developed the plan b mnemonic ( figure 6 ) which is used routinely by staff in our unit before commencing mobilization of a mechanically ventilated patient . preparation and planning are essential , as mobilization of an icu patient requires coordination of the patient , several staff , and equipment . timing of the mobilization episode needs to be negotiated with the bedside nurse and coordinated with other care needs ( eg , procedures and scans ) . in our experience , 3060 minutes is a reasonable time frame to allocate for mobilization of a ventilated patient , including preparation time . mobilization should be timed to coincide with medication peak effectiveness where applicable ( eg , analgesia and bronchodilators ) . equipment requirements may include portable monitoring , overhead ceiling - mounted tracking or portable sling lifters , gait harnesses , tilt table , gait aid ( eg , forearm support frame ) , a suitable recliner chair , and linen . if mobilizing away from the bed space , other equipment may include portable oxygen , portable suction , and either a portable ventilator or resuscitator bag . when using a portable ventilator , a spare oxygen cylinder should accompany the patient to guarantee adequate oxygen supply . the safe weight limit of all equipment should be checked with the patient s body weight . when using gait harnesses , patency of all lines and attachments should be checked prior to proceeding with and at multiple time points during mobilization . preparation of the awake ventilated patient should include provision of a clear explanation of the plan , process , goals , and rationale for mobilization , answering questions , and providing reassurance as required . a final review of the physical environment ( eg , the bed space ) completes the preparation phase . this must include a consideration of lines and attachments to ensure that there is adequate space to perform mobilization safely . if lines are to be removed imminently , consider delaying mobilization until after this has occurred . often , it is possible to rationalize attachments to reduce the number of staff required for safe mobilization . it is highly recommended to remove any unnecessary equipment and clutter out of the area . throughout the mobilization episode , it is imperative that there is a clear leader . in our unit , this is usually the physiotherapist , coordinating between the patient , the bedside nurse , support staff , and sometimes , family members as well . members of the mobilization team must have a clear understanding of their role ( eg , airway , attachments , patient , and frame ) with clear communication throughout the treatment . however , while the physiotherapist may typically lead the mobilization , the person responsible for the airway reserves the right to revert to a leadership role at any point , as airway safety must be the first priority . in this case , in our unit , the physiotherapist would temporarily transfer leadership to the bedside nurse who is usually responsible for the airway . these leadership transfers are articulated clearly so that the whole team is aware of the change to ensure patient safety . emergency equipment including emergency airway and ventilation equipment should be checked as part of routine nursing practices . discussion with the icu medical team prior to mobilization is recommended for any patient with an identified difficult airway ( eg , documented grade 3 or 4 modified cormack lehane score at the time of intubation ) or if there is any concern regarding airway stability . one staff member ( usually the bedside nurse ) is dedicated to the role of airway and is responsible for supporting the airway to prevent tube migration or displacement . for patients with a high sputum load , airway clearance is recommended prior to any mobilization . if mobilizing a patient with a high sputum load away from the bed space , portable suction must be available . the number of staff required to safely mobilize a critically ill patient is risk - assessed on the basis of the patient s functional assessment . the minimum number of staff required to mobilize a ventilated patient in our icu is two ( one bedside nurse for the airway and one physiotherapist for the patient ) , but this is only possible where the patient requires minimal physical support . more commonly , we require between three and five staff members to mobilize a ventilated patient and safely manage all the lines and attachments . it is essential that the mdt members are trained in safe manual handling and have a clear understanding of each other s roles . before mobilization , the leader should clearly articulate the backup plan , which may occur if the patient is unable to complete the planned mobilization episode . the patient should also be aware of the backup plan , so that they are aware of the possible change in direction midtreatment . examples of this include having the bed nearby , a chair , commode , or wheelchair to follow the patient if mobilizing away from the bed space or a gait harness to prevent a fall in a high - risk patient . sometimes the backup plan is simply to sit the patient back in the chair if the patient has insufficient standing ability to proceed . preparation and planning are essential , as mobilization of an icu patient requires coordination of the patient , several staff , and equipment . timing of the mobilization episode needs to be negotiated with the bedside nurse and coordinated with other care needs ( eg , procedures and scans ) . in our experience , 3060 minutes is a reasonable time frame to allocate for mobilization of a ventilated patient , including preparation time . mobilization should be timed to coincide with medication peak effectiveness where applicable ( eg , analgesia and bronchodilators ) . equipment requirements may include portable monitoring , overhead ceiling - mounted tracking or portable sling lifters , gait harnesses , tilt table , gait aid ( eg , forearm support frame ) , a suitable recliner chair , and linen . if mobilizing away from the bed space , other equipment may include portable oxygen , portable suction , and either a portable ventilator or resuscitator bag . when using a portable ventilator , a spare oxygen cylinder should accompany the patient to guarantee adequate oxygen supply . the safe weight limit of all equipment should be checked with the patient s body weight . when using gait harnesses , patency of all lines and attachments should be checked prior to proceeding with and at multiple time points during mobilization . preparation of the awake ventilated patient should include provision of a clear explanation of the plan , process , goals , and rationale for mobilization , answering questions , and providing reassurance as required . a final review of the physical environment ( eg , the bed space ) completes the preparation phase . this must include a consideration of lines and attachments to ensure that there is adequate space to perform mobilization safely . if lines are to be removed imminently , consider delaying mobilization until after this has occurred . often , it is possible to rationalize attachments to reduce the number of staff required for safe mobilization . it is highly recommended to remove any unnecessary equipment and clutter out of the area . throughout the mobilization episode , it is imperative that there is a clear leader . in our unit , this is usually the physiotherapist , coordinating between the patient , the bedside nurse , support staff , and sometimes , family members as well . members of the mobilization team must have a clear understanding of their role ( eg , airway , attachments , patient , and frame ) with clear communication throughout the treatment . however , while the physiotherapist may typically lead the mobilization , the person responsible for the airway reserves the right to revert to a leadership role at any point , as airway safety must be the first priority . in this case , in our unit , the physiotherapist would temporarily transfer leadership to the bedside nurse who is usually responsible for the airway . these leadership transfers are articulated clearly so that the whole team is aware of the change to ensure patient safety . emergency equipment including emergency airway and ventilation equipment should be checked as part of routine nursing practices . discussion with the icu medical team prior to mobilization is recommended for any patient with an identified difficult airway ( eg , documented grade 3 or 4 modified cormack lehane score at the time of intubation ) or if there is any concern regarding airway stability . one staff member ( usually the bedside nurse ) is dedicated to the role of airway and is responsible for supporting the airway to prevent tube migration or displacement . for patients with a high sputum load , airway clearance is recommended prior to any mobilization . if mobilizing a patient with a high sputum load away from the bed space , portable suction must be available . the number of staff required to safely mobilize a critically ill patient is risk - assessed on the basis of the patient s functional assessment . the minimum number of staff required to mobilize a ventilated patient in our icu is two ( one bedside nurse for the airway and one physiotherapist for the patient ) , but this is only possible where the patient requires minimal physical support . more commonly , we require between three and five staff members to mobilize a ventilated patient and safely manage all the lines and attachments . it is essential that the mdt members are trained in safe manual handling and have a clear understanding of each other s roles . before mobilization , the leader should clearly articulate the backup plan , which may occur if the patient is unable to complete the planned mobilization episode . the patient should also be aware of the backup plan , so that they are aware of the possible change in direction midtreatment . examples of this include having the bed nearby , a chair , commode , or wheelchair to follow the patient if mobilizing away from the bed space or a gait harness to prevent a fall in a high - risk patient . sometimes the backup plan is simply to sit the patient back in the chair if the patient has insufficient standing ability to proceed . we have described strategies to guide the practicalities of safe early mobilization of icu patients , including those who are unable to actively participate . this approach has been used successfully in our icu for more than 10 years with a very low incidence of adverse outcomes , ie , 1.1%.14 the two adverse outcomes in our audit were both instances of hypotension requiring intervention ( ie , return to bed , fluid loading , and transient increase in vasopressor requirements ) with no long - term consequences . this is consistent with other available safety data , including a recent review which reported an incidence of 4% adverse events with early mobilization in icu22 and a study of 5,267 icu physical therapy sessions where a physiological abnormality or a potential safety event occurred in 34 ( 0.6% ) sessions.23 progressive mobilization protocols have been published from the united states3,12,24 and the united kingdom.11 bassett et al12 published a goal - directed progressive mobility continuum including safety criteria . our clinical practice differs as no member of our mdt routinely performs passive range of motion exercises given the absence of evidence that these reduce or prevent contractures in critically ill patients.25,26 the absence of passive mobilization is consistent with surveyed australian intensive care physiotherapy practice.27 another important distinction from other protocols is that we aim to sit our patients out of bed for a minimum of 4 hours per day with pressure care performed in the chair . although bassett et al12 targets a frequency of mobilization at two to three times per day , we have opted to individualize the frequency of mobilization . many of our patients are unlikely to tolerate this frequency due to fatigue . fatigue was a limiting factor in a recent randomized controlled trial of a protocolized rehabilitation program commencing in icu , where exercise prescription was targeted to be delivered twice per day but was only feasible in 55% of potential sessions.28 despite the evidence in favor of early mobilization , substantial barriers exist in many icus , including our own . in 2008 , we audited our mobilization practice and found that patients were mobilized on 54% of patient days , with avoidable factors identified in 47% of cases where patients were not mobilized.14 specifically , the most common perceived barriers were femoral vascular access , ( particularly femoral dialysis catheters ) , timing of procedures , and patient agitation or low level of consciousness . more recent evidence indicates that femoral access should not prohibit early mobilization,29 and , in the case of dialysis catheters , mobilization may actually prolong filter life.30 other potential barriers to mobilization may include staffing , equipment , leadership , referral processes , delirium , sedation , and perceived lack of safety . strategies to overcome these barriers have been published to assist clinicians in increasing mobility in icu.10,31 our icu sedation practices are goal - directed to achieve a target richmond agitation sedation score.18 for patients in whom deep sedation is not indicated , sedation is targeted to achieve a richmond agitation oversedation may be a barrier to mobilization , may contribute to delirium , and compromise the patient s ability to participate in mobilization . review of sedation practices may be a key step in increasing mobilization in critically ill patients . a strategy to reduce heavy sedation and increase mobilization in medical icu patients has been shown to be safe , reduce delirium , improve function , and reduce icu and hospital length of stay.1 the optimization of sedation to facilitate mobilization clearly requires close multidisciplinary collaboration between medical , nursing , and physiotherapy staff . in our icu , this interaction and negotiation occurs frequently not just in handover meetings and ward rounds , but ad hoc across the day as issues arise . this open multidisciplinary communication requires mutual respect for each other s roles and expertise , and an understanding of the priorities of competing demands for care of acutely unwell patients . the description of our experience and approach to early mobilization in icu adds to the body of evidence by providing specific practical details of how to perform this safe and feasible intervention as a mdt . a limitation of the material presented in this paper is that the ideal timing , type , frequency , intensity , and duration of mobilization required to prevent or minimize functional impairments in icu survivors are yet to be established . our approach is a feasible bundle of care that promotes early mobilization in critically ill patients ; however , the effect of the individual components can not be currently demonstrated . given the heterogeneity of general icu presentations , individualized goal - directed prescription is warranted . clinicians need to make informed patient - centered decisions balancing the risks of prolonged bed rest against the benefits of early mobilization . due to the multidisciplinary nature of the icu environment , the decision to mobilize an icu patient should be shared between the physiotherapy , medical , and nursing staff . developing and sustaining an icu mdt culture that promotes , values , and prioritizes early mobilization is essential to translating evidence into clinical practice . numerous articles are available to assist in units developing an early mobilization culture.1,10,12,16,32,33 in our experience , early mobilization is both feasible and safe and is the result of a concerted commitment from the mdt to make early mobilization the norm , rather than the exception . it is also possible that , despite the contrast of our approach with other published guidelines , we are still too conservative , and the limits of early proactive rehabilitation in icu are yet to be established . early progressive mobilization is safe and feasible in critically ill patients and requires close collaboration of the mdt on a daily basis . it is our hope that the guided clinical reasoning and practical considerations described in this paper will provide tools that allow frontline clinical staff to implement early mobilization in the majority of critically ill patients in a safe and effective manner .

objectivesto describe our experience and the practical tools we have developed to facilitate early mobilization in the intensive care unit ( icu ) as a multidisciplinary team.backgrounddespite the evidence supporting early mobilization for improving outcomes for icu patients , recent international point - prevalence studies reveal that few patients are mobilized in the icu . existing guidelines rarely address the practical issues faced by multidisciplinary icu teams attempting to translate evidence into practice . we present a comprehensive strategy for safe mobilization utilized in our icu , incorporating the combined skills of medical , nursing , and physiotherapy staff to achieve safe outcomes and establish a culture which prioritizes this intervention.methodsa raft of tools and strategies are described to facilitate mobilization in icu by the multidisciplinary team . patients without safe unsupported sitting balance and without 3/5 ( oxford scale ) strength in the lower limbs commence phase 1 mobilization , including training of sitting balance and use of the tilt table . phase 2 mobilization involves supported or active weight - bearing , incorporating gait harnesses if necessary . the plan b mnemonic guides safe multidisciplinary mobilization of invasively ventilated patients and emphasizes the importance of a clearly articulated plan in delivering this valuable treatment as a team.discussionthese tools have been used over the past 5 years in a tertiary icu with a very low incidence of adverse outcomes ( < 2% ) . the tools and strategies described are useful not only to guide practical implementation of early mobilization , but also in the creation of a unit culture where icu staff prioritize early mobilization and collaborate daily to provide the best possible care.conclusionthese practical tools allow icu clinicians to safely and effectively implement early mobilization in critically ill patients . a genuinely multidisciplinary approach to safe mobilization in icu is key to its success in the long term .

Introduction Mobilization methods Bed rest Passive mobilization Active mobilization Mobilizing an invasively mechanically ventilated patient P preparation L leader A airway and emergency equipment N number of staff B backup plan Discussion Conclusion

early progressive mobilization of adult intensive care unit ( icu ) patients has been shown to be safe and feasible and1,2 result in reduced delirium,3 improved functional outcomes,4,5 reduced hospital length of stay,3,6 and reduced mortality in patients with acute respiratory failure.7 in a recent systematic review of physiotherapy in icu , stiller6 suggested that , given the evidence supporting the outcomes for early mobilization , icu physiotherapists should give priority to interventions aimed at early progressive mobilization . however , point - prevalence studies from germany8 and australia and new zealand9 have demonstrated a low incidence of mobilization of patients with an endotracheal tube ( 8% in germany and 0% in australia and new zealand ) . although there are several guidelines available that discuss the implementation of early mobilization in critically ill patients,1013 few papers address the practical challenges faced by clinicians attempting to translate this evidence into practice . we have been practicing early mobilization and rehabilitation for over a decade , and it is well embedded in our unit culture across the multidisciplinary team ( mdt ) ( physiotherapy , medical , nursing , and support staff14 ) . in this paper , we share the practical tools we have developed to educate and train staff in early mobilization across the spectrum of acuity , including patients with neurological diagnoses . prior to any mobilization episode , a comprehensive assessment and review of safety criteria should occur to minimize risk . this paper is intended to be used in conjunction with published expert consensus statements13,1517 and recommendations and is focused more on the practical implementation of mobilization once a safety assessment has been completed in collaboration with the mdt . we perform a daily assessment of all patients in the icu regarding their suitability for mobilization with the aim of achieving the highest level of mobilization possible each day . the physiotherapist will discuss any safety concerns with senior medical staff to determine whether these factors should preclude mobilization . in particular , the team must also consider whether the patient has sufficient respiratory and cardiovascular reserve to perform the proposed mobilization task and if acceptable limits of organ support can be established to facilitate mobilization ( eg , alteration of ventilator settings or increasing vasoactive infusions ) . if sedation can be weaned or barriers to mobilization overcome ( eg , timing of procedures or tests ) , this is prioritized by the mdt early in the day to facilitate mobilization . if any member of the mdt has concerns about whether mobilization should proceed , this is discussed openly with the icu physiotherapist and senior medical staff . for each patient , we clearly articulate and agree on target ranges for physiological parameters during mobilization , rather than arbitrary thresholds . these goals are collaboratively determined by the physiotherapist , bedside nurse , and patient following assessment . where patients are able to rate their perceived exertion,19 this can be helpful in guiding treatment intensity and has shown to be reliable in mechanically ventilated patients.20 for example , we may encourage a patient to work at a rated perceived exertion of 34 out of 10 while mobilizing around the icu and reassure them that it will feel challenging at this point in their recovery . once the decision to actively mobilize a patient is made , the next step requires the physiotherapist to complete a functional assessment , including a sitting balance and strength assessment . if the patient does not have at least 3/5 ( oxford scale ) strength in their lower limbs or safe unsupported sitting balance , the patient commences phase 1 mobilization phase 1 can involve sitting balance retraining ( eg , reaching and returning to midline from the bed or chair ) , strength training including the use of weights or slings , and/or treatment on the tilt table ( figure 3 ) . some patients in the passive mobilization group ( eg , patients with a neurological diagnosis ) may be assessed as suitable for phase 1 mobilization even if they are unable to participate in therapy or their level of consciousness fluctuates . a patient will remain in this phase until they achieve adequate sitting balance and lower limb strength to progress to phase 2 mobilization . if the patient is not able to stand with the assistance of two staff , they progress to the supported weight - bearing phase , which involves the use of a gait harness to facilitate mobilization ( figure 4 ) . for patients with several drains and attachments in the abdomen and thorax , we recommend noncircumferential gait harnesses ( figure 4 ) as these are less likely to result in compression or dislodgement of these items . this sometimes requires the patient to be lifted with a standard sling , and then lowered onto the gait harness either on the edge of the bed or in a chair . if the patient is able to stand with the assistance of two staff members , they proceed to the active weight - bearing phase ( figure 5 ) . patients may require the use of a gait aid ( eg , a forearm support frame ) , and some patients will require a sling hoist to sit out of bed , with more effective mobilization commencing from the chair . mobilization is progressive both within and between treatments to achieve functional goals ( figure 1 , phase 2 mobilization ) . for some chronically critically ill patients , following discussion and planning with the mdt , it has been possible to complete rehabilitation sessions outside of the bed space , including the balcony area adjacent to our icu and the rehabilitation gym . for example , we have had a mechanically ventilated patient complete a 30-minute therapy session in the rehabilitation gym , including treadmill training on a portable ventilator . we have developed the plan b mnemonic ( figure 6 ) which is used routinely by staff in our unit before commencing mobilization of a mechanically ventilated patient . preparation and planning are essential , as mobilization of an icu patient requires coordination of the patient , several staff , and equipment . timing of the mobilization episode needs to be negotiated with the bedside nurse and coordinated with other care needs ( eg , procedures and scans ) . in our experience , 3060 minutes is a reasonable time frame to allocate for mobilization of a ventilated patient , including preparation time . equipment requirements may include portable monitoring , overhead ceiling - mounted tracking or portable sling lifters , gait harnesses , tilt table , gait aid ( eg , forearm support frame ) , a suitable recliner chair , and linen . preparation of the awake ventilated patient should include provision of a clear explanation of the plan , process , goals , and rationale for mobilization , answering questions , and providing reassurance as required . often , it is possible to rationalize attachments to reduce the number of staff required for safe mobilization . in our unit , this is usually the physiotherapist , coordinating between the patient , the bedside nurse , support staff , and sometimes , family members as well . members of the mobilization team must have a clear understanding of their role ( eg , airway , attachments , patient , and frame ) with clear communication throughout the treatment . these leadership transfers are articulated clearly so that the whole team is aware of the change to ensure patient safety . discussion with the icu medical team prior to mobilization is recommended for any patient with an identified difficult airway lehane score at the time of intubation ) or if there is any concern regarding airway stability . for patients with a high sputum load , airway clearance is recommended prior to any mobilization . the number of staff required to safely mobilize a critically ill patient is risk - assessed on the basis of the patient s functional assessment . the minimum number of staff required to mobilize a ventilated patient in our icu is two ( one bedside nurse for the airway and one physiotherapist for the patient ) , but this is only possible where the patient requires minimal physical support . even patients who are sedated and unresponsive may still benefit from the high sitting position in an appropriate chair to potentially minimize orthostatic intolerance , which is known to occur after just 24 hours of bed rest.21 a portable sling lifter for mobilization is feasible and is a standard practice in our unit . once the decision to actively mobilize a patient is made , the next step requires the physiotherapist to complete a functional assessment , including a sitting balance and strength assessment . if the patient does not have at least 3/5 ( oxford scale ) strength in their lower limbs or safe unsupported sitting balance , the patient commences phase 1 mobilization phase 1 can involve sitting balance retraining ( eg , reaching and returning to midline from the bed or chair ) , strength training including the use of weights or slings , and/or treatment on the tilt table ( figure 3 ) . some patients in the passive mobilization group ( eg , patients with a neurological diagnosis ) may be assessed as suitable for phase 1 mobilization even if they are unable to participate in therapy or their level of consciousness fluctuates . a patient will remain in this phase until they achieve adequate sitting balance and lower limb strength to progress to phase 2 mobilization . if the patient is not able to stand with the assistance of two staff , they progress to the supported weight - bearing phase , which involves the use of a gait harness to facilitate mobilization ( figure 4 ) . for patients with several drains and attachments in the abdomen and thorax , we recommend noncircumferential gait harnesses ( figure 4 ) as these are less likely to result in compression or dislodgement of these items . this sometimes requires the patient to be lifted with a standard sling , and then lowered onto the gait harness either on the edge of the bed or in a chair . if the patient is able to stand with the assistance of two staff members , they proceed to the active weight - bearing phase ( figure 5 ) . patients may require the use of a gait aid ( eg , a forearm support frame ) , and some patients will require a sling hoist to sit out of bed , with more effective mobilization commencing from the chair . mobilization is progressive both within and between treatments to achieve functional goals ( figure 1 , phase 2 mobilization ) . for some chronically critically ill patients , following discussion and planning with the mdt , it has been possible to complete rehabilitation sessions outside of the bed space , including the balcony area adjacent to our icu and the rehabilitation gym . for example , we have had a mechanically ventilated patient complete a 30-minute therapy session in the rehabilitation gym , including treadmill training on a portable ventilator . we have developed the plan b mnemonic ( figure 6 ) which is used routinely by staff in our unit before commencing mobilization of a mechanically ventilated patient . preparation and planning are essential , as mobilization of an icu patient requires coordination of the patient , several staff , and equipment . in our experience , 3060 minutes is a reasonable time frame to allocate for mobilization of a ventilated patient , including preparation time . equipment requirements may include portable monitoring , overhead ceiling - mounted tracking or portable sling lifters , gait harnesses , tilt table , gait aid ( eg , forearm support frame ) , a suitable recliner chair , and linen . preparation of the awake ventilated patient should include provision of a clear explanation of the plan , process , goals , and rationale for mobilization , answering questions , and providing reassurance as required . often , it is possible to rationalize attachments to reduce the number of staff required for safe mobilization . it is highly recommended to remove any unnecessary equipment and clutter out of the area . in our unit , this is usually the physiotherapist , coordinating between the patient , the bedside nurse , support staff , and sometimes , family members as well . members of the mobilization team must have a clear understanding of their role ( eg , airway , attachments , patient , and frame ) with clear communication throughout the treatment . in this case , in our unit , the physiotherapist would temporarily transfer leadership to the bedside nurse who is usually responsible for the airway . for patients with a high sputum load , airway clearance is recommended prior to any mobilization . the number of staff required to safely mobilize a critically ill patient is risk - assessed on the basis of the patient s functional assessment . the minimum number of staff required to mobilize a ventilated patient in our icu is two ( one bedside nurse for the airway and one physiotherapist for the patient ) , but this is only possible where the patient requires minimal physical support . the patient should also be aware of the backup plan , so that they are aware of the possible change in direction midtreatment . preparation and planning are essential , as mobilization of an icu patient requires coordination of the patient , several staff , and equipment . timing of the mobilization episode needs to be negotiated with the bedside nurse and coordinated with other care needs ( eg , procedures and scans ) . in our experience , 3060 minutes is a reasonable time frame to allocate for mobilization of a ventilated patient , including preparation time . equipment requirements may include portable monitoring , overhead ceiling - mounted tracking or portable sling lifters , gait harnesses , tilt table , gait aid ( eg , forearm support frame ) , a suitable recliner chair , and linen . preparation of the awake ventilated patient should include provision of a clear explanation of the plan , process , goals , and rationale for mobilization , answering questions , and providing reassurance as required . often , it is possible to rationalize attachments to reduce the number of staff required for safe mobilization . in our unit , this is usually the physiotherapist , coordinating between the patient , the bedside nurse , support staff , and sometimes , family members as well . members of the mobilization team must have a clear understanding of their role ( eg , airway , attachments , patient , and frame ) with clear communication throughout the treatment . the number of staff required to safely mobilize a critically ill patient is risk - assessed on the basis of the patient s functional assessment . the minimum number of staff required to mobilize a ventilated patient in our icu is two ( one bedside nurse for the airway and one physiotherapist for the patient ) , but this is only possible where the patient requires minimal physical support . before mobilization , the leader should clearly articulate the backup plan , which may occur if the patient is unable to complete the planned mobilization episode . the patient should also be aware of the backup plan , so that they are aware of the possible change in direction midtreatment . we have described strategies to guide the practicalities of safe early mobilization of icu patients , including those who are unable to actively participate . this approach has been used successfully in our icu for more than 10 years with a very low incidence of adverse outcomes , ie , 1.1%.14 the two adverse outcomes in our audit were both instances of hypotension requiring intervention ( ie , return to bed , fluid loading , and transient increase in vasopressor requirements ) with no long - term consequences . this is consistent with other available safety data , including a recent review which reported an incidence of 4% adverse events with early mobilization in icu22 and a study of 5,267 icu physical therapy sessions where a physiological abnormality or a potential safety event occurred in 34 ( 0.6% ) sessions.23 progressive mobilization protocols have been published from the united states3,12,24 and the united kingdom.11 bassett et al12 published a goal - directed progressive mobility continuum including safety criteria . our clinical practice differs as no member of our mdt routinely performs passive range of motion exercises given the absence of evidence that these reduce or prevent contractures in critically ill patients.25,26 the absence of passive mobilization is consistent with surveyed australian intensive care physiotherapy practice.27 another important distinction from other protocols is that we aim to sit our patients out of bed for a minimum of 4 hours per day with pressure care performed in the chair . although bassett et al12 targets a frequency of mobilization at two to three times per day , we have opted to individualize the frequency of mobilization . many of our patients are unlikely to tolerate this frequency due to fatigue . fatigue was a limiting factor in a recent randomized controlled trial of a protocolized rehabilitation program commencing in icu , where exercise prescription was targeted to be delivered twice per day but was only feasible in 55% of potential sessions.28 despite the evidence in favor of early mobilization , substantial barriers exist in many icus , including our own . in 2008 , we audited our mobilization practice and found that patients were mobilized on 54% of patient days , with avoidable factors identified in 47% of cases where patients were not mobilized.14 specifically , the most common perceived barriers were femoral vascular access , ( particularly femoral dialysis catheters ) , timing of procedures , and patient agitation or low level of consciousness . more recent evidence indicates that femoral access should not prohibit early mobilization,29 and , in the case of dialysis catheters , mobilization may actually prolong filter life.30 other potential barriers to mobilization may include staffing , equipment , leadership , referral processes , delirium , sedation , and perceived lack of safety . strategies to overcome these barriers have been published to assist clinicians in increasing mobility in icu.10,31 our icu sedation practices are goal - directed to achieve a target richmond agitation sedation score.18 for patients in whom deep sedation is not indicated , sedation is targeted to achieve a richmond agitation oversedation may be a barrier to mobilization , may contribute to delirium , and compromise the patient s ability to participate in mobilization . review of sedation practices may be a key step in increasing mobilization in critically ill patients . a strategy to reduce heavy sedation and increase mobilization in medical icu patients has been shown to be safe , reduce delirium , improve function , and reduce icu and hospital length of stay.1 the optimization of sedation to facilitate mobilization clearly requires close multidisciplinary collaboration between medical , nursing , and physiotherapy staff . in our icu , this interaction and negotiation occurs frequently not just in handover meetings and ward rounds , but ad hoc across the day as issues arise . this open multidisciplinary communication requires mutual respect for each other s roles and expertise , and an understanding of the priorities of competing demands for care of acutely unwell patients . the description of our experience and approach to early mobilization in icu adds to the body of evidence by providing specific practical details of how to perform this safe and feasible intervention as a mdt . a limitation of the material presented in this paper is that the ideal timing , type , frequency , intensity , and duration of mobilization required to prevent or minimize functional impairments in icu survivors are yet to be established . our approach is a feasible bundle of care that promotes early mobilization in critically ill patients ; however , the effect of the individual components can not be currently demonstrated . clinicians need to make informed patient - centered decisions balancing the risks of prolonged bed rest against the benefits of early mobilization . due to the multidisciplinary nature of the icu environment , the decision to mobilize an icu patient should be shared between the physiotherapy , medical , and nursing staff . developing and sustaining an icu mdt culture that promotes , values , and prioritizes early mobilization is essential to translating evidence into clinical practice . numerous articles are available to assist in units developing an early mobilization culture.1,10,12,16,32,33 in our experience , early mobilization is both feasible and safe and is the result of a concerted commitment from the mdt to make early mobilization the norm , rather than the exception . it is also possible that , despite the contrast of our approach with other published guidelines , we are still too conservative , and the limits of early proactive rehabilitation in icu are yet to be established . early progressive mobilization is safe and feasible in critically ill patients and requires close collaboration of the mdt on a daily basis . it is our hope that the guided clinical reasoning and practical considerations described in this paper will provide tools that allow frontline clinical staff to implement early mobilization in the majority of critically ill patients in a safe and effective manner .

[ 2 + 3 ] cycloaddition reactions of nitroacetylene with allenyl - type three - atom components take place according to the polar , but a one - step mechanism . alternatively to cycloadducts , during the reaction between the aforementioned reagents , zwitterionic structures with extended nitroacetylenes belong because of the presence of a strongly electron withdrawing no2-group to a class of strongly electrophilic acetylenes . they are relatively unstable , and their physicochemistry is very poorly known . in particular , very little is known about the simple representative of this group of compounds parent nitroacetylene ( 1 ) . recently , the preliminary results of experimental studies related to nitroacetylene as a diels alder reaction component were published . however , no systematic studies of its participation in reactions of [ 2 + 3 ] cycloaddition have been performed to date . this work initiates research in this area ; in particular , within the study , complex , quantum - chemical studies of the [ 2 + 3 ] cycloaddition reaction of nitroacetylene ( 1 ) to selected allenyl - type three - atom components ( tacs ) . three such tacs were selected , often tested in recent years as components of [ 2 + 3 ] cycloaddition reactions with electrophilic ethylenes : benzonitrile n - oxide ( 2a ) [ 36 ] , phenyl azide ( 2b ) [ 68 ] , and phenyldiazomethane ( 2c ) [ 6 , 9 ] ( scheme 1 ) . reactions of these tacs with nitroacetylene are a potentially highly effective and selective method of synthesis of nitro - substituted , five - membered , unsaturated heterocycles that are valuable from the point of view of organic preparatory work [ 1012 ] . for this reason , identification of factors determining their course is so important . with this in mind , the following work was performed : ( 1 ) an analysis of nature of reagent interactions based on reactivity indices theory , and ( 2 ) simulations of a theoretically possible reaction path in the presence of a weakly polar ( toluene ) , and strongly polar medium ( nitromethane ) . it must be stressed here that the problematics of the [ 2 + 3 ] cycloaddition reaction of the aforementioned reagents is also very interesting from a mechanistic point of view . these reactions may also take place according to a one - step mechanism , as well as to a two - step mechanism , with a zwitterionic intermediate [ 1315 ] . the two - step mechanism is facilitated in this case by ( 1 ) the strongly electrophilic character of the dipolarophile and the nucleophilic nature of the tacs and ( 2 ) unequal screening of the reaction centres of both reagents . nitroacetylene ( 1 ) is characterized by high global electrophilicity [ 16 , 17 ] , exceeding 3 ev . using the scale proposed by domingo , it should be classified as belonging to the group of strong electrophiles . on the other hand , tacs 2a2c have a significantly weaker electrophilic nature ( < 1.6 ev)using the aforementioned scale they are classified as moderate electrophiles . the nucleophilic character of these compounds is indicated by values of the n indices . as can be concluded from the data summarized in table 1 , phenzyldiazomethane ( 2c ) is the strongest nucleophile in the analysed series , while benzonitrile n - oxide ( 2a ) is the weakest nucleophile . it should be noted that in cases of all reagent pairs , the difference in global electrophilicities exceeds 1.5 v. the title reactions are thus polar processes .table 1essential electronic properties of nitroacetylene ( 1 ) and tacs 2a2c global propertieslocal properties /a.u . /ev n / ev p x p y n x / ev n y / ev p + p + /ev /ev 1 0.17713.140.100.330.301.05 2a 0.14061.462.780.420.011.180.03 2b 0.13301.272.920.210.190.600.57 2c 0.12761.553.710.340.251.260.94 essential electronic properties of nitroacetylene ( 1 ) and tacs 2a2c next , it was decided to determine which of the theoretically possible directions of substrate transformations would be favoured by the electrophile nucleophile interactions . in the case of every single one of the cycloaddition reactions studied , analysis of local electronic properties allows one to state that the most electrophilically activated centre of the nitroacetylene molecule is the c carbon atom . local electrophilicity on the c carbon atom is more than three times weaker . on the other hand , the terminal x atom of tacs is the most nucleophilic activated centre in these species . if it is assumed that the reaction route is determined by an attack of the more strongly nucleophilic reaction centre of the tac on the more strongly electrophilic reaction centre of nitroacetylene , then the favoured direction of reagent transformation should always be determined by the a path ( see scheme 2 ) . taking into account the nature of the electrophile nucleophile interactions , reactions of nitroacetylene with tacs 2a2c must be recognized as polar processes . there are two possible variants of the conversion of reagents into adducts : ( a ) a one - step polar mechanism and ( b ) two - step zwitterionic mechanism . in the first case , one should expect a transition state ( ts ) in the energy profile , in the second two tss connected by a valley corresponding to the zwitterionic intermediate ( respectively , 5 or 6 , see scheme 2 ) . as suggested by quantum - chemical calculations , in weakly polar toluene ( = 2.38 ) , the reaction of nitroacetylene with benzonitrile n - oxide takes place regardless of the regioisomeric path as a one - step process . it should be noted , that lms are exclusively enthalpic in character because gibbs free energy gap between suitable intermediate and reactants is always greater than zero ( g > 0 ) due to the entropic factor ( ts ) . only then does the system start heading towards the activation barrier . by analysing barrier heights on paths a and b , it must be said that both regioisomeric substrate transformation routes are possible from the kinetic point of view . however , the favoured path is the one leading finally to 4-nitroisoxazole ( 3a ) . this conclusion is in agreement with forecasts based on reactivity indices analysis . a similar picture of 1 + 2a reaction is provided by calculations at more advanced levels of theory b3lyp/6 - 31+g(d ) , b3lyp/6 - 311g(d ) , and b3lyp/6 - 311+g(d ) ( fig . 1enthalpy profiles for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level enthalpy profiles for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level dft calculations also indicate a one - step mechanism of cycloaddition reaction of nitroacetylene to tacs 2b and 2c . the performed simulations suggest , however , a different reaction regioselectivity than would result from reactivity index analysis . in particular , in the case of cycloaddition reactions using phenyl azide ( 2b ) , both regioisomeric reaction channels should be permitted , but path b will be favoured . however , in the case of a cycloaddition reaction using phenyldiazomethane , path a must be treated as kinetically forbidden . dft calculations indicate that the only permitted cycloaddition channel is the path leading to the 4c adduct . analysing conversion routes for reagent pairs 1 + 2b , however , these are not the expected zwitterions 5 and 6 with cyclic conformation ( see . scheme 3 ) but zwitterions with extended their conversion to adducts can be executed via a step of dissociation into individual reagents and ( in the next step ) a stage of cycloaddition according to a one - step mechanism . 2enthalpy profiles for reaction between nitroacetylene ( 1 ) and phenylazide ( 2b ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level enthalpy profiles for reaction between nitroacetylene ( 1 ) and phenylazide ( 2b ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level introduction of a more polar medium of nitromethane ( = 38.20 ) into the reaction mixture does not result in qualitative changes in energy profiles of the reaction . however , their quantitative description does change . in particular , activation barriers in cases of relatively less polar cycloaddition reactions 1 + 2a 3a/4a and 1 + 2b 3b/4b increase . in case of the most polar of the considered cycloaddition reactions ( 1 + 2c 3c/4c ) , activation barriers become slightly lower in a more polar medium . however , in the case of all reactions leading to zwitterions 7 and 8 an increase in the polarity of the reaction medium facilitates the lowering of the activation barrier . however , this lowering is not significant enough to treat these processes as permitted kinetically . within the area of located pre - reaction complexes lm distances between reaction centres still remain far outside the range typical for bonds in intermediate states . medium polarity the nature of a charge - transfer complex ( ct ) [ see global electron density transfer indices ( gedt ) in table 2].table 2selected properties of critical structures for reactions between nitroacetylene ( 1 ) and tacs 2a2c according to b3lyp/6 - 31g(d ) calculationssolventreactionstructureinteratomic distancesl gedt / e x - c x - c y - c y - c r / l r / l r / l r / l toluene ( = 2.38 ) 1 + 2a lm a 3.1034.246 ts a 2.1300.3922.3920.3400.050.11 3a 1.3251.441 lm b 3.3285.598 ts b 2.3210.2482.2190.4600.210.11 4a 1.3251.441 1 + 2b lm a 1.4411.441 ts a 2.1170.4332.2410.3610.070.05 3b 1.3511.367 lm b 4.2237.214 ts b 2.2790.3172.1310.4210.100.08 4b 1.3541.350 lm c 4.741 ts c 1.7280.6990.26 7b 1.329 lm d 3.507 ts d 1.5490.8800.21 8b 1.383 1 + 2c lm a 4.1063.872 ts a 2.2850.4012.3350.4380.040.20 3c 1.4291.495 ts b 2.5130.2372.2200.5130.280.21 4c 1.4261.493 ts c 1.8450.6190.28 7c 1.336 ts d 1.6230.7980.36 8c 1.350nitromethane ( = 38.20 ) 1 + 2a lm a 3.1224.287 ts a 2.1060.4102.4090.3260.080.13 3a 1.3241.439 lm b 3.3965.636 ts b 2.3200.2682.2040.4590.190.12 4a 1.3391.431 1 + 2b lm a 5.7704.195 ts a 2.1030.4432.2480.3540.090.07 3b 1.3511.366 lm b 3.3065.770 ts b 2.3120.2942.1060.4380.140.10 4b 1.3551.348 lm c 4.675 ts c 1.7770.7020.29 7b 1.3690.29 lm d 3.615 ts d 1.5610.8800.33 8b 1.3940.32 1 + 2c lm a 5.4418.076 ts a 2.2190.4462.3880.4010.040.21 3c 1.4281.494 ts b 2.5960.1762.2000.5260.350.27 4c 1.4241.492 ts c 1.9150.5850.30 7c 1.3530.25 ts d 1.6210.8190.40 8c 1.3730.31 selected properties of critical structures for reactions between nitroacetylene ( 1 ) and tacs 2a2c according to b3lyp/6 - 31g(d ) calculations analysing geometrical parameters of ts leading to cycloadducts 3 and 4 , it can be noted that the advancement stage of new bonds depends on reagent nature and to a smaller extent on medium polarity . so , in the case of a reaction with benzonitrile n - oxide in toluene , the bond at the carbon atom introduced from nitroacetylene always is more advanced at the ts ( fig . formation of bonds in the area of the less favoured energetically tsb takes place in a more asynchronous manner ( l > 0.2 ) . both tss have a polar nature , which is reflected in the value of gedt index ( see table 2).fig . 3key structures for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level key structures for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level in case of a similar reaction with phenyl azide in the same reaction medium , both analysed tss show a similar level of asynchronicity . the more energetically favourable tsb is the more polar one of the two . on the other hand , the nature of tss of the reaction using phenyldiazomethane shows complete difference . in particular , the less energetically favourable tsa is characterized by an almost ideal synchronicity of new bond formation and a very weak polar character . on the other hand , in the area of tsb new bonds are formed in a strongly asynchronous manner , accompanied by a strong charge - transfer effect towards the dipolarophile substructure . the asynchronicity of this ts is the greatest among all considered critical structures . with the introduction of a stronger polar medium of nitromethane into the reaction medium , key parameters of the analysed structures do not change significantly . in the area of most tss , asynchronicity of formation of new bonds increases . it is always the bond with the more nucleophilic atom of the x 1,3-dipole ( fig . progress of this bond is always greater than 50 % ( see l values in table 2 ) . it should be noted that tss on paths leading to zwitterions 7 and 8 are always more polar than competitive units on paths leading to cycloadducts 3 and 4 . also in the case of these structures , the polarity increase of the reaction medium does not change their nature substantially . only their polarity becomes more pronounced ( see gedt values in table 2 ) . taking into account the nature of the electrophile nucleophile interactions , reactions of nitroacetylene with tacs 2a2c must be recognized as polar processes . there are two possible variants of the conversion of reagents into adducts : ( a ) a one - step polar mechanism and ( b ) two - step zwitterionic mechanism . in the first case , one should expect a transition state ( ts ) in the energy profile , in the second two tss connected by a valley corresponding to the zwitterionic intermediate ( respectively , 5 or 6 , see scheme 2 ) . as suggested by quantum - chemical calculations , in weakly polar toluene ( = 2.38 ) , the reaction of nitroacetylene with benzonitrile n - oxide takes place regardless of the regioisomeric path as a one - step process . it should be noted , that lms are exclusively enthalpic in character because gibbs free energy gap between suitable intermediate and reactants is always greater than zero ( g > 0 ) due to the entropic factor ( ts ) . only then does the system start heading towards the activation barrier . by analysing barrier heights on paths a and b , it must be said that both regioisomeric substrate transformation routes are possible from the kinetic point of view . however , the favoured path is the one leading finally to 4-nitroisoxazole ( 3a ) . a similar picture of 1 + 2a reaction is provided by calculations at more advanced levels of theory b3lyp/6 - 31+g(d ) , b3lyp/6 - 311g(d ) , and b3lyp/6 - 311+g(d ) ( fig . 1enthalpy profiles for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level enthalpy profiles for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level dft calculations also indicate a one - step mechanism of cycloaddition reaction of nitroacetylene to tacs 2b and 2c . the performed simulations suggest , however , a different reaction regioselectivity than would result from reactivity index analysis . in particular , in the case of cycloaddition reactions using phenyl azide ( 2b ) , both regioisomeric reaction channels should be permitted , but path b will be favoured . however , in the case of a cycloaddition reaction using phenyldiazomethane , path a must be treated as kinetically forbidden . dft calculations indicate that the only permitted cycloaddition channel is the path leading to the 4c adduct . analysing conversion routes for reagent pairs 1 + 2b , paths leading to zwitterionic structures were found ( fig . 2 ) . however , these are not the expected zwitterions 5 and 6 with cyclic conformation ( see . scheme 3 ) but zwitterions with extended their conversion to adducts can be executed via a step of dissociation into individual reagents and ( in the next step ) a stage of cycloaddition according to a one - step mechanism . 2enthalpy profiles for reaction between nitroacetylene ( 1 ) and phenylazide ( 2b ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level enthalpy profiles for reaction between nitroacetylene ( 1 ) and phenylazide ( 2b ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level introduction of a more polar medium of nitromethane ( = 38.20 ) into the reaction mixture does not result in qualitative changes in energy profiles of the reaction . however , their quantitative description does change . in particular , activation barriers in cases of relatively less polar cycloaddition reactions 1 + 2a 3a/4a and 1 + 2b 3b/4b increase . in case of the most polar of the considered cycloaddition reactions ( 1 + 2c 3c/4c ) , activation barriers become slightly lower in a more polar medium . however , in the case of all reactions leading to zwitterions 7 and 8 an increase in the polarity of the reaction medium facilitates the lowering of the activation barrier . however , this lowering is not significant enough to treat these processes as permitted kinetically . within the area of located pre - reaction complexes lm distances between reaction centres still remain far outside the range typical for bonds in intermediate states . none of them has , however regardless of reaction medium polarity the nature of a charge - transfer complex ( ct ) [ see global electron density transfer indices ( gedt ) in table 2].table 2selected properties of critical structures for reactions between nitroacetylene ( 1 ) and tacs 2a2c according to b3lyp/6 - 31g(d ) calculationssolventreactionstructureinteratomic distancesl gedt / e x - c x - c y - c y - c r / l r / l r / l r / l toluene ( = 2.38 ) 1 + 2a lm a 3.1034.246 ts a 2.1300.3922.3920.3400.050.11 3a 1.3251.441 lm b 3.3285.598 ts b 2.3210.2482.2190.4600.210.11 4a 1.3251.441 1 + 2b lm a 1.4411.441 ts a 2.1170.4332.2410.3610.070.05 3b 1.3511.367 lm b 4.2237.214 ts b 2.2790.3172.1310.4210.100.08 4b 1.3541.350 lm c 4.741 ts c 1.7280.6990.26 7b 1.329 lm d 3.507 ts d 1.5490.8800.21 8b 1.383 1 + 2c lm a 4.1063.872 ts a 2.2850.4012.3350.4380.040.20 3c 1.4291.495 ts b 2.5130.2372.2200.5130.280.21 4c 1.4261.493 ts c 1.8450.6190.28 7c 1.336 ts d 1.6230.7980.36 8c 1.350nitromethane ( = 38.20 ) 1 + 2a lm a 3.1224.287 ts a 2.1060.4102.4090.3260.080.13 3a 1.3241.439 lm b 3.3965.636 ts b 2.3200.2682.2040.4590.190.12 4a 1.3391.431 1 + 2b lm a 5.7704.195 ts a 2.1030.4432.2480.3540.090.07 3b 1.3511.366 lm b 3.3065.770 ts b 2.3120.2942.1060.4380.140.10 4b 1.3551.348 lm c 4.675 ts c 1.7770.7020.29 7b 1.3690.29 lm d 3.615 ts d 1.5610.8800.33 8b 1.3940.32 1 + 2c lm a 5.4418.076 ts a 2.2190.4462.3880.4010.040.21 3c 1.4281.494 ts b 2.5960.1762.2000.5260.350.27 4c 1.4241.492 ts c 1.9150.5850.30 7c 1.3530.25 ts d 1.6210.8190.40 8c 1.3730.31 selected properties of critical structures for reactions between nitroacetylene ( 1 ) and tacs 2a2c according to b3lyp/6 - 31g(d ) calculations new bonds are formed after the system reaches the ts . analysing geometrical parameters of ts leading to cycloadducts 3 and 4 , it can be noted that the advancement stage of new bonds depends on reagent nature and to a smaller extent on medium polarity . so , in the case of a reaction with benzonitrile n - oxide in toluene , the bond at the carbon atom introduced from nitroacetylene always is more advanced at the ts ( fig . 3 ) . formation of bonds in the area of the less favoured energetically tsb takes place in a more asynchronous manner ( l > 0.2 ) . both tss have a polar nature , which is reflected in the value of gedt index ( see table 2).fig . 3key structures for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level key structures for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level in case of a similar reaction with phenyl azide in the same reaction medium , both analysed tss show a similar level of asynchronicity . the more energetically favourable tsb is the more polar one of the two . on the other hand , in particular , the less energetically favourable tsa is characterized by an almost ideal synchronicity of new bond formation and a very weak polar character . on the other hand , in the area of tsb new bonds are formed in a strongly asynchronous manner , accompanied by a strong charge - transfer effect towards the dipolarophile substructure . the asynchronicity of this ts is the greatest among all considered critical structures . with the introduction of a stronger polar medium of nitromethane into the reaction medium , key parameters of the analysed structures do not change significantly . in the area of most tss , asynchronicity of formation of new bonds increases . it is always the bond with the more nucleophilic atom of the x 1,3-dipole ( fig . progress of this bond is always greater than 50 % ( see l values in table 2 ) . it should be noted that tss on paths leading to zwitterions 7 and 8 are always more polar than competitive units on paths leading to cycloadducts 3 and 4 . also in the case of these structures , the polarity increase of the reaction medium does not change their nature substantially . only their polarity becomes more pronounced ( see gedt values in table 2 ) . dft calculations for various theory levels show that [ 2 + 3 ] cycloaddition reactions of nitroacetylene with allenyl - type tacs take place according to the polar mechanism . this is not , however , the expected two - step , zwitterionic mechanism , but a one - step mechanism . zwitterionic structures with the extended conformation may theoretically form along competitive paths . however , this route is supported by neither kinetic nor thermodynamic factors . despite the clearly polar nature of the reactions discussed , the influence of the polarity of the reaction medium on their kinetics and the nature of critical structures is relatively small . all calculations reported in this paper were performed on zeus supercomputer in the cyfronet computational centre in cracow . global and local electronic properties of reactants were estimated according to the equations described earlier . in particular , the electronic chemical potentials ( ) and chemical hardness ( ) were evaluated in terms of one - electron energies of fmo ( ehomo and elumo ) using the equations:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu \approx ( e_{\text{homo } } + e_{\text{lumo } } ) /2\quad next , the values of and were then used for the calculation of global electrophilicity ( ) [ 16 , 17 ] according to the formula:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \omega = \mu^{2 } /2\eta $ $ \end{document}=2/2and the global nucleophilicity ( n ) of tacs can be expressed in terms of equation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n = e_{{{\text{homo}}\ ; ( { \text{tac ) } } } } - e_{{{\text{homo}}\;\left ( { \text{tetracyanoethene } } \right ) } } . the local electrophilicity ( k ) condensed to atom k was calculated by projecting the index onto any reaction centre k in the molecule by using parr function pk+ : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \omega_{\text{k } } = { \text{p}}^ { + } _ { \text{k } } \cdot\omega . $ $ \end{document}k = pk+. the local nucleophilicity ( nk ) condensed to atom k was calculated using global nucleophilicity n and parr function pk according to the formula:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{\text{k } } = { \text{p}}^ { - } _ { \text{k } } \cdot\,n . reactivity indexes calculated on this way are collected in table 1 . for the simulation of the reaction paths hybrid functional b3lyp with the 6 - 31g(d ) , basis set included in the gaussian 09 package it was found previously that the b3lyp/6 - 31g(d ) calculations illustrate well the structure of tss in polar [ 2 + 3 ] cycloadditions involving conjugated nitroalkenes [ 14 , 23 , 24 ] . the critical points on reaction paths were localized in an analogous manner as in the case of the previously analysed [ 2 + 3 ] cycloadditions of ( z)-c , n - diphenylnitrone with gem - dinitroethene . in particular , for structure optimization of the reactants and the reaction products the berny algorithm was applied . the tss were verified by diagonalization of the hessian matrix and by analysis of the intrinsic reaction coordinates ( irc ) . in addition , similar simulations using more advanced b3lyp/6 - 31+g(d ) , b3lyp/6 - 311g(d ) , as well as b3lyp/6 - 311+g(d ) theoretical levels were performed . all calculations were carried out for the simulated presence of toluene or nitromethane as the reaction medium . for optimized structures the thermochemical data for the temperature t = 298 k and pressure p = 1 atm were computed using vibrational analysis data . global electron density transfer ( gedt ) was calculated according to the formula:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ gedt = - \varsigma q_{\text{a } } , $ $ \end{document}gedt=-qa , where qa is the net mulliken charge and the sum is taken over all the atoms of dipolarophile . indexes of -bonds development ( l ) were calculated according to formula : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ l_{\text{a - b } } = 1 - \frac{{r_{{{\text{a}}\text { - } { \text{b}}}}^{\text{ts } } - r_{{{\text{a}}\text { - } { \text{b}}}}^{\text{p } } } } { { r_{{{\text{a}}\text { - } { \text{b}}}}^{\text{p } } } } , $ $ \end{document}la - b=1-ra - bts - ra - bpra - bp , where rabts is the distance between the reaction centres a and b at the ts and rabp is the same distance at the corresponding product . the kinetic parameters as well as essential properties of critical structures are displayed in tables 2 , 3 , 4.table 3eyring parameters for reactions between nitroacetylene ( 1 ) and tacs 2a-2c according to b3lyp/6 - 31g(d ) calculations1,3-dipoletransitiontoluene ( = 2.38)nitromethane ( = 38.20)h / kj mol g / kj mol s / j mol k h / kj mol g / kj mol s / j mol k 2a 1 + 2a lm a 13.821.8119.27.126.4112.1 1 + 2a ts a 41.492.9172.445.695.0166.5 1 + 2a 3a 351.5287.4214.2347.7284.5212.1 1 + 2a lm b 7.928.5122.24.230.5117.2 1 + 2a ts b 46.095.8167.446.496.2166.9 1 + 2a 4a 348.9287.9204.6347.3278.2231.4 2b 1 + 2b lm a 2.924.392.01.724.386.2 1 + 2b ts a 59.4109.6168.261.5111.7168.6 1 + 2b 3b 298.7238.1202.5299.6239.3202.1 1 + 2b lm b 5.023.094.62.926.498.3 1 + 2b ts b 59.0108.4164.858.2107.1164.8 1 + 2b 4b 327.2266.1205.4332.2271.5204.6 1 + 2b lm c 5.421.891.62.923.087.0 1 + 2b ts c 125.1169.9150.2111.3157.7155.6 1 + 2b 7b 72.8120.5160.766.1114.6163.2 1 + 2b lm d 1.326.894.1 1 + 2b ts d 154.8205.9171.1152.7205.0174.9 1 + 2b 8b 136.0186.6169.5128.9179.5169.9 2c 1 + 2c lm a 5.030.1118.45.919.785.4 1 + 2c ts a 37.788.3169.538.187.9167.4 1 + 2c 3c 231.0172.8194.6231.8172.4199.6 1 + 2c ts b 33.582.4164.428.577.0164.0 1 + 2c 3c 233.5176.1192.5238.1180.3193.7 1 + 2c ts c 63.6108.4151.050.695.8151.9 1 + 2c 7c 9.241.0168.617.232.6166.9 1 + 2c ts d 109.2161.9176.697.9150.6177.8 1 + 2c 8c 66.9115.9164.859.4107.9162.3table 4eyring parameters for reactions between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31+g(d ) , b3lyp/6 - 311g(d ) , and b3lyp/6 - 311+g(d ) theory levelstheory leveltransitionh / kj mol g / kj mol s / j mol k b3lyp/6 - 31+g(d ) 1 + 2a lm a 10.018.093.3 1 + 2a ts a 48.595.8158.6 1 + 2a 3a 333.5270.7211.7 1 + 2a lm b 2.932.6119.7 1 + 2a ts b 53.1102.5165.3 1 + 2a 4a 332.2271.1204.2b3lyp/6 - 311g(d ) 1 + 2a lm a 11.719.7105.4 1 + 2a ts a 49.8100.0168.2 1 + 2a 3a 319.7256.5211.3 1 + 2a lm b 7.126.8112.5 1 + 2a ts b 55.6104.6164.8 1 + 2a 4a 318.4257.7204.2b3lyp/6 - 311+g(d ) 1 + 2a lm a 10.017.692.0 1 + 2a ts a 53.699.2154.0 1 + 2a 3a 310.9248.5209.2 1 + 2a lm b 0.826.892.5 1 + 2a ts b 59.0107.5163.6 1 + 2a 4a 309.6248.1205.4 eyring parameters for reactions between nitroacetylene ( 1 ) and tacs 2a-2c according to b3lyp/6 - 31g(d ) calculations eyring parameters for reactions between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31+g(d ) , b3lyp/6 - 311g(d ) , and b3lyp/6 - 311+g(d ) theory levels

abstract[2 + 3 ] cycloaddition reactions of nitroacetylene with allenyl - type three - atom components take place according to the polar , but a one - step mechanism . alternatively to cycloadducts , during the reaction between the aforementioned reagents , zwitterionic structures with extended conformation may be formally created . however , this route is supported by neither kinetic nor thermodynamic factors.graphical abstract

Abstract Graphical Abstract Introduction Results and discussion Reaction profiles Key structures Conclusion Computational details

[ 2 + 3 ] cycloaddition reactions of nitroacetylene with allenyl - type three - atom components take place according to the polar , but a one - step mechanism . alternatively to cycloadducts , during the reaction between the aforementioned reagents , zwitterionic structures with extended nitroacetylenes belong because of the presence of a strongly electron withdrawing no2-group to a class of strongly electrophilic acetylenes . however , no systematic studies of its participation in reactions of [ 2 + 3 ] cycloaddition have been performed to date . this work initiates research in this area ; in particular , within the study , complex , quantum - chemical studies of the [ 2 + 3 ] cycloaddition reaction of nitroacetylene ( 1 ) to selected allenyl - type three - atom components ( tacs ) . three such tacs were selected , often tested in recent years as components of [ 2 + 3 ] cycloaddition reactions with electrophilic ethylenes : benzonitrile n - oxide ( 2a ) [ 36 ] , phenyl azide ( 2b ) [ 68 ] , and phenyldiazomethane ( 2c ) [ 6 , 9 ] ( scheme 1 ) . it must be stressed here that the problematics of the [ 2 + 3 ] cycloaddition reaction of the aforementioned reagents is also very interesting from a mechanistic point of view . these reactions may also take place according to a one - step mechanism , as well as to a two - step mechanism , with a zwitterionic intermediate [ 1315 ] . the two - step mechanism is facilitated in this case by ( 1 ) the strongly electrophilic character of the dipolarophile and the nucleophilic nature of the tacs and ( 2 ) unequal screening of the reaction centres of both reagents . if it is assumed that the reaction route is determined by an attack of the more strongly nucleophilic reaction centre of the tac on the more strongly electrophilic reaction centre of nitroacetylene , then the favoured direction of reagent transformation should always be determined by the a path ( see scheme 2 ) . taking into account the nature of the electrophile nucleophile interactions , reactions of nitroacetylene with tacs 2a2c must be recognized as polar processes . there are two possible variants of the conversion of reagents into adducts : ( a ) a one - step polar mechanism and ( b ) two - step zwitterionic mechanism . as suggested by quantum - chemical calculations , in weakly polar toluene ( = 2.38 ) , the reaction of nitroacetylene with benzonitrile n - oxide takes place regardless of the regioisomeric path as a one - step process . 1enthalpy profiles for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level enthalpy profiles for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level dft calculations also indicate a one - step mechanism of cycloaddition reaction of nitroacetylene to tacs 2b and 2c . in particular , in the case of cycloaddition reactions using phenyl azide ( 2b ) , both regioisomeric reaction channels should be permitted , but path b will be favoured . scheme 3 ) but zwitterions with extended their conversion to adducts can be executed via a step of dissociation into individual reagents and ( in the next step ) a stage of cycloaddition according to a one - step mechanism . 2enthalpy profiles for reaction between nitroacetylene ( 1 ) and phenylazide ( 2b ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level enthalpy profiles for reaction between nitroacetylene ( 1 ) and phenylazide ( 2b ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level introduction of a more polar medium of nitromethane ( = 38.20 ) into the reaction mixture does not result in qualitative changes in energy profiles of the reaction . however , in the case of all reactions leading to zwitterions 7 and 8 an increase in the polarity of the reaction medium facilitates the lowering of the activation barrier . however , this lowering is not significant enough to treat these processes as permitted kinetically . medium polarity the nature of a charge - transfer complex ( ct ) [ see global electron density transfer indices ( gedt ) in table 2].table 2selected properties of critical structures for reactions between nitroacetylene ( 1 ) and tacs 2a2c according to b3lyp/6 - 31g(d ) calculationssolventreactionstructureinteratomic distancesl gedt / e x - c x - c y - c y - c r / l r / l r / l r / l toluene ( = 2.38 ) 1 + 2a lm a 3.1034.246 ts a 2.1300.3922.3920.3400.050.11 3a 1.3251.441 lm b 3.3285.598 ts b 2.3210.2482.2190.4600.210.11 4a 1.3251.441 1 + 2b lm a 1.4411.441 ts a 2.1170.4332.2410.3610.070.05 3b 1.3511.367 lm b 4.2237.214 ts b 2.2790.3172.1310.4210.100.08 4b 1.3541.350 lm c 4.741 ts c 1.7280.6990.26 7b 1.329 lm d 3.507 ts d 1.5490.8800.21 8b 1.383 1 + 2c lm a 4.1063.872 ts a 2.2850.4012.3350.4380.040.20 3c 1.4291.495 ts b 2.5130.2372.2200.5130.280.21 4c 1.4261.493 ts c 1.8450.6190.28 7c 1.336 ts d 1.6230.7980.36 8c 1.350nitromethane ( = 38.20 ) 1 + 2a lm a 3.1224.287 ts a 2.1060.4102.4090.3260.080.13 3a 1.3241.439 lm b 3.3965.636 ts b 2.3200.2682.2040.4590.190.12 4a 1.3391.431 1 + 2b lm a 5.7704.195 ts a 2.1030.4432.2480.3540.090.07 3b 1.3511.366 lm b 3.3065.770 ts b 2.3120.2942.1060.4380.140.10 4b 1.3551.348 lm c 4.675 ts c 1.7770.7020.29 7b 1.3690.29 lm d 3.615 ts d 1.5610.8800.33 8b 1.3940.32 1 + 2c lm a 5.4418.076 ts a 2.2190.4462.3880.4010.040.21 3c 1.4281.494 ts b 2.5960.1762.2000.5260.350.27 4c 1.4241.492 ts c 1.9150.5850.30 7c 1.3530.25 ts d 1.6210.8190.40 8c 1.3730.31 selected properties of critical structures for reactions between nitroacetylene ( 1 ) and tacs 2a2c according to b3lyp/6 - 31g(d ) calculations analysing geometrical parameters of ts leading to cycloadducts 3 and 4 , it can be noted that the advancement stage of new bonds depends on reagent nature and to a smaller extent on medium polarity . 3key structures for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level key structures for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level in case of a similar reaction with phenyl azide in the same reaction medium , both analysed tss show a similar level of asynchronicity . taking into account the nature of the electrophile nucleophile interactions , reactions of nitroacetylene with tacs 2a2c must be recognized as polar processes . there are two possible variants of the conversion of reagents into adducts : ( a ) a one - step polar mechanism and ( b ) two - step zwitterionic mechanism . as suggested by quantum - chemical calculations , in weakly polar toluene ( = 2.38 ) , the reaction of nitroacetylene with benzonitrile n - oxide takes place regardless of the regioisomeric path as a one - step process . 1enthalpy profiles for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level enthalpy profiles for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level dft calculations also indicate a one - step mechanism of cycloaddition reaction of nitroacetylene to tacs 2b and 2c . in particular , in the case of cycloaddition reactions using phenyl azide ( 2b ) , both regioisomeric reaction channels should be permitted , but path b will be favoured . scheme 3 ) but zwitterions with extended their conversion to adducts can be executed via a step of dissociation into individual reagents and ( in the next step ) a stage of cycloaddition according to a one - step mechanism . 2enthalpy profiles for reaction between nitroacetylene ( 1 ) and phenylazide ( 2b ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level enthalpy profiles for reaction between nitroacetylene ( 1 ) and phenylazide ( 2b ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level introduction of a more polar medium of nitromethane ( = 38.20 ) into the reaction mixture does not result in qualitative changes in energy profiles of the reaction . however , in the case of all reactions leading to zwitterions 7 and 8 an increase in the polarity of the reaction medium facilitates the lowering of the activation barrier . however , this lowering is not significant enough to treat these processes as permitted kinetically . 3key structures for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level key structures for reaction between nitroacetylene ( 1 ) and benzonitrile n - oxide ( 2a ) in toluene according to dft calculations at b3lyp/6 - 31g(d ) theory level in case of a similar reaction with phenyl azide in the same reaction medium , both analysed tss show a similar level of asynchronicity . dft calculations for various theory levels show that [ 2 + 3 ] cycloaddition reactions of nitroacetylene with allenyl - type tacs take place according to the polar mechanism . this is not , however , the expected two - step , zwitterionic mechanism , but a one - step mechanism . zwitterionic structures with the extended conformation may theoretically form along competitive paths . however , this route is supported by neither kinetic nor thermodynamic factors . global and local electronic properties of reactants were estimated according to the equations described earlier . in particular , the electronic chemical potentials ( ) and chemical hardness ( ) were evaluated in terms of one - electron energies of fmo ( ehomo and elumo ) using the equations:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu \approx ( e_{\text{homo } } + e_{\text{lumo } } ) /2\quad next , the values of and were then used for the calculation of global electrophilicity ( ) [ 16 , 17 ] according to the formula:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \omega = \mu^{2 } /2\eta $ $ \end{document}=2/2and the global nucleophilicity ( n ) of tacs can be expressed in terms of equation:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n = e_{{{\text{homo}}\ ; ( { \text{tac ) } } } } - e_{{{\text{homo}}\;\left ( { \text{tetracyanoethene } } \right ) } } . the local nucleophilicity ( nk ) condensed to atom k was calculated using global nucleophilicity n and parr function pk according to the formula:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n_{\text{k } } = { \text{p}}^ { - } _ { \text{k } } \cdot\,n . for the simulation of the reaction paths hybrid functional b3lyp with the 6 - 31g(d ) , basis set included in the gaussian 09 package it was found previously that the b3lyp/6 - 31g(d ) calculations illustrate well the structure of tss in polar [ 2 + 3 ] cycloadditions involving conjugated nitroalkenes [ 14 , 23 , 24 ] . the critical points on reaction paths were localized in an analogous manner as in the case of the previously analysed [ 2 + 3 ] cycloadditions of ( z)-c , n - diphenylnitrone with gem - dinitroethene . global electron density transfer ( gedt ) was calculated according to the formula:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ gedt = - \varsigma q_{\text{a } } , $ $ \end{document}gedt=-qa , where qa is the net mulliken charge and the sum is taken over all the atoms of dipolarophile . indexes of -bonds development ( l ) were calculated according to formula : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ l_{\text{a - b } } = 1 - \frac{{r_{{{\text{a}}\text { - } { \text{b}}}}^{\text{ts } } - r_{{{\text{a}}\text { - } { \text{b}}}}^{\text{p } } } } { { r_{{{\text{a}}\text { - } { \text{b}}}}^{\text{p } } } } , $ $ \end{document}la - b=1-ra - bts - ra - bpra - bp , where rabts is the distance between the reaction centres a and b at the ts and rabp is the same distance at the corresponding product .

nearly 25 million americans have diabetes , and if current trends persist , more than one in three american adults are projected to have diabetes by 2050 ( 13 ) . coronary heart disease and stroke are the leading causes of excess morbidity , mortality , and cost in adults with diabetes ( 4 ) . advances in medical care and improvements in cardiovascular ( cv ) risk factor control have resulted in the reduction of cv events and deaths among adults with diabetes ( 2,3 ) . at the population level , however , increased diabetes prevalence and more years lived with diabetes offset much of the improvement in rates of cv events and mortality such that the absolute burden of cv events in those with diabetes remains high . major risk factors for cv events are well known , and effective drugs and behavioral interventions are available to manage blood pressure ( bp ) , ldl cholesterol ( ldl - c ) , glucose , and tobacco use . estimating the prevalence of inadequately controlled risk factors and the proportion of major cv events and all - cause mortality associated with each of these uncontrolled cv risk factors could help prioritize clinical and public health strategies to further reduce the burden of cv disease ( cvd ) in adults with diabetes . because risk factor prevalence , risk factor association with cv events , and impact of treatment of uncontrolled risk factors on cv events differ depending on whether an adult with diabetes has cvd , we conducted separate analyses of those with versus without cvd at baseline . the surveillance , prevention , and management of diabetes mellitus ( supreme - dm ) datalink was developed in a network of 11 large , integrated health care organizations in the hmo research network in the united states ( 5 ) . members of these health systems include 16 million persons who receive insurance through a variety of mechanisms , including commercial plans , self - pay , medicare , medicaid , and other federal- or state - supported insurance programs . health systems participating in supreme - dm include geisinger health system ( pennsylvania ) , group health ( washington ) , healthpartners ( minnesota and wisconsin ) , henry ford health system ( michigan ) , marshfield clinic ( wisconsin ) , and kaiser permanente regions in colorado ( kpco ) , northern california ( kpnc ) , southern california ( kpsc ) , hawaii ( kphi ) , georgia ( kpse ) , and the northwest ( oregon and washington [ kpnw ] ) . research units embedded in these health care organizations developed a distributed virtual data warehouse that contains information on demographics , pharmacy , laboratory , diagnoses , and procedures from outpatient and inpatient encounters extracted from their respective electronic medical record and administrative data ( claims ) systems ( 6 ) . mortality data were extracted from administrative registries , state death registries , and the national death index ( in two health systems ) and were available with a 1-year lag . these data were used to construct the supreme - dm datalink , the largest and most clinically detailed diabetes population ever assembled in the united states outside the u.s . department of veterans affairs ( 5 ) . to be included in the analyses reported here , subjects had to 1 ) be at least 20 years old ; 2 ) be insured ; 3 ) have received care in one of the 11 participating health systems between 1 january 2005 and 31 december 2010 ; 4 ) have an assigned diabetes identification date ; 5 ) have had at least 6 months of continuous enrollment before the cohort entry date ; and 6 ) have had one or more laboratory measurements of fasting plasma glucose , a1c , or ldl - c and bp . the cohort entry date was defined as the latest of 1 ) the diabetes identification date , 2 ) 1 january 2005 , or 3 ) the date the patient turned 20 years old . study criteria for diabetes required either one or more inpatient diabetes diagnosis codes ( icd-9 clinical modification 250.x , 357.2 , 366.41 , 362.01362.07 ) or any combination of two or more of the following events on separate days no more than 2 years apart : 1 ) a1c 6.5% ( 48 mmol / mol ) , 2 ) fasting plasma glucose 126 mg / dl , 3 ) random plasma glucose 200 mg / dl , 4 ) outpatient visit diabetes diagnosis code ( same codes as that used for inpatients ) , or 5 ) any filled prescription for a glucose - lowering medication . patients prescribed metformin or thiazolidinediones with no other indicator of diabetes were not included because these agents could be used for other conditions . patients were assigned a diabetes identification date defined as the second date on which they met study criteria for diabetes . we extracted baseline data within 1 year of the cohort entry date for each subject . when multiple baseline clinical measures were available , we selected the measure closest to the cohort entry date , except baseline bp , which was averaged from all outpatient readings within 1 year of the cohort entry date . we defined inadequate baseline control following american diabetes association guidelines ( 10,11)ldl - c 100 mg / dl and a1c 7% ( 53 mmol / mol)and the eighth joint national committee 2014 guidelines ( 11)systolic bp 140 mmhg or diastolic bp 90 mmhg or current smoking . additional variables included age ; sex ; bmi ( kilograms per meters squared ) ; hdl cholesterol ( hdl - c ) ; treatment with antihypertensive medications , statins , insulin , or other glucose - lowering drugs ; incident diabetes status ; and preexisting comorbidities . to be classified as having incident diabetes in our cohort , a patient was required to have 18 months of antecedent enrollment with no evidence of diabetes before the diabetes identification date ( 5 ) . baseline status for comorbid conditions was assigned based on two or more outpatient diagnosis codes or one or more inpatient diagnosis codes on or before the cohort entry date for chronic kidney disease ( ckd ; icd-9 code 585.xx ) , cvd ( icd-9 codes 410414.xx and 429.2 ) , heart failure ( hf ; icd-9 codes 428428.9 ) , hemorrhagic stroke ( icd-9 codes 430432.9 ) , ischemic stroke ( icd-9 codes 433434.91 ) , and transient ischemic attack ( icd-9 code 435.xx ) . multiple imputation was used for missing data on a1c , ldl - c , and hdl - c following previous work using the supreme - dm cohort ( 12 ) . major cv events were ascertained based on the date of primary hospital discharge icd-9 diagnosis codes as follows : 1 ) myocardial infarction ( mi)/acute coronary syndrome ( acs ) ( icd-9 codes 410.0410.91 , 411.1 , 411.8 ) , 2 ) ischemic and hemorrhagic stroke ( icd-9 codes 433434.91 , 430432.9 ) , or 3 ) hf ( icd-9 codes 428428.9 ) . these events were defined as the first occurrence 7 days or more after the cohort entry date ( to avoid inpatient stays starting before the cohort entry date ) . all - cause mortality was ascertained through 31 december 2010 , independent of health insurance enrollment at the time of death . time to event was calculated as days elapsed from the cohort entry date + 7 days to the hospital discharge date associated with the given event . follow - up time was censored 1 ) when disenrolled from health insurance for more than 90 days , 2 ) at the time of death , or 3 ) at the end of the study period ( 31 december 2012 ) . follow - up time after death was censored at 31 december 2010 to accommodate lag in the currency of death index data . models to predict hospitalization for mi / acs , stroke , hf , and all - cause mortality were constructed with generalized linear models using poisson distribution and log link . a two - step analysis was implemented to evaluate the inclusion of baseline covariates in the prediction model . in the first step , the associations of individual baseline variables with each major cv event and with all - cause mortality were evaluated in separate models for subjects with and without cvd at baseline , using only age and sex as covariates . individual baseline variables included uncontrolled risk factors ( bp , ldl - c , a1c , and tobacco status ) , medication use variables , bmi , hdl - c , incident diabetes , presence of ckd , race / ethnicity , and study site . nonmonotonic associations of continuous variables with cv events or all - cause mortality were explored . in the second step , all baseline factors associated with a major cv event or with all - cause mortality in step 1 and with p < 0.05 as main effects were retained for inclusion in the final models . to estimate the population - attributable fraction for each uncontrolled risk factor in adults with diabetes with and without baseline cvd , we calculated the average attributable fraction ( aaf ) , as described by rckinger et al . the aaf is preferred to alternative measures of population - attributable fraction because of its properties of additivity and symmetry ( 14,15 ) . aaf was evaluated for inadequately controlled risk factors ( bp , ldl - c , a1c , and tobacco use ) in the same poisson model that included the other risk factors age , sex , hdl - c , ckd , incident ( vs. prevalent ) diabetes , and medication use variables as main effects for adults with diabetes with or without baseline cvd . cis for aaf point estimates were estimated using delta method approximations , as described by jewell ( 16 ) . to evaluate whether the lag between the cohort entry date and the diabetes identification date created an immortal time bias , we conducted a sensitivity analysis by restricting the analysis to those who were identified in the datalink after 2005 . in addition , we estimated the nondifferential misclassification of cv events on estimates of aaf following the work by vogel and gefeller ( 17 ) . this study was reviewed in advance , approved , and monitored by the kpco institutional review board ( irb ) , and each participating site either ceded oversight to the kpco irb or received approval and oversight from their local irb . the surveillance , prevention , and management of diabetes mellitus ( supreme - dm ) datalink was developed in a network of 11 large , integrated health care organizations in the hmo research network in the united states ( 5 ) . members of these health systems include 16 million persons who receive insurance through a variety of mechanisms , including commercial plans , self - pay , medicare , medicaid , and other federal- or state - supported insurance programs . health systems participating in supreme - dm include geisinger health system ( pennsylvania ) , group health ( washington ) , healthpartners ( minnesota and wisconsin ) , henry ford health system ( michigan ) , marshfield clinic ( wisconsin ) , and kaiser permanente regions in colorado ( kpco ) , northern california ( kpnc ) , southern california ( kpsc ) , hawaii ( kphi ) , georgia ( kpse ) , and the northwest ( oregon and washington [ kpnw ] ) . research units embedded in these health care organizations developed a distributed virtual data warehouse that contains information on demographics , pharmacy , laboratory , diagnoses , and procedures from outpatient and inpatient encounters extracted from their respective electronic medical record and administrative data ( claims ) systems ( 6 ) . mortality data were extracted from administrative registries , state death registries , and the national death index ( in two health systems ) and were available with a 1-year lag . these data were used to construct the supreme - dm datalink , the largest and most clinically detailed diabetes population ever assembled in the united states outside the u.s . to be included in the analyses reported here , subjects had to 1 ) be at least 20 years old ; 2 ) be insured ; 3 ) have received care in one of the 11 participating health systems between 1 january 2005 and 31 december 2010 ; 4 ) have an assigned diabetes identification date ; 5 ) have had at least 6 months of continuous enrollment before the cohort entry date ; and 6 ) have had one or more laboratory measurements of fasting plasma glucose , a1c , or ldl - c and bp . the cohort entry date was defined as the latest of 1 ) the diabetes identification date , 2 ) 1 january 2005 , or 3 ) the date the patient turned 20 years old . study criteria for diabetes required either one or more inpatient diabetes diagnosis codes ( icd-9 clinical modification 250.x , 357.2 , 366.41 , 362.01362.07 ) or any combination of two or more of the following events on separate days no more than 2 years apart : 1 ) a1c 6.5% ( 48 mmol / mol ) , 2 ) fasting plasma glucose 126 mg / dl , 3 ) random plasma glucose 200 mg / dl , 4 ) outpatient visit diabetes diagnosis code ( same codes as that used for inpatients ) , or 5 ) any filled prescription for a glucose - lowering medication . patients prescribed metformin or thiazolidinediones with no other indicator of diabetes were not included because these agents could be used for other conditions . patients were assigned a diabetes identification date defined as the second date on which they met study criteria for diabetes . we extracted baseline data within 1 year of the cohort entry date for each subject . when multiple baseline clinical measures were available , we selected the measure closest to the cohort entry date , except baseline bp , which was averaged from all outpatient readings within 1 year of the cohort entry date . we defined inadequate baseline control following american diabetes association guidelines ( 10,11)ldl - c 100 mg / dl and a1c 7% ( 53 mmol / mol)and the eighth joint national committee 2014 guidelines ( 11)systolic bp 140 mmhg or diastolic bp 90 mmhg or current smoking . additional variables included age ; sex ; bmi ( kilograms per meters squared ) ; hdl cholesterol ( hdl - c ) ; treatment with antihypertensive medications , statins , insulin , or other glucose - lowering drugs ; incident diabetes status ; and preexisting comorbidities . to be classified as having incident diabetes in our cohort , a patient was required to have 18 months of antecedent enrollment with no evidence of diabetes before the diabetes identification date ( 5 ) . baseline status for comorbid conditions was assigned based on two or more outpatient diagnosis codes or one or more inpatient diagnosis codes on or before the cohort entry date for chronic kidney disease ( ckd ; icd-9 code 585.xx ) , cvd ( icd-9 codes 410414.xx and 429.2 ) , heart failure ( hf ; icd-9 codes 428428.9 ) , hemorrhagic stroke ( icd-9 codes 430432.9 ) , ischemic stroke ( icd-9 codes 433434.91 ) , and transient ischemic attack ( icd-9 code 435.xx ) . multiple imputation was used for missing data on a1c , ldl - c , and hdl - c following previous work using the supreme - dm cohort ( 12 ) . major cv events were ascertained based on the date of primary hospital discharge icd-9 diagnosis codes as follows : 1 ) myocardial infarction ( mi)/acute coronary syndrome ( acs ) ( icd-9 codes 410.0410.91 , 411.1 , 411.8 ) , 2 ) ischemic and hemorrhagic stroke ( icd-9 codes 433434.91 , 430432.9 ) , or 3 ) hf ( icd-9 codes 428428.9 ) . these events were defined as the first occurrence 7 days or more after the cohort entry date ( to avoid inpatient stays starting before the cohort entry date ) . all - cause mortality was ascertained through 31 december 2010 , independent of health insurance enrollment at the time of death . time to event was calculated as days elapsed from the cohort entry date + 7 days to the hospital discharge date associated with the given event . follow - up time was censored 1 ) when disenrolled from health insurance for more than 90 days , 2 ) at the time of death , or 3 ) at the end of the study period ( 31 december 2012 ) . follow - up time after death was censored at 31 december 2010 to accommodate lag in the currency of death index data . models to predict hospitalization for mi / acs , stroke , hf , and all - cause mortality were constructed with generalized linear models using poisson distribution and log link . a two - step analysis was implemented to evaluate the inclusion of baseline covariates in the prediction model . in the first step , the associations of individual baseline variables with each major cv event and with all - cause mortality were evaluated in separate models for subjects with and without cvd at baseline , using only age and sex as covariates . individual baseline variables included uncontrolled risk factors ( bp , ldl - c , a1c , and tobacco status ) , medication use variables , bmi , hdl - c , incident diabetes , presence of ckd , race / ethnicity , and study site . nonmonotonic associations of continuous variables with cv events or all - cause mortality were explored . in the second step , all baseline factors associated with a major cv event or with all - cause mortality in step 1 and with p < 0.05 as main effects were retained for inclusion in the final models . to estimate the population - attributable fraction for each uncontrolled risk factor in adults with diabetes with and without baseline cvd , we calculated the average attributable fraction ( aaf ) , as described by rckinger et al . the aaf is preferred to alternative measures of population - attributable fraction because of its properties of additivity and symmetry ( 14,15 ) . aaf was evaluated for inadequately controlled risk factors ( bp , ldl - c , a1c , and tobacco use ) in the same poisson model that included the other risk factors age , sex , hdl - c , ckd , incident ( vs. prevalent ) diabetes , and medication use variables as main effects for adults with diabetes with or without baseline cvd . cis for aaf point estimates were estimated using delta method approximations , as described by jewell ( 16 ) . to evaluate whether the lag between the cohort entry date and the diabetes identification date created an immortal time bias , we conducted a sensitivity analysis by restricting the analysis to those who were identified in the datalink after 2005 . in addition , we estimated the nondifferential misclassification of cv events on estimates of aaf following the work by vogel and gefeller ( 17 ) . this study was reviewed in advance , approved , and monitored by the kpco institutional review board ( irb ) , and each participating site either ceded oversight to the kpco irb or received approval and oversight from their local irb . baseline characteristics of the 859,617 study subjects with diabetes and with or without cvd are presented in table 1 . mean follow up was 59 months , and 4,233,786 person - years of observation for cv events were included in the analysis . mean age was 59 years ( sd 14 years ) ; 48% were female , 45% were white , and 31% had cvd . compared with those without baseline cvd , those with baseline cvd had better control of smoking ( 8.0% vs. 9.8% ) , a1c 7% ( 53 mmol / mol ) ( 42% vs. 53% ) , and ldl - c 100 mg / dl ( 38% vs. 58% ) , and they had similar proportions of subjects with systolic / diastolic bp 140/90 mmhg ( 23% vs. 21% ) . baseline characteristics of 859,617 adults with diabetes with and without cvd receiving care from 2005 to 2010 at 11 u.s . health care organizations and prevalence of inadequate control of bp ( systolic / diastolic 140/90 mmhg ) , ldl - c ( 100 mg / dl ) , a1c ( 8% [ 64 mmol / mol ] ) , and current smoking the associations of inadequately controlled baseline a1c , bp , ldl - c , and tobacco use with subsequent cv events and all - cause mortality are presented in table 2 . cv events and all - cause mortality also were associated with sex , age , race / ethnicity , ckd , hdl - c , incident diabetes status , and medication use variables ( table 1 ) . however , major cv events and all - cause mortality were not significantly associated with baseline bmi ( data not shown ) . in subjects without baseline cvd , all inadequately controlled cv risk factors were associated with higher risk of mi / acs , stroke , and hf , but only smoking and bp were associated with all - cause mortality . a similar pattern emerged in subjects with baseline cvd : all inadequately controlled cv risk factors were associated with an increase in mi / acs and stroke . current smoking , bp , and a1c ( but not ldl - c ) were associated with an increase in hf . rate ratios ( 95% cis ) of inadequate controlled risk factors with cv events and all - cause mortality in adults with diabetes with and without baseline cvd estimates adjusted for age , sex , race / ethnicity , diabetes medication , statins , bp medication , ckd at baseline , incident diabetes at baseline , low hdl - c ( 40 mg / dl [ men]/50 mg / dl [ women ] ) , and all other inadequately controlled factors as described in the table . rates of major cv events and all - cause mortality in adults with diabetes and cvd were substantially higher than in those with diabetes but no cvd ( table 3 ) . five - year cv event rates per 100 person - years for adults with diabetes and cvd versus those without cvd were 6.0 vs. 1.7 for mi / acs , 5.3 vs. 1.5 for stroke , 8.4 vs. 1.2 for hf , 18.1 vs. 4.0 for any cv event , and 23.5 vs. 5.0 for all - cause mortality . in those without baseline cvd , 34% of major cv events and 7% of all deaths were associated with inadequately controlled bp , ldl - c , a1c , or tobacco use . similarly , in those with baseline cvd , 11% of major cv events and 3% of all deaths were associated with inadequately controlled risk factors . five - year event rates and all - cause mortality and percent of preventable cv events among adults with diabetes with and without baseline cvd table 4 quantifies the contribution of specific uncontrolled risk factors to cv events and deaths . in adults with diabetes and without cvd , inadequately controlled ldl - c was associated with 20% of observed mi / acs and 14% of strokes . inadequately controlled bp was associated with 12% of strokes and hf events , whereas inadequately controlled a1c was associated with 10% of hf events . in subjects with diabetes and cvd , the proportion of major cv events associated with smoking ranged from nearly 1% ( hf ) to 3% ( mi / acs ) . inadequate control of ldl - c or bp was associated with only a small proportion of cv events ( < 4% ) , and only smoking was associated with a small proportion of deaths ( 3% ) . aafs ( 95% cis ) of inadequately controlled risk factors for major cv events and all - cause mortality among adults with diabetes with and without baseline cvd estimates adjusted for age , sex , race / ethnicity , diabetes medication , statins , bp medication , ckd at baseline , incident diabetes at baseline , hdl - c 40 mg / dl ( men)/50 mg / dl ( women ) , elevated ldl - c ( 100 mg / dl ) , elevated a1c ( 8% [ 64 mmol / mol ] ) , elevated systolic / diastolic bp ( 140/90 mmhg ) . sensitivity analysis exploring the immortal time bias caused by the lag between cohort entry date and diabetes identification date revealed a 1% relative difference in event rates and all - cause mortality and no discernible bias on measures of association ( rate ratios ) or aaf ( data not shown ) when the analysis was repeated in those identified in the datalink after 2005 . to estimate the effect of misclassification of cv events in the attributable fractions , we assumed a sensitivity of 0.95 and a specificity of 0.995 based on previous work ( 1820 ) . this resulted in a relative reduction ranging from 24% ( mi / acs ) to 30% ( congestive hf ) in adults with diabetes and no baseline cvd and from 6% ( congestive hf ) to 9% ( stroke ) in adults with diabetes and baseline cvd . despite sustained and quite impressive improvements in the control of glucose , bp , and lipids as well as smoking cessation in those with diabetes in the last decade in the united states , a substantial proportion of individuals have one or more of these factors that remain out of control ( 21 ) . our findings suggest that better management of these risk factors could lead to substantial further reductions in cv events and deaths among those with diabetes . for example , among those without cvd , the attributable risk fraction data suggest that about 1 in 3 major cv events and fewer than 1 in 10 deaths are associated with uncontrolled risk factors , including hypertension , dyslipidemia , smoking , and poor glucose control . although the fraction of cv events attributable to inadequate risk factor control is lower among those with cvd ( 11% ) than those without cvd ( 34% ) , the overall rates of major cv events are nearly five times higher in patients with cvd than in those without it . in addition , only a portion of the cv risk of patients with diabetes is preventable , even with optimal control of these major cv risk factors . thus primary prevention of diabetes is a critically important strategy to minimize the impact of diabetes on cv events and mortality , even though implementation of effective interventions to prevent diabetes can be challenging ( 2225 ) . the necessity of this approach is underscored by the observed number of cv events per 100 person - years in 2010 : 14 in those with diabetes versus 8 in an age- and sex - matched sample without diabetes in the supreme - dm cohort ( j.r.d . furthermore , the total direct medical cost related to these cv events is magnified because costs of cv events in adults with diabetes are twice the costs of events in those without diabetes during the 12-month period initiated by the event ( 26 ) . nonetheless , once a person has diabetes , strenuous efforts to prevent or delay cvd onset substantially lowers the risk of major cv events and are well justified ( 27 ) . our results benchmark control of cv risk factors in adults with diabetes receiving care at 11 health care organizations and enable comparison with national data . compared with data from the national health and nutrition examination survey ( nhanes ) for 2007 to 2010 ( 21 ) , we found a lower prevalence of controlled risk factors : a1c ( < 7% , 53 mmol / mol ) , 50% vs. 52% ; ldl - c ( < 100 mg / dl ) , 48% vs. 56% ; and systolic / diastolic bp ( < 130/80 mmhg ) , 47% vs. 51% , as well as a similar prevalence ( 21% vs. 19% ) of those reaching all three goals . our results differ from the nhanes in that we identified people with incident diabetes , 24% of whom have not been targeted for optimal risk factor control . a key finding in our study was the increased cv risk for a1c 9% ( 75 mmol / mol ) , especially in those with no cvd , and no increased risk for a1c 77.9% compared with 6.56.9% . this finding is congruent with the results of the action to control cardiovascular risk in diabetes ( accord ) trial and action in diabetes and vascular disease : preterax and diamicron mr controlled evaluation ( advance ) and support current guidelines that recommend personalizing an a1c goal between < 7% ( 53 mmol / mol ) and < 8% ( 64 mmol / mol ) in patients with diabetes ( 28 ) . our data also are congruent with results from the steno-2 study , which demonstrated a major reduction in cv events and cv mortality with aggressive bp and ldl - c management , with a median achieved a1c of about 7.8% ( 29 ) . our results and those of previous studies ( 30 ) suggest that the impact of a1c on cv events accelerates as a1c rises above 8% . the lack of an association of elevated bp and hf in those with baseline cvd may be explained by the relatively short follow - up period in our study . consistent with other studies of adults with diabetes , we observed no independent association of bmi with cv events or all - cause mortality ( 3133 ) , although others have found a u - shaped association in adults with incident diabetes ( 34 ) . unfortunately , management of low hdl in those with diabetes is clinically challenging because of a paucity of evidence that existing therapies that increase hdl are effective in reducing cv events or deaths ( 3538 ) . our analysis did not include other novel cv risk factors such as hs - crp , coronary artery calcium , bp variability , sleep disorders , or chronic psychological stress ( 39,40 ) . these factors either were not available in our data or were available only in a selected group of patients . the substantial residual cv risk not attributable to optimal management of bp , ldl - c , a1c , and tobacco raises the possibility that unidentified genetic , metabolic , or psychosocial risk factors may affect risk . several epidemiological studies have evaluated cv event attributable fractions using methods similar to ours or by combining published relative risk estimates with population - based estimates of cv risk factor prevalence in various populations ( 13,4145 ) . a taiwanese study found that the proportion of all - cause mortality and cvd - related mortality attributable to metabolic syndrome in the general population was 11.6% and 39.2% among men and 18.6% and 44.4% among women , respectively , and that central obesity in women and hypertension in men accounted for the highest number of deaths . in a study using 20052006 nhanes data , rckinger et al . ( 13 ) concluded that , for subjects aged 40 years and older , hypertension accounted for 15.7% of cv events , followed by smoking ( 11.4% ) and total cholesterol ( 10% ) , with an aggregate of 44% of cv events attributable to uncontrolled bp , total cholesterol , hdl , or smoking combined , whereas diabetes accounted for 5.4% . first , data for this study were obtained from routine care settings with varying time intervals and considerable missing data . to compensate for this , we did multivariate imputation for a1c , ldl - c , and hdl - c in about 5% of the sample . second , classification of diabetes status is necessarily imperfect , although sensitivity is estimated at 91% and has a positive predictive value of 94% ( 46 ) . accurately distinguishing between type 1 and type 2 diabetes using electronic diagnosis data or duration of diabetes is not possible ( 7 ) . third , cv events were based on primary hospital discharge diagnoses and were not adjudicated . however , hospital discharge diagnoses for major cv events are highly accurate ( 1820 ) . using the primary discharge diagnosis moreover , cv events based on primary hospital discharge diagnoses are thought to have near - perfect specificity , which greatly improves the accuracy of aafs . fifth , the aaf is a well - recognized method of quantifying attributable risk that provides a valid estimate of the causal effect of improved risk factor control on health outcomes , but results using lagged cross - sectional analysis of observational data should be interpreted with caution . in addition , this method does not take into account nonlinearity , which may operate with some cv risk factors . in the present analysis another limitation is that we assessed risk factors and comorbid conditions only upon entry into the cohort ; thus changes in risk factor control during follow - up were not reflected in the results . finally , although we studied more than 800,000 adults with diabetes and had more than four million person - years of follow - up observation , our results may not apply to all segments of the u.s . these limitations must be weighed against the strengths of this study , which provides contemporary community - based estimates of attributable risk percentage for cv events and total mortality that reflect current levels of a1c , bp , ldl - c , and tobacco control in u.s . underscore the importance of the primary prevention of type 2 diabetes and 2 ) indicate that substantial additional reductions in major cv events and all - cause mortality in those with diabetes may be achieved through more effective control of glucose , bp , ldl - c , and smoking .

objectivethe objective of this study was to assess the incidence of major cardiovascular ( cv ) hospitalization events and all - cause deaths among adults with diabetes with or without cv disease ( cvd ) associated with inadequately controlled glycated hemoglobin ( a1c ) , high ldl cholesterol ( ldl - c ) , high blood pressure ( bp ) , and current smoking.research design and methodsstudy subjects included 859,617 adults with diabetes enrolled for more than 6 months during 20052011 in a network of 11 u.s . integrated health care organizations . inadequate risk factor control was classified as ldl - c 100 mg / dl , a1c 7% ( 53 mmol / mol ) , bp 140/90 mm hg , or smoking . major cv events were based on primary hospital discharge diagnoses for myocardial infarction ( mi ) and acute coronary syndrome ( acs ) , stroke , or heart failure ( hf ) . five - year incidence rates , rate ratios , and average attributable fractions were estimated using multivariable poisson regression models.resultsmean ( sd ) age at baseline was 59 ( 14 ) years ; 48% of subjects were female , 45% were white , and 31% had cvd . mean follow - up was 59 months . event rates per 100 person - years for adults with diabetes and cvd versus those without cvd were 6.0 vs. 1.7 for mi / acs , 5.3 vs. 1.5 for stroke , 8.4 vs. 1.2 for hf , 18.1 vs. 40 for all cv events , and 23.5 vs. 5.0 for all - cause mortality . the percentages of cv events and deaths associated with inadequate risk factor control were 11% and 3% , respectively , for those with cvd and 34% and 7% , respectively , for those without cvd.conclusionsadditional attention to traditional cv risk factors could yield further substantive reductions in cv events and mortality in adults with diabetes .

Introduction Research Design and Methods Data Sources Study Population Risk Factor Definitions CV Events and All-Cause Mortality Statistical Analysis Protection of Human Research Subjects Results Conclusions

advances in medical care and improvements in cardiovascular ( cv ) risk factor control have resulted in the reduction of cv events and deaths among adults with diabetes ( 2,3 ) . at the population level , however , increased diabetes prevalence and more years lived with diabetes offset much of the improvement in rates of cv events and mortality such that the absolute burden of cv events in those with diabetes remains high . major risk factors for cv events are well known , and effective drugs and behavioral interventions are available to manage blood pressure ( bp ) , ldl cholesterol ( ldl - c ) , glucose , and tobacco use . estimating the prevalence of inadequately controlled risk factors and the proportion of major cv events and all - cause mortality associated with each of these uncontrolled cv risk factors could help prioritize clinical and public health strategies to further reduce the burden of cv disease ( cvd ) in adults with diabetes . because risk factor prevalence , risk factor association with cv events , and impact of treatment of uncontrolled risk factors on cv events differ depending on whether an adult with diabetes has cvd , we conducted separate analyses of those with versus without cvd at baseline . the surveillance , prevention , and management of diabetes mellitus ( supreme - dm ) datalink was developed in a network of 11 large , integrated health care organizations in the hmo research network in the united states ( 5 ) . study criteria for diabetes required either one or more inpatient diabetes diagnosis codes ( icd-9 clinical modification 250.x , 357.2 , 366.41 , 362.01362.07 ) or any combination of two or more of the following events on separate days no more than 2 years apart : 1 ) a1c 6.5% ( 48 mmol / mol ) , 2 ) fasting plasma glucose 126 mg / dl , 3 ) random plasma glucose 200 mg / dl , 4 ) outpatient visit diabetes diagnosis code ( same codes as that used for inpatients ) , or 5 ) any filled prescription for a glucose - lowering medication . we defined inadequate baseline control following american diabetes association guidelines ( 10,11)ldl - c 100 mg / dl and a1c 7% ( 53 mmol / mol)and the eighth joint national committee 2014 guidelines ( 11)systolic bp 140 mmhg or diastolic bp 90 mmhg or current smoking . baseline status for comorbid conditions was assigned based on two or more outpatient diagnosis codes or one or more inpatient diagnosis codes on or before the cohort entry date for chronic kidney disease ( ckd ; icd-9 code 585.xx ) , cvd ( icd-9 codes 410414.xx and 429.2 ) , heart failure ( hf ; icd-9 codes 428428.9 ) , hemorrhagic stroke ( icd-9 codes 430432.9 ) , ischemic stroke ( icd-9 codes 433434.91 ) , and transient ischemic attack ( icd-9 code 435.xx ) . major cv events were ascertained based on the date of primary hospital discharge icd-9 diagnosis codes as follows : 1 ) myocardial infarction ( mi)/acute coronary syndrome ( acs ) ( icd-9 codes 410.0410.91 , 411.1 , 411.8 ) , 2 ) ischemic and hemorrhagic stroke ( icd-9 codes 433434.91 , 430432.9 ) , or 3 ) hf ( icd-9 codes 428428.9 ) . models to predict hospitalization for mi / acs , stroke , hf , and all - cause mortality were constructed with generalized linear models using poisson distribution and log link . in the first step , the associations of individual baseline variables with each major cv event and with all - cause mortality were evaluated in separate models for subjects with and without cvd at baseline , using only age and sex as covariates . individual baseline variables included uncontrolled risk factors ( bp , ldl - c , a1c , and tobacco status ) , medication use variables , bmi , hdl - c , incident diabetes , presence of ckd , race / ethnicity , and study site . in the second step , all baseline factors associated with a major cv event or with all - cause mortality in step 1 and with p < 0.05 as main effects were retained for inclusion in the final models . to estimate the population - attributable fraction for each uncontrolled risk factor in adults with diabetes with and without baseline cvd , we calculated the average attributable fraction ( aaf ) , as described by rckinger et al . aaf was evaluated for inadequately controlled risk factors ( bp , ldl - c , a1c , and tobacco use ) in the same poisson model that included the other risk factors age , sex , hdl - c , ckd , incident ( vs. prevalent ) diabetes , and medication use variables as main effects for adults with diabetes with or without baseline cvd . the surveillance , prevention , and management of diabetes mellitus ( supreme - dm ) datalink was developed in a network of 11 large , integrated health care organizations in the hmo research network in the united states ( 5 ) . study criteria for diabetes required either one or more inpatient diabetes diagnosis codes ( icd-9 clinical modification 250.x , 357.2 , 366.41 , 362.01362.07 ) or any combination of two or more of the following events on separate days no more than 2 years apart : 1 ) a1c 6.5% ( 48 mmol / mol ) , 2 ) fasting plasma glucose 126 mg / dl , 3 ) random plasma glucose 200 mg / dl , 4 ) outpatient visit diabetes diagnosis code ( same codes as that used for inpatients ) , or 5 ) any filled prescription for a glucose - lowering medication . we defined inadequate baseline control following american diabetes association guidelines ( 10,11)ldl - c 100 mg / dl and a1c 7% ( 53 mmol / mol)and the eighth joint national committee 2014 guidelines ( 11)systolic bp 140 mmhg or diastolic bp 90 mmhg or current smoking . baseline status for comorbid conditions was assigned based on two or more outpatient diagnosis codes or one or more inpatient diagnosis codes on or before the cohort entry date for chronic kidney disease ( ckd ; icd-9 code 585.xx ) , cvd ( icd-9 codes 410414.xx and 429.2 ) , heart failure ( hf ; icd-9 codes 428428.9 ) , hemorrhagic stroke ( icd-9 codes 430432.9 ) , ischemic stroke ( icd-9 codes 433434.91 ) , and transient ischemic attack ( icd-9 code 435.xx ) . major cv events were ascertained based on the date of primary hospital discharge icd-9 diagnosis codes as follows : 1 ) myocardial infarction ( mi)/acute coronary syndrome ( acs ) ( icd-9 codes 410.0410.91 , 411.1 , 411.8 ) , 2 ) ischemic and hemorrhagic stroke ( icd-9 codes 433434.91 , 430432.9 ) , or 3 ) hf ( icd-9 codes 428428.9 ) . models to predict hospitalization for mi / acs , stroke , hf , and all - cause mortality were constructed with generalized linear models using poisson distribution and log link . in the first step , the associations of individual baseline variables with each major cv event and with all - cause mortality were evaluated in separate models for subjects with and without cvd at baseline , using only age and sex as covariates . individual baseline variables included uncontrolled risk factors ( bp , ldl - c , a1c , and tobacco status ) , medication use variables , bmi , hdl - c , incident diabetes , presence of ckd , race / ethnicity , and study site . in the second step , all baseline factors associated with a major cv event or with all - cause mortality in step 1 and with p < 0.05 as main effects were retained for inclusion in the final models . to estimate the population - attributable fraction for each uncontrolled risk factor in adults with diabetes with and without baseline cvd , we calculated the average attributable fraction ( aaf ) , as described by rckinger et al . aaf was evaluated for inadequately controlled risk factors ( bp , ldl - c , a1c , and tobacco use ) in the same poisson model that included the other risk factors age , sex , hdl - c , ckd , incident ( vs. prevalent ) diabetes , and medication use variables as main effects for adults with diabetes with or without baseline cvd . mean follow up was 59 months , and 4,233,786 person - years of observation for cv events were included in the analysis . mean age was 59 years ( sd 14 years ) ; 48% were female , 45% were white , and 31% had cvd . compared with those without baseline cvd , those with baseline cvd had better control of smoking ( 8.0% vs. 9.8% ) , a1c 7% ( 53 mmol / mol ) ( 42% vs. 53% ) , and ldl - c 100 mg / dl ( 38% vs. 58% ) , and they had similar proportions of subjects with systolic / diastolic bp 140/90 mmhg ( 23% vs. 21% ) . baseline characteristics of 859,617 adults with diabetes with and without cvd receiving care from 2005 to 2010 at 11 u.s . health care organizations and prevalence of inadequate control of bp ( systolic / diastolic 140/90 mmhg ) , ldl - c ( 100 mg / dl ) , a1c ( 8% [ 64 mmol / mol ] ) , and current smoking the associations of inadequately controlled baseline a1c , bp , ldl - c , and tobacco use with subsequent cv events and all - cause mortality are presented in table 2 . cv events and all - cause mortality also were associated with sex , age , race / ethnicity , ckd , hdl - c , incident diabetes status , and medication use variables ( table 1 ) . however , major cv events and all - cause mortality were not significantly associated with baseline bmi ( data not shown ) . in subjects without baseline cvd , all inadequately controlled cv risk factors were associated with higher risk of mi / acs , stroke , and hf , but only smoking and bp were associated with all - cause mortality . a similar pattern emerged in subjects with baseline cvd : all inadequately controlled cv risk factors were associated with an increase in mi / acs and stroke . current smoking , bp , and a1c ( but not ldl - c ) were associated with an increase in hf . rate ratios ( 95% cis ) of inadequate controlled risk factors with cv events and all - cause mortality in adults with diabetes with and without baseline cvd estimates adjusted for age , sex , race / ethnicity , diabetes medication , statins , bp medication , ckd at baseline , incident diabetes at baseline , low hdl - c ( 40 mg / dl [ men]/50 mg / dl [ women ] ) , and all other inadequately controlled factors as described in the table . rates of major cv events and all - cause mortality in adults with diabetes and cvd were substantially higher than in those with diabetes but no cvd ( table 3 ) . five - year cv event rates per 100 person - years for adults with diabetes and cvd versus those without cvd were 6.0 vs. 1.7 for mi / acs , 5.3 vs. 1.5 for stroke , 8.4 vs. 1.2 for hf , 18.1 vs. 4.0 for any cv event , and 23.5 vs. 5.0 for all - cause mortality . in those without baseline cvd , 34% of major cv events and 7% of all deaths were associated with inadequately controlled bp , ldl - c , a1c , or tobacco use . similarly , in those with baseline cvd , 11% of major cv events and 3% of all deaths were associated with inadequately controlled risk factors . five - year event rates and all - cause mortality and percent of preventable cv events among adults with diabetes with and without baseline cvd table 4 quantifies the contribution of specific uncontrolled risk factors to cv events and deaths . in adults with diabetes and without cvd , inadequately controlled ldl - c was associated with 20% of observed mi / acs and 14% of strokes . in subjects with diabetes and cvd , the proportion of major cv events associated with smoking ranged from nearly 1% ( hf ) to 3% ( mi / acs ) . inadequate control of ldl - c or bp was associated with only a small proportion of cv events ( < 4% ) , and only smoking was associated with a small proportion of deaths ( 3% ) . aafs ( 95% cis ) of inadequately controlled risk factors for major cv events and all - cause mortality among adults with diabetes with and without baseline cvd estimates adjusted for age , sex , race / ethnicity , diabetes medication , statins , bp medication , ckd at baseline , incident diabetes at baseline , hdl - c 40 mg / dl ( men)/50 mg / dl ( women ) , elevated ldl - c ( 100 mg / dl ) , elevated a1c ( 8% [ 64 mmol / mol ] ) , elevated systolic / diastolic bp ( 140/90 mmhg ) . sensitivity analysis exploring the immortal time bias caused by the lag between cohort entry date and diabetes identification date revealed a 1% relative difference in event rates and all - cause mortality and no discernible bias on measures of association ( rate ratios ) or aaf ( data not shown ) when the analysis was repeated in those identified in the datalink after 2005 . this resulted in a relative reduction ranging from 24% ( mi / acs ) to 30% ( congestive hf ) in adults with diabetes and no baseline cvd and from 6% ( congestive hf ) to 9% ( stroke ) in adults with diabetes and baseline cvd . our findings suggest that better management of these risk factors could lead to substantial further reductions in cv events and deaths among those with diabetes . for example , among those without cvd , the attributable risk fraction data suggest that about 1 in 3 major cv events and fewer than 1 in 10 deaths are associated with uncontrolled risk factors , including hypertension , dyslipidemia , smoking , and poor glucose control . although the fraction of cv events attributable to inadequate risk factor control is lower among those with cvd ( 11% ) than those without cvd ( 34% ) , the overall rates of major cv events are nearly five times higher in patients with cvd than in those without it . the necessity of this approach is underscored by the observed number of cv events per 100 person - years in 2010 : 14 in those with diabetes versus 8 in an age- and sex - matched sample without diabetes in the supreme - dm cohort ( j.r.d . furthermore , the total direct medical cost related to these cv events is magnified because costs of cv events in adults with diabetes are twice the costs of events in those without diabetes during the 12-month period initiated by the event ( 26 ) . our results benchmark control of cv risk factors in adults with diabetes receiving care at 11 health care organizations and enable comparison with national data . compared with data from the national health and nutrition examination survey ( nhanes ) for 2007 to 2010 ( 21 ) , we found a lower prevalence of controlled risk factors : a1c ( < 7% , 53 mmol / mol ) , 50% vs. 52% ; ldl - c ( < 100 mg / dl ) , 48% vs. 56% ; and systolic / diastolic bp ( < 130/80 mmhg ) , 47% vs. 51% , as well as a similar prevalence ( 21% vs. 19% ) of those reaching all three goals . a key finding in our study was the increased cv risk for a1c 9% ( 75 mmol / mol ) , especially in those with no cvd , and no increased risk for a1c 77.9% compared with 6.56.9% . this finding is congruent with the results of the action to control cardiovascular risk in diabetes ( accord ) trial and action in diabetes and vascular disease : preterax and diamicron mr controlled evaluation ( advance ) and support current guidelines that recommend personalizing an a1c goal between < 7% ( 53 mmol / mol ) and < 8% ( 64 mmol / mol ) in patients with diabetes ( 28 ) . our data also are congruent with results from the steno-2 study , which demonstrated a major reduction in cv events and cv mortality with aggressive bp and ldl - c management , with a median achieved a1c of about 7.8% ( 29 ) . consistent with other studies of adults with diabetes , we observed no independent association of bmi with cv events or all - cause mortality ( 3133 ) , although others have found a u - shaped association in adults with incident diabetes ( 34 ) . the substantial residual cv risk not attributable to optimal management of bp , ldl - c , a1c , and tobacco raises the possibility that unidentified genetic , metabolic , or psychosocial risk factors may affect risk . a taiwanese study found that the proportion of all - cause mortality and cvd - related mortality attributable to metabolic syndrome in the general population was 11.6% and 39.2% among men and 18.6% and 44.4% among women , respectively , and that central obesity in women and hypertension in men accounted for the highest number of deaths . ( 13 ) concluded that , for subjects aged 40 years and older , hypertension accounted for 15.7% of cv events , followed by smoking ( 11.4% ) and total cholesterol ( 10% ) , with an aggregate of 44% of cv events attributable to uncontrolled bp , total cholesterol , hdl , or smoking combined , whereas diabetes accounted for 5.4% . third , cv events were based on primary hospital discharge diagnoses and were not adjudicated . using the primary discharge diagnosis moreover , cv events based on primary hospital discharge diagnoses are thought to have near - perfect specificity , which greatly improves the accuracy of aafs . in the present analysis another limitation is that we assessed risk factors and comorbid conditions only upon entry into the cohort ; thus changes in risk factor control during follow - up were not reflected in the results . finally , although we studied more than 800,000 adults with diabetes and had more than four million person - years of follow - up observation , our results may not apply to all segments of the u.s . these limitations must be weighed against the strengths of this study , which provides contemporary community - based estimates of attributable risk percentage for cv events and total mortality that reflect current levels of a1c , bp , ldl - c , and tobacco control in u.s . underscore the importance of the primary prevention of type 2 diabetes and 2 ) indicate that substantial additional reductions in major cv events and all - cause mortality in those with diabetes may be achieved through more effective control of glucose , bp , ldl - c , and smoking .

on 25 september 2015 , the united nations approved a set of 17 goals and 169 targets to end poverty , protect the planet , and ensure prosperity for all as part of a new sustainable development agenda . these so - called sustainable development goals ( sdgs ) aim to transform our world , and each goal has specific targets to be achieved by 2030 . the goals and targets are setting out a supremely ambitious and transformational vision to stimulate action over the next 15 years in areas of critical importance for humanity and the planet : people , planet , prosperity , peace and partnership . sdgs are the result of first consensus among world leaders reached across such a broad and universal policy agenda . sdgs build upon the achievements of millennium development goals ( mdgs ) , approved at the beginning of 3 millennium to foster universal development . however , the progress towards mdgs has been uneven , particularly in less developed countries , where some mdgs remain off track , in particular , those related to maternal , newborn and child health and to reproductive health . seeking to complete what mdgs did not achieve , particularly in reaching the most vulnerable , sdgs envisage a world free of poverty , hunger , disease , fear and violence , and desire a universal literacy , equitable and universal access to quality education at all levels , to health care and social protection among all global citizens . it presents a good opportunity to bring all countries and citizens of the planet together to take the bold and transformative steps to shift the world on to a sustainable and resilient path , aiming to improve the lives of human beings anywhere they live , in a way that no one will be left behind . for the goals to be achieved , everyone needs to do their part : governments , academia , the private sector , civil society and all people . we conducted document analysis to identify the main literature around uhc and ncds in the format of sdgs and created a full report of the two days conference on sustainable health development . to contribute to exploring to the academia s role in reaching sdgs , the 1 international conference on sustainable health development , was held simultaneously with commemoration of the 50 anniversary of the establishment of school of public health ( sph ) , tehran university of medical sciences , on 2425 april 2016 , in tehran , i.r . iran . in line with goal 3 of sdgs : ensure healthy lives and promote well - being for all at all ages , appreciating the great need for more efforts to fully eradicate a wide range of diseases and address many different persistent and emerging health issues , and on the basis of the priorities of the health system of the islamic republic of iran , the conference provided an opportunity to study and discuss various aspects of universal health coverage ( uhc ) , as well as global action plans for prevention and control of non - communicable diseases ( ncds ) , both of which are among the targets of sdg 3 , and explain the role of research and higher education institutions in this regard . attracting over 700 participants , the 2 days event accommodated six specific panels with 39 speakers from policy makers , managers , academics , and researchers at the global , international and national levels , who discussed in the presence of above 700 participants , the special role of academic public health institutes in education , research and service provision in the two above - mentioned areas . traditionally , many developing countries , and also a number of currently - developed countries , did not have a strong health system in place for provision of health services . therefore , health - related academic institutes , i.e. sphs were in charge of providing some health services to the public . for instance , sph at tehran university of medical sciences has been in charge of conducting some specific viral and parasitology lab tests for the public . the gradual strengthening of health systems in many countries , has resulted a less prominent role for academia in service delivery , but a more pronounced focus on advocacy , community sensitization , i.e. community - based initiatives ( cbis ) and promoting health in the societies . because of ever - improving health technologies and the significant attention to financial risk protection and uhc , a number of countries have implemented a high cost approach to provide specialized treatment services . the conference covered three areas : a ) uhc and social determinants of health , b ) primary health care and uhc , and c ) essential health functions and uhc . the key question was whether these three issues have been properly addressed in the implementation activities of the world health systems in the area of uhc and possible ways that public health academia could address these . by definition , public health involves all aspects of prevention as well as all fundamental services , which have demographical dimensions . conventionally , academic public health institutes have focused on the issues of promotion , primary prevention and primary health care , hence , leading uhc to deal mostly with primary prevention rather than palliation services . the panel addressed the role of academic public health institutes in providing health care , rehabilitation and palliative services , as well as the distinction between schools of public health and other higher education institutes in the fields of medicine , rehabilitation , and other disciplines . in addition , the appropriate structural model of academic public health institutes in addressing managerial and leadership aspects of sdgs was discussed , with special focus on the workforce needed for uhc . two topics of a ) filling the gap between research and higher education institutions and the decision making environment and b ) networking and areas of cooperation between academic public health institutes were the main topics of this panel . in fact , the differences between the decision - making environment and the academic environment are a well - known phenomenon , which has drawn global attention . the discussion introduced successful models and shared experiences in this area , from the eastern mediterranean region , as well as other parts of the world . finding mutual areas of interest for collaboration among academic public health institutes and agreeing upon practical ways to move this initiative forward , which was discussed in more details during the specific meeting of academic public health institutes of eastern mediterranean region as the side event on 3 day of the conference , was the main aim of this panel . the academic public health institutes have an important role to play in prevention and control of ncds . however , the academic community has been sporadic and heterogeneous in terms of its interest and exposure to ncds - related programs . it seems that it is more the individuals within the institutions , rather than the institutions themselves , who have been involved in ncds - related research or policy drafting . this panel discussed the mechanisms for institutionalizing the ways that the individual ncds focal points can work jointly together , so that the ncds academia becomes more recognizable and perceptible . preparing the academic public health institutes to undertake greater responsibility in ncd - related works , especially when there is inevitable increase in attention and funding from the global , regional and the national agencies , was also discussed . in particular , the panel recommended the formation of a number of specialized research centers on ncds , e.g. mental health , which could be affiliated with sphs , to work on focused research and education on ncds on below areas : research , policy analysis and policy makingadvocacy , partnership and leadership e.g. partnering with other stakeholderssupport healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives.health system strengthening for early detection and management of ncds . research , policy analysis and policy making advocacy , partnership and leadership e.g. partnering with other stakeholders support healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives . ncds are multi - factorial and their prevention and control needs strong multi - sectoral collaborations . this panel discussed the role of a wide range of stakeholders , i.e. ministry of health and other state ministries ; academia ; non - governmental and civil society organizations ; private sectors ; as well as external and international agencies , in prevention and control of ncds and ways to strengthen their involvement in this regard . this panel provided an overview of environmental challenges , i.e. water scarcity , poor air quality , global warming , waste management , climate change , the link between environmental health issues and uhc , and the need for academic public health institutes to address such challenges to reach sustainable development . to contribute to exploring to the academia s role in reaching sdgs , the 1 international conference on sustainable health development , was held simultaneously with commemoration of the 50 anniversary of the establishment of school of public health ( sph ) , tehran university of medical sciences , on 2425 april 2016 , in tehran , i.r . iran . in line with goal 3 of sdgs : ensure healthy lives and promote well - being for all at all ages , appreciating the great need for more efforts to fully eradicate a wide range of diseases and address many different persistent and emerging health issues , and on the basis of the priorities of the health system of the islamic republic of iran , the conference provided an opportunity to study and discuss various aspects of universal health coverage ( uhc ) , as well as global action plans for prevention and control of non - communicable diseases ( ncds ) , both of which are among the targets of sdg 3 , and explain the role of research and higher education institutions in this regard . attracting over 700 participants , the 2 days event accommodated six specific panels with 39 speakers from policy makers , managers , academics , and researchers at the global , international and national levels , who discussed in the presence of above 700 participants , the special role of academic public health institutes in education , research and service provision in the two above - mentioned areas . traditionally , many developing countries , and also a number of currently - developed countries , did not have a strong health system in place for provision of health services . therefore , health - related academic institutes , i.e. sphs were in charge of providing some health services to the public . for instance , sph at tehran university of medical sciences has been in charge of conducting some specific viral and parasitology lab tests for the public . the gradual strengthening of health systems in many countries , has resulted a less prominent role for academia in service delivery , but a more pronounced focus on advocacy , community sensitization , i.e. community - based initiatives ( cbis ) and promoting health in the societies . because of ever - improving health technologies and the significant attention to financial risk protection and uhc , a number of countries have implemented a high cost approach to provide specialized treatment services . the conference covered three areas : a ) uhc and social determinants of health , b ) primary health care and uhc , and c ) essential health functions and uhc . the key question was whether these three issues have been properly addressed in the implementation activities of the world health systems in the area of uhc and possible ways that public health academia could address these . traditionally , many developing countries , and also a number of currently - developed countries , did not have a strong health system in place for provision of health services . therefore , health - related academic institutes , i.e. sphs were in charge of providing some health services to the public . for instance , sph at tehran university of medical sciences has been in charge of conducting some specific viral and parasitology lab tests for the public . the gradual strengthening of health systems in many countries , has resulted a less prominent role for academia in service delivery , but a more pronounced focus on advocacy , community sensitization , i.e. community - based initiatives ( cbis ) and promoting health in the societies . because of ever - improving health technologies and the significant attention to financial risk protection and uhc , a number of countries have implemented a high cost approach to provide specialized treatment services . the conference covered three areas : a ) uhc and social determinants of health , b ) primary health care and uhc , and c ) essential health functions and uhc . the key question was whether these three issues have been properly addressed in the implementation activities of the world health systems in the area of uhc and possible ways that public health academia could address these . by definition , public health involves all aspects of prevention as well as all fundamental services , which have demographical dimensions . conventionally , academic public health institutes have focused on the issues of promotion , primary prevention and primary health care , hence , leading uhc to deal mostly with primary prevention rather than palliation services . the panel addressed the role of academic public health institutes in providing health care , rehabilitation and palliative services , as well as the distinction between schools of public health and other higher education institutes in the fields of medicine , rehabilitation , and other disciplines . in addition , the appropriate structural model of academic public health institutes in addressing managerial and leadership aspects of sdgs was discussed , with special focus on the workforce needed for uhc . two topics of a ) filling the gap between research and higher education institutions and the decision making environment and b ) networking and areas of cooperation between academic public health institutes were the main topics of this panel . in fact , the differences between the decision - making environment and the academic environment are a well - known phenomenon , which has drawn global attention . the discussion introduced successful models and shared experiences in this area , from the eastern mediterranean region , as well as other parts of the world . finding mutual areas of interest for collaboration among academic public health institutes and agreeing upon practical ways to move this initiative forward , which was discussed in more details during the specific meeting of academic public health institutes of eastern mediterranean region as the side event on 3 day of the conference , was the main aim of this panel . two topics of a ) filling the gap between research and higher education institutions and the decision making environment and b ) networking and areas of cooperation between academic public health institutes were the main topics of this panel . in fact , the differences between the decision - making environment and the academic environment are a well - known phenomenon , which has drawn global attention . the discussion introduced successful models and shared experiences in this area , from the eastern mediterranean region , as well as other parts of the world . finding mutual areas of interest for collaboration among academic public health institutes and agreeing upon practical ways to move this initiative forward , which was discussed in more details during the specific meeting of academic public health institutes of eastern mediterranean region as the side event on 3 day of the conference , was the main aim of this panel . the academic public health institutes have an important role to play in prevention and control of ncds . however , the academic community has been sporadic and heterogeneous in terms of its interest and exposure to ncds - related programs . it seems that it is more the individuals within the institutions , rather than the institutions themselves , who have been involved in ncds - related research or policy drafting . this panel discussed the mechanisms for institutionalizing the ways that the individual ncds focal points can work jointly together , so that the ncds academia becomes more recognizable and perceptible . preparing the academic public health institutes to undertake greater responsibility in ncd - related works , especially when there is inevitable increase in attention and funding from the global , regional and the national agencies , was also discussed . in particular , the panel recommended the formation of a number of specialized research centers on ncds , e.g. mental health , which could be affiliated with sphs , to work on focused research and education on ncds on below areas : research , policy analysis and policy makingadvocacy , partnership and leadership e.g. partnering with other stakeholderssupport healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives.health system strengthening for early detection and management of ncds . research , policy analysis and policy making advocacy , partnership and leadership e.g. partnering with other stakeholders support healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives . health system strengthening for early detection and management of ncds . ncds are multi - factorial and their prevention and control needs strong multi - sectoral collaborations . this panel discussed the role of a wide range of stakeholders , i.e. ministry of health and other state ministries ; academia ; non - governmental and civil society organizations ; private sectors ; as well as external and international agencies , in prevention and control of ncds and ways to strengthen their involvement in this regard . this panel provided an overview of environmental challenges , i.e. water scarcity , poor air quality , global warming , waste management , climate change , the link between environmental health issues and uhc , and the need for academic public health institutes to address such challenges to reach sustainable development . the academic public health institutes have an important role to play in prevention and control of ncds . however , the academic community has been sporadic and heterogeneous in terms of its interest and exposure to ncds - related programs . it seems that it is more the individuals within the institutions , rather than the institutions themselves , who have been involved in ncds - related research or policy drafting . this panel discussed the mechanisms for institutionalizing the ways that the individual ncds focal points can work jointly together , so that the ncds academia becomes more recognizable and perceptible . preparing the academic public health institutes to undertake greater responsibility in ncd - related works , especially when there is inevitable increase in attention and funding from the global , regional and the national agencies , was also discussed . in particular , the panel recommended the formation of a number of specialized research centers on ncds , e.g. mental health , which could be affiliated with sphs , to work on focused research and education on ncds on below areas : research , policy analysis and policy makingadvocacy , partnership and leadership e.g. partnering with other stakeholderssupport healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives.health system strengthening for early detection and management of ncds . research , policy analysis and policy making advocacy , partnership and leadership e.g. partnering with other stakeholders support healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives . health system strengthening for early detection and management of ncds . ncds are multi - factorial and their prevention and control needs strong multi - sectoral collaborations . this panel discussed the role of a wide range of stakeholders , i.e. ministry of health and other state ministries ; academia ; non - governmental and civil society organizations ; private sectors ; as well as external and international agencies , in prevention and control of ncds and ways to strengthen their involvement in this regard . this panel provided an overview of environmental challenges , i.e. water scarcity , poor air quality , global warming , waste management , climate change , the link between environmental health issues and uhc , and the need for academic public health institutes to address such challenges to reach sustainable development . in line with sustainable development goal 3 , the 2012 un general assembly resolution on universal health coverage ( uhc ) a/67/l.36 , world health assembly resolution wha67.23 and who global action plan for the prevention and control of ncds 20132020 ( resolution wha66.10 ) , the 1 international conference on sustainable health development approved a resolution in its final session . the resolution , which is meant to be a manifest for participating academic public health institutes in the conference , recognized : uhc to deliver equitable opportunities for the highest attainable standards of physical , social and mental health;the importance of non - communicable diseases ( ncds ) in disabling and killing people , their considerable burden to peoples health , and the importance of having national , robust and evidence - based plans and policies for prevention and control of ncds;the specific and central role of academic public health institutes in provision of useful and need - based education , research and service to address the challenges of public health , as well as reach sustainable development;the great need of reconsidering the role of academic public health institutes in the context of low - middle income countries to respond to the emerging challenges of public health;that public health policies are only meaningful when they are translated into action by regulation , budget allocations , purchasing and procurement , monitoring , evaluation and supervision;that global and national action is required to find a right solution to support priority settings for health that ensure alignment , participation , transparency , empowerment , nondiscrimination and accountability resulted by a synergistic collaboration of stakeholders . uhc to deliver equitable opportunities for the highest attainable standards of physical , social and mental health ; the importance of non - communicable diseases ( ncds ) in disabling and killing people , their considerable burden to peoples health , and the importance of having national , robust and evidence - based plans and policies for prevention and control of ncds ; the specific and central role of academic public health institutes in provision of useful and need - based education , research and service to address the challenges of public health , as well as reach sustainable development ; the great need of reconsidering the role of academic public health institutes in the context of low - middle income countries to respond to the emerging challenges of public health ; that public health policies are only meaningful when they are translated into action by regulation , budget allocations , purchasing and procurement , monitoring , evaluation and supervision ; that global and national action is required to find a right solution to support priority settings for health that ensure alignment , participation , transparency , empowerment , nondiscrimination and accountability resulted by a synergistic collaboration of stakeholders . the resolution called on all academic public health institutes across the world , and the global public health arena to : take progressive action to achieve the health related sdgs and their indicators by nationalizing the goals within their countries.facilitate exchange of faculty members , scholars and other researchers among different academic public health institutes in order to share and learn various experiences , as well as franchising a spectrum of projects and activities.develop , strengthen and sustain collaborative research on similar problems in different countries within a region and beyond it.deploy joint implementation of public health practices and interventions to enable governments to achieve uhc and program and implement national plans for prevention and control of ncds.share best practices of each academic public health institute and contextualize them for integration into the health system.increase communication and exchanges regarding the sdgs to achieve a unified vision about the goals and indicators.assist and play a fundamental role in the interpretation of sdgs , i.e. uhc and ncds in the context of their own countries.establish objective - driven networks to fulfill the above - mentioned vision . take progressive action to achieve the health related sdgs and their indicators by nationalizing the goals within their countries . facilitate exchange of faculty members , scholars and other researchers among different academic public health institutes in order to share and learn various experiences , as well as franchising a spectrum of projects and activities . develop , strengthen and sustain collaborative research on similar problems in different countries within a region and beyond it . deploy joint implementation of public health practices and interventions to enable governments to achieve uhc and program and implement national plans for prevention and control of ncds . share best practices of each academic public health institute and contextualize them for integration into the health system . increase communication and exchanges regarding the sdgs to achieve a unified vision about the goals and indicators . assist and play a fundamental role in the interpretation of sdgs , i.e. uhc and ncds in the context of their own countries . establish objective - driven networks to fulfill the above - mentioned vision . in line with sustainable development goal 3 , the 2012 un general assembly resolution on universal health coverage ( uhc ) a/67/l.36 , world health assembly resolution wha67.23 and who global action plan for the prevention and control of ncds 20132020 ( resolution wha66.10 ) , the 1 international conference on sustainable health development approved a resolution in its final session . the resolution , which is meant to be a manifest for participating academic public health institutes in the conference , recognized : uhc to deliver equitable opportunities for the highest attainable standards of physical , social and mental health;the importance of non - communicable diseases ( ncds ) in disabling and killing people , their considerable burden to peoples health , and the importance of having national , robust and evidence - based plans and policies for prevention and control of ncds;the specific and central role of academic public health institutes in provision of useful and need - based education , research and service to address the challenges of public health , as well as reach sustainable development;the great need of reconsidering the role of academic public health institutes in the context of low - middle income countries to respond to the emerging challenges of public health;that public health policies are only meaningful when they are translated into action by regulation , budget allocations , purchasing and procurement , monitoring , evaluation and supervision;that global and national action is required to find a right solution to support priority settings for health that ensure alignment , participation , transparency , empowerment , nondiscrimination and accountability resulted by a synergistic collaboration of stakeholders . uhc to deliver equitable opportunities for the highest attainable standards of physical , social and mental health ; the importance of non - communicable diseases ( ncds ) in disabling and killing people , their considerable burden to peoples health , and the importance of having national , robust and evidence - based plans and policies for prevention and control of ncds ; the specific and central role of academic public health institutes in provision of useful and need - based education , research and service to address the challenges of public health , as well as reach sustainable development ; the great need of reconsidering the role of academic public health institutes in the context of low - middle income countries to respond to the emerging challenges of public health ; that public health policies are only meaningful when they are translated into action by regulation , budget allocations , purchasing and procurement , monitoring , evaluation and supervision ; that global and national action is required to find a right solution to support priority settings for health that ensure alignment , participation , transparency , empowerment , nondiscrimination and accountability resulted by a synergistic collaboration of stakeholders . the resolution called on all academic public health institutes across the world , and the global public health arena to : take progressive action to achieve the health related sdgs and their indicators by nationalizing the goals within their countries.facilitate exchange of faculty members , scholars and other researchers among different academic public health institutes in order to share and learn various experiences , as well as franchising a spectrum of projects and activities.develop , strengthen and sustain collaborative research on similar problems in different countries within a region and beyond it.deploy joint implementation of public health practices and interventions to enable governments to achieve uhc and program and implement national plans for prevention and control of ncds.share best practices of each academic public health institute and contextualize them for integration into the health system.increase communication and exchanges regarding the sdgs to achieve a unified vision about the goals and indicators.assist and play a fundamental role in the interpretation of sdgs , i.e. uhc and ncds in the context of their own countries.establish objective - driven networks to fulfill the above - mentioned vision . take progressive action to achieve the health related sdgs and their indicators by nationalizing the goals within their countries . facilitate exchange of faculty members , scholars and other researchers among different academic public health institutes in order to share and learn various experiences , as well as franchising a spectrum of projects and activities . develop , strengthen and sustain collaborative research on similar problems in different countries within a region and beyond it . deploy joint implementation of public health practices and interventions to enable governments to achieve uhc and program and implement national plans for prevention and control of ncds . share best practices of each academic public health institute and contextualize them for integration into the health system . increase communication and exchanges regarding the sdgs to achieve a unified vision about the goals and indicators . assist and play a fundamental role in the interpretation of sdgs , i.e. uhc and ncds in the context of their own countries . establish objective - driven networks to fulfill the above - mentioned vision . academic public health institutes must invest in transforming their conventional approach in research , education , advocacy and service provision , should they decide to play a meaningful role in reaching sustainable health development . multidisciplinary partnerships with communities , institutions , and policy - makers , is an essential step that academic public health institutes need to take for establishment of a sound basis for effective and sustainable solutions to priority population health challenges and related health system problems . as the global agenda for health by 2030 , sdgs have created an evidence - based , universal and comprehensive platform for more meaningful multi - sectoral collaboration in this regard . academic public health institutes should seize the opportunity to establish their role in backing sustainable health development within their home countries . this requires fundamental changes in the infrastructure , administrative skills , and leadership capacities necessary to empower faculty and staff , which may be fundamentally different from traditional approaches to address public health challenges . academic public health institutes must pioneer in finding innovative approaches to respond to the challenges of sustainable health development . this requires modern approaches to funding , education , teaching , research priority setting and advocacy , which in turn need novel strategies in collaboration and constructive partnerships among academic public health institutes from low , middle and high - income countries . the opportunity of sdgs has created a common forum to fulfill such a mission by the academic public health institutes from across the globe . with a motto of leaving nobody behind , and considering partnership as their core , sdgs should be effectively and cooperatively utilized by the academic public health institutes from all settings for improving the health and wellbeing of citizens of the planet through sustainable health development .

background : sustainable development goals ( sdgs ) aim to transform our world , and each goal has specific targets to be achieved by 2030 . for the goals to be achieved , everyone needs to do their part : governments , academia , the private sector and all people . this paper summarizes the main evidence - based recommendations made by excellent academics and scholars who discussed their experiences and views during the conference to respond to the challenges of sustainable health development.methods:to contribute to exploring to the academia s role in reaching sdgs , the 1st international conference on sustainable health development was held at tehran university of medical sciences , on 2425 april 2016 , in tehran , iran.results:in line with goal 3 of sdgs : ensure healthy lives and promote well - being for all at all ages , the conference discussed various aspects of universal health coverage ( uhc ) , as well as global action plans for prevention and control of non - communicable diseases ( ncds ) , and explained the special role of academic public health institutes in education , research and service provision in the two above - mentioned areas.conclusion:to fulfill the requirements of sdgs , modern approaches to funding , education , teaching , research priority setting and advocacy , which in turn need novel strategies in collaboration and constructive partnerships among academic public health institutes from low , middle and high - income countries , are essential .

Introduction Methods Results Sustainable Health Development as the main agenda of academic public health institutes A- The role of academic public health institutes in UHC UHC and health services The special role of academic public health institutes in capacity building of human resources and education UHC and research B- The role of academic public health institutes in prevention and control of NCDs Role of academia in prevention and control of NCDs: identification, involvement and institutionalization NCDs and other sectors NCDs and Environmental Health Discussion The resolution of conference: Together, geared up towards sustainable health development Conclusion

on 25 september 2015 , the united nations approved a set of 17 goals and 169 targets to end poverty , protect the planet , and ensure prosperity for all as part of a new sustainable development agenda . these so - called sustainable development goals ( sdgs ) aim to transform our world , and each goal has specific targets to be achieved by 2030 . seeking to complete what mdgs did not achieve , particularly in reaching the most vulnerable , sdgs envisage a world free of poverty , hunger , disease , fear and violence , and desire a universal literacy , equitable and universal access to quality education at all levels , to health care and social protection among all global citizens . for the goals to be achieved , everyone needs to do their part : governments , academia , the private sector , civil society and all people . we conducted document analysis to identify the main literature around uhc and ncds in the format of sdgs and created a full report of the two days conference on sustainable health development . to contribute to exploring to the academia s role in reaching sdgs , the 1 international conference on sustainable health development , was held simultaneously with commemoration of the 50 anniversary of the establishment of school of public health ( sph ) , tehran university of medical sciences , on 2425 april 2016 , in tehran , i.r . in line with goal 3 of sdgs : ensure healthy lives and promote well - being for all at all ages , appreciating the great need for more efforts to fully eradicate a wide range of diseases and address many different persistent and emerging health issues , and on the basis of the priorities of the health system of the islamic republic of iran , the conference provided an opportunity to study and discuss various aspects of universal health coverage ( uhc ) , as well as global action plans for prevention and control of non - communicable diseases ( ncds ) , both of which are among the targets of sdg 3 , and explain the role of research and higher education institutions in this regard . attracting over 700 participants , the 2 days event accommodated six specific panels with 39 speakers from policy makers , managers , academics , and researchers at the global , international and national levels , who discussed in the presence of above 700 participants , the special role of academic public health institutes in education , research and service provision in the two above - mentioned areas . for instance , sph at tehran university of medical sciences has been in charge of conducting some specific viral and parasitology lab tests for the public . by definition , public health involves all aspects of prevention as well as all fundamental services , which have demographical dimensions . conventionally , academic public health institutes have focused on the issues of promotion , primary prevention and primary health care , hence , leading uhc to deal mostly with primary prevention rather than palliation services . the panel addressed the role of academic public health institutes in providing health care , rehabilitation and palliative services , as well as the distinction between schools of public health and other higher education institutes in the fields of medicine , rehabilitation , and other disciplines . in addition , the appropriate structural model of academic public health institutes in addressing managerial and leadership aspects of sdgs was discussed , with special focus on the workforce needed for uhc . two topics of a ) filling the gap between research and higher education institutions and the decision making environment and b ) networking and areas of cooperation between academic public health institutes were the main topics of this panel . the discussion introduced successful models and shared experiences in this area , from the eastern mediterranean region , as well as other parts of the world . finding mutual areas of interest for collaboration among academic public health institutes and agreeing upon practical ways to move this initiative forward , which was discussed in more details during the specific meeting of academic public health institutes of eastern mediterranean region as the side event on 3 day of the conference , was the main aim of this panel . the academic public health institutes have an important role to play in prevention and control of ncds . partnering with other stakeholderssupport healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives.health system strengthening for early detection and management of ncds . partnering with other stakeholders support healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives . ministry of health and other state ministries ; academia ; non - governmental and civil society organizations ; private sectors ; as well as external and international agencies , in prevention and control of ncds and ways to strengthen their involvement in this regard . water scarcity , poor air quality , global warming , waste management , climate change , the link between environmental health issues and uhc , and the need for academic public health institutes to address such challenges to reach sustainable development . to contribute to exploring to the academia s role in reaching sdgs , the 1 international conference on sustainable health development , was held simultaneously with commemoration of the 50 anniversary of the establishment of school of public health ( sph ) , tehran university of medical sciences , on 2425 april 2016 , in tehran , i.r . in line with goal 3 of sdgs : ensure healthy lives and promote well - being for all at all ages , appreciating the great need for more efforts to fully eradicate a wide range of diseases and address many different persistent and emerging health issues , and on the basis of the priorities of the health system of the islamic republic of iran , the conference provided an opportunity to study and discuss various aspects of universal health coverage ( uhc ) , as well as global action plans for prevention and control of non - communicable diseases ( ncds ) , both of which are among the targets of sdg 3 , and explain the role of research and higher education institutions in this regard . attracting over 700 participants , the 2 days event accommodated six specific panels with 39 speakers from policy makers , managers , academics , and researchers at the global , international and national levels , who discussed in the presence of above 700 participants , the special role of academic public health institutes in education , research and service provision in the two above - mentioned areas . for instance , sph at tehran university of medical sciences has been in charge of conducting some specific viral and parasitology lab tests for the public . for instance , sph at tehran university of medical sciences has been in charge of conducting some specific viral and parasitology lab tests for the public . by definition , public health involves all aspects of prevention as well as all fundamental services , which have demographical dimensions . conventionally , academic public health institutes have focused on the issues of promotion , primary prevention and primary health care , hence , leading uhc to deal mostly with primary prevention rather than palliation services . the panel addressed the role of academic public health institutes in providing health care , rehabilitation and palliative services , as well as the distinction between schools of public health and other higher education institutes in the fields of medicine , rehabilitation , and other disciplines . in addition , the appropriate structural model of academic public health institutes in addressing managerial and leadership aspects of sdgs was discussed , with special focus on the workforce needed for uhc . two topics of a ) filling the gap between research and higher education institutions and the decision making environment and b ) networking and areas of cooperation between academic public health institutes were the main topics of this panel . in fact , the differences between the decision - making environment and the academic environment are a well - known phenomenon , which has drawn global attention . finding mutual areas of interest for collaboration among academic public health institutes and agreeing upon practical ways to move this initiative forward , which was discussed in more details during the specific meeting of academic public health institutes of eastern mediterranean region as the side event on 3 day of the conference , was the main aim of this panel . two topics of a ) filling the gap between research and higher education institutions and the decision making environment and b ) networking and areas of cooperation between academic public health institutes were the main topics of this panel . finding mutual areas of interest for collaboration among academic public health institutes and agreeing upon practical ways to move this initiative forward , which was discussed in more details during the specific meeting of academic public health institutes of eastern mediterranean region as the side event on 3 day of the conference , was the main aim of this panel . the academic public health institutes have an important role to play in prevention and control of ncds . preparing the academic public health institutes to undertake greater responsibility in ncd - related works , especially when there is inevitable increase in attention and funding from the global , regional and the national agencies , was also discussed . partnering with other stakeholderssupport healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives.health system strengthening for early detection and management of ncds . partnering with other stakeholders support healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives . ministry of health and other state ministries ; academia ; non - governmental and civil society organizations ; private sectors ; as well as external and international agencies , in prevention and control of ncds and ways to strengthen their involvement in this regard . water scarcity , poor air quality , global warming , waste management , climate change , the link between environmental health issues and uhc , and the need for academic public health institutes to address such challenges to reach sustainable development . the academic public health institutes have an important role to play in prevention and control of ncds . partnering with other stakeholderssupport healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives.health system strengthening for early detection and management of ncds . partnering with other stakeholders support healthy lifestyle and reduction of major ncds risk factors by incorporating health promotional activities in academic public health institutes endeavors , including community - based initiatives . ministry of health and other state ministries ; academia ; non - governmental and civil society organizations ; private sectors ; as well as external and international agencies , in prevention and control of ncds and ways to strengthen their involvement in this regard . water scarcity , poor air quality , global warming , waste management , climate change , the link between environmental health issues and uhc , and the need for academic public health institutes to address such challenges to reach sustainable development . in line with sustainable development goal 3 , the 2012 un general assembly resolution on universal health coverage ( uhc ) a/67/l.36 , world health assembly resolution wha67.23 and who global action plan for the prevention and control of ncds 20132020 ( resolution wha66.10 ) , the 1 international conference on sustainable health development approved a resolution in its final session . the resolution , which is meant to be a manifest for participating academic public health institutes in the conference , recognized : uhc to deliver equitable opportunities for the highest attainable standards of physical , social and mental health;the importance of non - communicable diseases ( ncds ) in disabling and killing people , their considerable burden to peoples health , and the importance of having national , robust and evidence - based plans and policies for prevention and control of ncds;the specific and central role of academic public health institutes in provision of useful and need - based education , research and service to address the challenges of public health , as well as reach sustainable development;the great need of reconsidering the role of academic public health institutes in the context of low - middle income countries to respond to the emerging challenges of public health;that public health policies are only meaningful when they are translated into action by regulation , budget allocations , purchasing and procurement , monitoring , evaluation and supervision;that global and national action is required to find a right solution to support priority settings for health that ensure alignment , participation , transparency , empowerment , nondiscrimination and accountability resulted by a synergistic collaboration of stakeholders . uhc to deliver equitable opportunities for the highest attainable standards of physical , social and mental health ; the importance of non - communicable diseases ( ncds ) in disabling and killing people , their considerable burden to peoples health , and the importance of having national , robust and evidence - based plans and policies for prevention and control of ncds ; the specific and central role of academic public health institutes in provision of useful and need - based education , research and service to address the challenges of public health , as well as reach sustainable development ; the great need of reconsidering the role of academic public health institutes in the context of low - middle income countries to respond to the emerging challenges of public health ; that public health policies are only meaningful when they are translated into action by regulation , budget allocations , purchasing and procurement , monitoring , evaluation and supervision ; that global and national action is required to find a right solution to support priority settings for health that ensure alignment , participation , transparency , empowerment , nondiscrimination and accountability resulted by a synergistic collaboration of stakeholders . the resolution called on all academic public health institutes across the world , and the global public health arena to : take progressive action to achieve the health related sdgs and their indicators by nationalizing the goals within their countries.facilitate exchange of faculty members , scholars and other researchers among different academic public health institutes in order to share and learn various experiences , as well as franchising a spectrum of projects and activities.develop , strengthen and sustain collaborative research on similar problems in different countries within a region and beyond it.deploy joint implementation of public health practices and interventions to enable governments to achieve uhc and program and implement national plans for prevention and control of ncds.share best practices of each academic public health institute and contextualize them for integration into the health system.increase communication and exchanges regarding the sdgs to achieve a unified vision about the goals and indicators.assist and play a fundamental role in the interpretation of sdgs , i.e. uhc and ncds in the context of their own countries.establish objective - driven networks to fulfill the above - mentioned vision . facilitate exchange of faculty members , scholars and other researchers among different academic public health institutes in order to share and learn various experiences , as well as franchising a spectrum of projects and activities . deploy joint implementation of public health practices and interventions to enable governments to achieve uhc and program and implement national plans for prevention and control of ncds . assist and play a fundamental role in the interpretation of sdgs , i.e. in line with sustainable development goal 3 , the 2012 un general assembly resolution on universal health coverage ( uhc ) a/67/l.36 , world health assembly resolution wha67.23 and who global action plan for the prevention and control of ncds 20132020 ( resolution wha66.10 ) , the 1 international conference on sustainable health development approved a resolution in its final session . the resolution , which is meant to be a manifest for participating academic public health institutes in the conference , recognized : uhc to deliver equitable opportunities for the highest attainable standards of physical , social and mental health;the importance of non - communicable diseases ( ncds ) in disabling and killing people , their considerable burden to peoples health , and the importance of having national , robust and evidence - based plans and policies for prevention and control of ncds;the specific and central role of academic public health institutes in provision of useful and need - based education , research and service to address the challenges of public health , as well as reach sustainable development;the great need of reconsidering the role of academic public health institutes in the context of low - middle income countries to respond to the emerging challenges of public health;that public health policies are only meaningful when they are translated into action by regulation , budget allocations , purchasing and procurement , monitoring , evaluation and supervision;that global and national action is required to find a right solution to support priority settings for health that ensure alignment , participation , transparency , empowerment , nondiscrimination and accountability resulted by a synergistic collaboration of stakeholders . uhc to deliver equitable opportunities for the highest attainable standards of physical , social and mental health ; the importance of non - communicable diseases ( ncds ) in disabling and killing people , their considerable burden to peoples health , and the importance of having national , robust and evidence - based plans and policies for prevention and control of ncds ; the specific and central role of academic public health institutes in provision of useful and need - based education , research and service to address the challenges of public health , as well as reach sustainable development ; the great need of reconsidering the role of academic public health institutes in the context of low - middle income countries to respond to the emerging challenges of public health ; that public health policies are only meaningful when they are translated into action by regulation , budget allocations , purchasing and procurement , monitoring , evaluation and supervision ; that global and national action is required to find a right solution to support priority settings for health that ensure alignment , participation , transparency , empowerment , nondiscrimination and accountability resulted by a synergistic collaboration of stakeholders . the resolution called on all academic public health institutes across the world , and the global public health arena to : take progressive action to achieve the health related sdgs and their indicators by nationalizing the goals within their countries.facilitate exchange of faculty members , scholars and other researchers among different academic public health institutes in order to share and learn various experiences , as well as franchising a spectrum of projects and activities.develop , strengthen and sustain collaborative research on similar problems in different countries within a region and beyond it.deploy joint implementation of public health practices and interventions to enable governments to achieve uhc and program and implement national plans for prevention and control of ncds.share best practices of each academic public health institute and contextualize them for integration into the health system.increase communication and exchanges regarding the sdgs to achieve a unified vision about the goals and indicators.assist and play a fundamental role in the interpretation of sdgs , i.e. uhc and ncds in the context of their own countries.establish objective - driven networks to fulfill the above - mentioned vision . facilitate exchange of faculty members , scholars and other researchers among different academic public health institutes in order to share and learn various experiences , as well as franchising a spectrum of projects and activities . deploy joint implementation of public health practices and interventions to enable governments to achieve uhc and program and implement national plans for prevention and control of ncds . assist and play a fundamental role in the interpretation of sdgs , i.e. academic public health institutes must invest in transforming their conventional approach in research , education , advocacy and service provision , should they decide to play a meaningful role in reaching sustainable health development . multidisciplinary partnerships with communities , institutions , and policy - makers , is an essential step that academic public health institutes need to take for establishment of a sound basis for effective and sustainable solutions to priority population health challenges and related health system problems . academic public health institutes should seize the opportunity to establish their role in backing sustainable health development within their home countries . this requires fundamental changes in the infrastructure , administrative skills , and leadership capacities necessary to empower faculty and staff , which may be fundamentally different from traditional approaches to address public health challenges . academic public health institutes must pioneer in finding innovative approaches to respond to the challenges of sustainable health development . this requires modern approaches to funding , education , teaching , research priority setting and advocacy , which in turn need novel strategies in collaboration and constructive partnerships among academic public health institutes from low , middle and high - income countries . the opportunity of sdgs has created a common forum to fulfill such a mission by the academic public health institutes from across the globe . with a motto of leaving nobody behind , and considering partnership as their core , sdgs should be effectively and cooperatively utilized by the academic public health institutes from all settings for improving the health and wellbeing of citizens of the planet through sustainable health development .

autism spectrum disorders ( asd ) are a complex group of severe neurodevelopmental disorders that affect over 1% of children in the united states . typical symptoms of autism include impairments in social interaction , deficits in verbal and nonverbal communication , and repetitive behaviors and restricted interests . although the exact etiology of the disorder has not been identified , a link between altered immune responses and asd was first recognized nearly 40 years ago . neurobiological studies in asd have highlighted pathways involved in neural development , synapse plasticity , structural brain abnormalities , cognition , and behavior . in addition , abnormalities in the cellular immune response have also been reported in children with autism ; in particular , reduced cytotoxic activity and elevated levels of selected proinflammatory cytokines produced by peripheral blood mononuclear cells , such as tumor necrosis factor ( tnf- ) and il1 , have been shown to disrupt neurodevelopment [ 3 , 4 ] . cytokines are polypeptides that are 825 kda in size and include interleukins , chemokines , interferons , tumor necrosis factors ( tnfs ) , and growth factors . cytokines have been shown to regulate the growth and cell proliferation of neuronal tissue and to modulate host responses to infection , injury , inflammation , and diseases of uncertain etiology . the dynamic expression of different cytokines can modify immune system function ; for example , increased levels of interferon - gamma ( ifn- ) and il-12 can induce inflammation , whereas increased production of tgf- can negatively regulate inflammation . a number of recent studies have demonstrated that the levels of various inflammatory cytokines differ in the blood mononuclear cells , serum , plasma , brain tissue , and cerebrospinal fluid of autistic subjects compared with normal subjects , which might impair immune capacity in the central nervous system ( cns ) and stimulate the production and activation of microglia in the brain [ 7 , 8 ] . microglia are the unique resident immune cells of the cns ; they act as primary mediators of inflammation , participating in immune surveillance of the cns and synaptic pruning during normal neurodevelopment . mounting evidence indicates that chronic microglial activation might also contribute to the development and progression of neurodegenerative disorders . active microglia can induce the production of pro - inflammatory cytokines such as il-1 , il-6 , and tnf- , which are typically intended to prevent further damage to brain tissue . however , abnormally activated microglia can sometimes be toxic to neurons and other glial cells . microglia cell activation is a prominent feature of autism , and there is a complex interaction between microglial cells and cytokines . wei et al . found that il-6 elevation can modulate autism - like behaviors through impairments of synapse formation , dendritic spine development , and neuronal circuit balance . in this review , we focused on several types of proinflammatory and anti - inflammatory cytokines that might affect different cell signal pathways and play a role in the pathogenic mechanism that might be responsible for autism ( table 1 ) . interleukin-1 ( il-1 ) consists of two different proteins , il-1 and il-1 , which exert their proinflammatory effects by binding to type - i il-1 receptors ( il-1 ri ) , whereas the type ii il-1 receptor ( il-1rii ) is thought to act as a decoy receptor that does not participate in active il-1 signaling [ 9 , 10 ] . il-1 is produced , processed , and secreted from activated immune cells and plays a major role in the initiation of local and systemic inflammatory processes . signaling mediated by il-1ri can activate the myeloid differentiation response gene 88 ( myd88 ) and tumor necrosis factor - associated factor 6 ( traf6 ) , leading to the activation of a number of different kinases , including il-1 receptor associated kinases 1 and 4 ( irak1 , irak4 ) , transforming growth factor -activated kinase ( tak ) , and members of the mitogen activated protein kinase ( mapk ) cascade , including p38 , c - jun n - terminal kinases ( jnk ) , and extracellular signal - regulated kinases 1/2 ( erk1/2 ) . in 1991 , singh et al . first measured the serum levels of soluble il-1 and found that the level did not differ between the subjects with autism and the control subjects . ( 2002 ) demonstrated that there is also no difference in the il-1 receptor between autism and the control subjects . however , in vitro , jyonouchi and his group observed that stimulated peripheral blood mononuclear cells ( pbmcs ) from children with autism secrete significantly higher amounts of soluble il-1 compared with normal controls . in an animal model , maternal exposure to poly-(i : c ) or lipopolysaccharide ( lps ) can induce a significant increase of il-1 in amniotic fluid , placentas , and fetal brains . il-1 has also been shown to play a key role in mediating severe placental damage and neurodevelopmental anomalies in offspring [ 15 , 16 ] . il-1 showed the highest concentration levels in fetal brains and was the only cytokine that was significantly upregulated 24 h after maternal poly ( i : c ) injection , suggesting that il-1 may have a deleterious impact on central nervous system development . however , these studies did not evaluate the social behavior of offspring , so further study is needed to determine whether there is a link between the development of autism and elevated il-1 levels . x - linked interleukin-1 receptor accessory protein like-1 ( il1rapl1 ) is a member of the interleukin-1 receptor family and is similar to the interleukin-1 accessory proteins . ( 2008 ) first reported the function of the resulting truncated il1rapl1 protein , which is severely altered in hippocampal neurons , by measuring its effect on neurite outgrowth activity . the mutations in il1rapl1 cause a spectrum of neurological impairments ranging from mental retardation to high - functioning autism . pavlowsky and colleagues also reported a novel partner of il1rapl1 , psd-95 , a major scaffold protein of excitatory synapses , and showed that il1rapl1 regulates the dendritic spine number and psd-95 localization to synapses . further investigation showed that il-1-induced activation of the jnk pathway in neurons is mediated by il1rapl1 . this finding indicates that a novel pathophysiological mechanism underlying cognitive impairment could be associated with alterations of the jnk pathway in response to il-1 and hence lead to the mislocalization of psd-95 , which subsequently result in abnormal synaptic organization and function . interleukin-2 ( il-2 ) is a well - known cytokine that plays an important role in multiple immunoregulatory functions related to t - cells in peripheral and cns [ 20 , 21 ] . the il-2 receptor consists of three subunits , il-2r , ( il-2r ) , and ( il-2r ) , which can deliver biochemical signals to the cell interior . they affect different signal pathways , such as the mapk pathway , the phosphoinositide 3-kinase ( pi3k ) pathway , and the jak - stat pathway . il-2 in the brain is potentially produced by neurons and astrocytes and is widely distributed throughout the brain along with its receptors . there is a large body of literature indicating that il-2 may be involved in cns development , normal brain physiology , and homeostatic repair mechanisms as well as brain dysfunction and neurodegenerative processes . for instance , it has been shown that prolonged exposure to il-2 in cancer patients can result in cognitive dysfunction , altered behavior , and other negative neuropsychiatric side effects . in addition , il-2 levels have been found to be elevated in the postmortem brains of patients with alzheimer 's disease compared with normal control subjects . singh et al . found that serum concentrations of soluble il-2 were significantly higher in autistic children compared with normal controls . in addition , they showed that stimulated peripheral blood mononuclear cells ( pbmcs ) from children with autism secrete significantly higher amounts of il-2 , whereas soluble il-2 receptor levels did not differ between autistic and control subjects . however , more recent studies have reported that il-2 levels did not differ significantly between subjects with autism and control groups [ 25 , 26 ] . vojdani and colleagues measured nk cell activity in 1027 blood samples from autistic children obtained from ten clinics and compared the results to those of 113 healthy controls . in addition , they cultured lymphocytes from autistic children with low or high nk cell activity and with or without il-2 and determined that the induction of nk cell activity by il-2 was more pronounced in a subgroup with very low nk cell activity . il-6 is characterized as a 26 kda glycoprotein that can trigger cellular responses that mediate inflammation , neurogenesis , gliogenesis , cell growth , cell survival , myelination , and demyelination in the cns [ 28 , 29 ] . the il-6 receptor protein complex consists of one nonsignaling , membrane - associated subunit ( il-6r ) and two gp130 subunits responsible for signal transduction [ 30 , 31 ] . ligand binding of this protein complex results in the homodimerization of gp130 and activates multiple signaling mechanisms , including jak / stat , pi3k / akt , and the ras / raf / mapk pathways [ 3134 ] . recently , the role of maternal immune activation ( mia ) has become a hot topic in autism research . several groups have found that the developing fetus 's exposure to maternal cytokines precipitates neurological , immunological , and behavioral abnormalities in the offspring [ 35 , 36 ] . maternal injection with il-6 alone is sufficient to cause abnormal behavior in the offspring following maternal poly-(i : c ) injection or respiratory infection . conversely , il-6 inhibition was sufficient to attenuate the behavioral deficits caused by mia [ 35 , 37 ] . in our laboratory , we found that mice with elevated il-6 in the brain display many autistic features , including impaired cognitive abilities , learning deficits , abnormal anxiety traits , and habituations and decreased social interactions . we also examined the development of synapses and found that il-6 elevation altered excitatory and inhibitory synaptic formations , disrupted the balance of excitatory / inhibitory synaptic transmissions , and resulted in an abnormal change in the shape , length and distributing pattern of dendritic spines . these findings suggest that il-6 elevation in the brain could mediate autistic - like behaviors , possibly through the disruption of the neural circuitry balance and impairments of synaptic plasticity . hsiao and patterson confirmed that mia elevates il-6 protein and mrna expression in the placenta and found that maternally derived il-6 mediates jak / stat3 pathway activation specifically in the spongiotrophoblast layer , a fetal compartment of the placenta , which results in the expression of acute - phase genes . furthermore , hsiao and patterson also demonstrated an il-6-dependent dysregulation of the growth hormone / insulin - like growth factor ( gh - igf ) axis in mia placentas that was characterized by decreased levels of gh and igf1 mrna with corresponding decreases in placental igf1 and igf - binding protein 3 ( igfbp3 ) . singh first examined the plasma levels of il-6 and found no significant differences between subjects with autism and controls . however , vargas ' group demonstrated that il-6 was increased in autistic brains . in our recent studies , we also found that il-6 was significantly increased in the cerebella of autistic subjects . the cerebellum was suggested as a main focus of neuroinflammation in autism , and the selective vulnerability of the purkinje cells may play a role in the etiopathogenesis of autism . in further studies , we investigated how il-6 affects neural cell development and function by transfecting cultured mouse cerebellar granule cells with an il-6 viral expression vector . we demonstrated that il-6 overexpression in granule cells caused impairments in granule cell adhesion and migration but had little effect on the formation of dendritic spines or granule cell apoptosis . these findings suggest that il-6 not only plays an important role in the etiology of autism , but may provide a potential biological marker that enables the early diagnosis of the disorder and earlier therapeutic intervention ( figure 1 ) . because il-6 can attenuate abnormal behavior caused by mia , it might be a potential therapy target in the future . the chemokines are a family of small cytokines that play an important role in the development of lymphocytes , including their recruitment and trafficking to specific tissue compartments . in addition to acting as key chemotactic factors in the immune system , chemokines and their receptors are critical for dictating the movement of leukocytes into the cns in both healthy and diseased individuals . chemokines have been shown to regulate neuronal cell migration , proliferation , and neuronal cell differentiation and are involved in the communication between neurons and microglia [ 43 , 44 ] . monocyte chemoattractant protein 1 ( mcp-1/ccl2 ) is known to mediate monocyte and t - cell activation and trafficking into areas of tissue injury . in the cns , mcp-1 can modulate the recruitment of myeloid cells to injury or inflammation sites , and it is increased in brain in instances of ischemia , alzheimer 's disease , and experimental autoimmune encephalomyelitis . different groups have measured mcp-1 levels in the brain , csf , and plasma of individuals with autism and found that mcp-1 was elevated in individuals with autism . elevated mcp-1 production was associated with higher aberrant behavior scores and more impaired developmental and adaptive function [ 8 , 42 ] . abdallah and colleagues examined the chemokine levels in amniotic fluid ( af ) samples from individuals diagnosed with autism and controls . in an mia model , mcp-1 was significantly upregulated after the injection of poly-(i : c ) . mcp-1 can enhance neuronal excitability and synaptic transmission in hippocampal neurons and can modulate neuronal physiology [ 47 , 48 ] . taken together , these findings suggest that elevated mcp-1 in the brain might be linked to microglial activation and to the recruitment of monocytes / macrophages to areas of neurodegeneration . osteopontin ( opn ) is a 60 kda phosphoprotein that plays different roles , including initiating inflammation , cell adhesion , chemotaxis , immune responses , and protection against apoptosis , depending on its intracellular or extracellular localization . opn is also involved in regulating chronic inflammatory and autoimmune diseases , including multiple sclerosis ( ms ) , rheumatoid arthritis , inflammatory bowel disease , systemic lupus erythematosus , and type i diabetes [ 5053 ] . recently , studies have demonstrated that opn also plays a role in the development of neurodegenerative diseases , such as parkinson 's and alzheimer 's disease . however , opn can trigger neuronal toxicity and death in some contexts and can function as a neuroprotectant in others . al - ayadhi and mostafa ( 2011 ) measured serum opn levels using elisa in 42 autistic children and 42 matched healthy children . the authors found that the autistic children had significantly higher serum opn levels compared with the healthy controls , and the autistic children 's serum opn levels had significant positive correlations with their childhood autism rating scale ( cars ) scores . however , it is difficult to determine the exact roles of specific chemokines during neurodevelopment because of the high degree of interaction between different chemokines and their receptors . at present however , a number of studies have reported abnormal chemokine production in the brain , csf , and/or plasma of autistic individuals , suggesting that chemokines might be involved in aberrant neuronal development that could lead to altered early brain development and function . it is a member of a group of cytokines that stimulate the acute phase reaction . tnf- is produced mainly by activated macrophages ( m1 ) , although it can be produced by other cell types , such as cd4 + lymphocytes and nk cells . tnf can bind two receptors , tnf receptor type 1 ( tnf - r1 ) and tnf receptor type 2 ( tnf - r2 ) . binding with ligand , tnf- can activate nf-b , mapk , and the apoptosis signaling pathway . in 1996 , singh demonstrated that plasma tnf- did not significantly differ between autistic subjects and normal controls . however , jyonouchi and collaborators tested 71 autistic children aged 214 years and compared them with healthy siblings and other controls . their study showed that pbmcs activated by lps produced higher levels of tnf- , il-1 , and/or il-6 in most autistic children ( 83.1% ) compared with the control group . the investigators concluded that a majority of the autistic children in the group , especially those with increased tnf- , exhibited excessive or poorly regulated innate immune responses . ( 2007 ) detected elevated tnf- in the cerebrospinal fluid of autistic children . in 2009 , our lab found that tnf- was significantly increased in the brains of autistic subjects . furthermore , ashwood and colleagues used polyhydroxyalkanoates ( pha ) and tetanus to stimulate pbmcs from autistic subjects and controls to compare group - associated cellular responses . tnf- was specifically selected to activate the classical nf-b signaling cascade of the innate immune system and to stimulate the nuclear translocation of nf-b in primary cortical neurons . nf-b dna binding activity was significantly increased in the peripheral blood samples of children with autism . nf-b has also been shown to be aberrantly expressed in the orbitofrontal cortex in patients with autism , which indicates that nf-b could be part of a putative molecular cascade leading to inflammation in brain regions associated with the behavioral and clinical symptoms of autism . however , our laboratory results show that the expression of nf-b ( p65 ) and the phosphorylation / activation of nf-b ( p65 ) at ser536 are not significantly changed in the cerebellum and cortex of both autistic subjects and btbr mice in an autism model . these findings imply that nf-b may be involved in the abnormal inflammatory response processes suggested in autistic brain but do not play an important part . our results suggest that tnf- might affect the progress of autism through another pathway , such as the mapk / jnk pathway . ifn- is critical for innate and adaptive immunity against viral and intracellular bacterial infections , and it plays a role in tumor control . cellular responses to ifn- are activated through its interaction with a heterodimeric receptor consisting of interferon gamma receptor 1 ( ifngr1 ) and interferon gamma receptor 2 ( ifngr2 ) . aberrant ifn- expression is associated with a number of autoinflammatory and autoimmune diseases . in 1996 , singh found significantly elevated ifn- levels in the plasma of 20 autistic children compared with 20 healthy controls . recently , our laboratory also demonstrated that the concentration of ifn- was significantly increased in the brains of autistic subjects compared with normal control subjects . another study detected elevated serum ifn- in women who had given birth to a child who was later diagnosed with autism . ( 2004 ) demonstrated that the plasma levels of ifn- did not differ between the autistic group and control group , but they did correlate positively with plasma nitric oxide measures in the autistic group . it is known that under normal circumstances , pregnancy shifts the maternal immune system toward a more tolerant stage , causing an overall decrease in proinflammatory cytokine trajectories in the innate and adaptive arms of the immune system and an increase in counter regulatory cytokines . mothers of children with autism demonstrated increased levels of the inflammatory cytokine ifn- , which may indicate an atypical immune state during gestation . furthermore , in addition to maternal abnormality of ifn- , there is also elevated ifn- in autistic children . these findings support the hypothesis that ifn- is involved in mia and plays a role in the pathologic mechanism that may be responsible for autism . tgf-1 is one of the most important immune regulators that can effectively control diverse aspects of the immune response . it plays an important role in autoimmune diseases such as myasthenia gravis - related ophthalmoparesis , autoimmune diabetes , and ms [ 6770 ] . in the cns , tgf-1 is widely recognized as an injury - related cytokine specifically associated with astrocyte scar formation in response to brain injury . in recent years , emerging data regarding tgf-1 and its signaling molecules have suggested that , in addition to its role in brain injury , tgf-1 might be a crucial regulator of cns development . considering the importance of tgf1 in immune response and nervous system development , a number of studies have investigated whether tgf-1 alters in autistic subjects . vargas et al . found that tgf-1 was significantly increased in certain cell line specimens from autistic subjects . however , decreased tgf-1 levels have been observed in the plasma of autistic individuals [ 67 , 72 ] . in addition , there were significant correlations between clinical scores and tgf1 levels ; lower tgf1 levels were associated with reduced adaptive behaviors and worse behavioral symptoms . these findings suggest that immune responses in autism may be inappropriately regulated because of reductions in tgf1 , and such immune dysregulation may predispose individuals to the development of possible autoimmune responses and/or adverse neuroimmune interactions during critical windows in development . in an animal study , depino and colleagues found that overexpression of tgf-1 during postnatal can lead to asd - related behaviors , such as decreased social interaction , increased self - grooming , and depression - related behaviors . however , adult hippocampal levels of murine tgf-1 were reduced in animals that were injected with adtgf at pd14 compared with adbgal - injected mice . in addition , the study found that asd - related behaviors correlate with the long - term downregulation of tgf-1 and il-6 expression in the dentate gyrus and with decreases in the mrna levels of the synaptic protein neuroligin 3 and the number of reelin - positive neurons in the dentate gyrus . conversely , chronic expression of tgf-1 during adulthood leads to transient opposite effects on abnormal social behaviors : it increases social interaction and decreases repetitive behavior . these results indicate that hippocampal tgf-1 plays an important role in the programming and modulation of social interaction , repetitive behavior , and depression - related behavior . egf is a growth factor that stimulates cell growth , proliferation , and differentiation by binding to its receptor egfr . egf can be detected in all regions of the developing and adult brain and in a majority of the neurons and developing astrocytes . egf has neurotrophic effects on cultured cortical neurons and stimulates neurite outgrowth in dopaminergic neuron cultures . transgenic mice lacking the egf receptor ( egfr ) develop neurodegenerative disease and die within the first month of life . researchers examined the association of egf , tgf-1 , and hgf genes with autism in a trio association study using dna samples from families recruited to the autism genetic resource exchange . their results revealed a significant haplotypic association between egf and autism , but no significant snp or haplotypic associations were observed for tgf-1 or hgf . these results suggest a possible role of egf in the pathogenesis of autism . in 2007 , a research group measured serum levels of egf in 17 male subjects with high - functioning autism and 18 age - matched healthy male subjects . they found that the serum egf levels in the subjects with high - functioning autism were significantly lower compared with those of the normal control subjects . however , there were no correlations between serum egf levels and clinical variables in the subjects with autism . by contrast , other researchers found that the serum egf levels in subjects with autism were significantly higher than those of normal control subjects . there were also no correlations between serum egf levels and clinical variables in the subjects with autism [ 79 , 80 ] . bdnf is a member of the nerve growth factor family ( neurotrophins ) ; it contributes to prenatal and postnatal brain development . bdnf supports neuronal survival , maintains various neuronal activities , modulates neurotransmitter release , participates in neuronal plasticity , and mediates long - term potentiation and memory fixation . nelson and miyazaki reported higher bdnf levels in archived samples of neonatal blood and serum obtained from autistic subjects compared with normal controls [ 83 , 84 ] . however , in a carefully constructed large - sample study , the results demonstrated that the mean levels of bdnf were significantly lower in the serum of 0- to 9-year - old autistic children compared with age - matched healthy controls or teenagers or adults . our study found that the concentrations of bdnf and bcl2 were reduced in the frontal cerebral cortex of autistic subjects compared with age - matched controls . we also found that the expression and activation of akt kinase , which regulates bcl2 , are significantly decreased in the autistic brain . the downregulation of akt may result from a decreased concentration of bdnf , which is the growth factor that modulates akt activities . we thus reason that the downregulation of the bdnf - akt - bcl2 antiapoptotic signaling pathway in the autistic brain could be one of the underlying mechanisms responsible for the pathogenesis of autism . granulocyte - macrophage colony - stimulating factor ( gm - csf ) is a cytokine that regulates the survival , proliferation , differentiation , and functional activity of many myeloid hemopoietic cells ; it also regulates dendritic cell and t - cell function . ashwood and colleagues investigated adaptive cellular immune function in 66 children with a confirmed diagnosis of asd and 73 confirmed typically developing ( td ) controls aged 2 to 5 years . in this study , the in vitro stimulation of pbmc with pha and tetanus was used to compare group - associated cellular responses . the results showed that gm - csf , tnf- , and il-13 production were significantly increased , whereas il-12p40 was decreased following pha stimulation in autism relative to td controls . in addition , the study also found that induced cytokine production was associated with altered behaviors in autistic children : increased proinflammatory or t(h)1 cytokines were associated with greater impairments in the core features of autism and more aberrant behaviors . by contrast , the production of gm - csf and t(h)2 cytokines was associated with better cognitive and adaptive function . our laboratory also found that gm - csf was elevated in the brains of autistic patients compared with controls . however , a study by manzardo 's group showed that the gm - csf levels were significantly lower in the plasma of children with autism compared with unrelated siblings without autism . gm - csf can upregulate neuronal receptors on cd34 + hematopoietic stem progenitor cells to enhance responses to neurotransmitters , stimulate neurite growth , and promote axonal regeneration [ 8991 ] . gm - csf can also promote neuronal differentiation of adult neuronal stem cells and act as a neuronal growth factor in the brain . by contrast , gm - csf receptors are present on microglia , astrocytes , neurons , and oligodendrocytes . the blockade of gm - csf receptors with anti - gm - csf antibodies suppresses microglia activity . these findings suggest that the reduction of microglia activity by gm - csf receptor blockade may have important anti - inflammatory effects and implications for therapy . further investigation of the role of gm - csf in immune responses and neurodevelopment in autism is warranted . the studies published to date that have explored possible cytokines abnormalities in autism have been limited by small sample sizes , study design issues , and conflicting results . although many inconsistencies and controversies exist within these studies , the available data indicate a possible relationship between cytokine alterations and autism . however , systematic investigations of the neuroimmunological factors in autism are needed and will provide a better understanding of the underlying mechanisms responsible for the pathogenesis of autism , thus improving diagnosis and therapy .

autism is a disorder of neurobiological origin characterized by problems in communication and social skills and repetitive behavior . after more than six decades of research , the etiology of autism remains unknown , and no biomarkers have been proven to be characteristic of autism . a number of studies have shown that the cytokine levels in the blood , brain , and cerebrospinal fluid ( csf ) of autistic subjects differ from that of healthy individuals ; for example , a series of studies suggests that interleukin-6 ( il-6 ) , tumor necrosis factor- ( tnf- ) , and interferon- ( ifn- ) are significantly elevated in different tissues in autistic subjects . however , the expression of some cytokines , such as il-1 , il-2 , transforming growth factor- ( tgf- ) , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) , is controversial , and different studies have found various results in different tissues . in this review , we focused on several types of proinflammatory and anti - inflammatory cytokines that might affect different cell signal pathways and play a role in the pathophysiological mechanism of autistic spectrum disorders .

1. Introduction 2. Cytokines and Autism 3. Conclusion

autism spectrum disorders ( asd ) are a complex group of severe neurodevelopmental disorders that affect over 1% of children in the united states . typical symptoms of autism include impairments in social interaction , deficits in verbal and nonverbal communication , and repetitive behaviors and restricted interests . although the exact etiology of the disorder has not been identified , a link between altered immune responses and asd was first recognized nearly 40 years ago . neurobiological studies in asd have highlighted pathways involved in neural development , synapse plasticity , structural brain abnormalities , cognition , and behavior . in addition , abnormalities in the cellular immune response have also been reported in children with autism ; in particular , reduced cytotoxic activity and elevated levels of selected proinflammatory cytokines produced by peripheral blood mononuclear cells , such as tumor necrosis factor ( tnf- ) and il1 , have been shown to disrupt neurodevelopment [ 3 , 4 ] . cytokines are polypeptides that are 825 kda in size and include interleukins , chemokines , interferons , tumor necrosis factors ( tnfs ) , and growth factors . cytokines have been shown to regulate the growth and cell proliferation of neuronal tissue and to modulate host responses to infection , injury , inflammation , and diseases of uncertain etiology . the dynamic expression of different cytokines can modify immune system function ; for example , increased levels of interferon - gamma ( ifn- ) and il-12 can induce inflammation , whereas increased production of tgf- can negatively regulate inflammation . a number of recent studies have demonstrated that the levels of various inflammatory cytokines differ in the blood mononuclear cells , serum , plasma , brain tissue , and cerebrospinal fluid of autistic subjects compared with normal subjects , which might impair immune capacity in the central nervous system ( cns ) and stimulate the production and activation of microglia in the brain [ 7 , 8 ] . active microglia can induce the production of pro - inflammatory cytokines such as il-1 , il-6 , and tnf- , which are typically intended to prevent further damage to brain tissue . microglia cell activation is a prominent feature of autism , and there is a complex interaction between microglial cells and cytokines . in this review , we focused on several types of proinflammatory and anti - inflammatory cytokines that might affect different cell signal pathways and play a role in the pathogenic mechanism that might be responsible for autism ( table 1 ) . interleukin-1 ( il-1 ) consists of two different proteins , il-1 and il-1 , which exert their proinflammatory effects by binding to type - i il-1 receptors ( il-1 ri ) , whereas the type ii il-1 receptor ( il-1rii ) is thought to act as a decoy receptor that does not participate in active il-1 signaling [ 9 , 10 ] . il-1 is produced , processed , and secreted from activated immune cells and plays a major role in the initiation of local and systemic inflammatory processes . signaling mediated by il-1ri can activate the myeloid differentiation response gene 88 ( myd88 ) and tumor necrosis factor - associated factor 6 ( traf6 ) , leading to the activation of a number of different kinases , including il-1 receptor associated kinases 1 and 4 ( irak1 , irak4 ) , transforming growth factor -activated kinase ( tak ) , and members of the mitogen activated protein kinase ( mapk ) cascade , including p38 , c - jun n - terminal kinases ( jnk ) , and extracellular signal - regulated kinases 1/2 ( erk1/2 ) . il-1 has also been shown to play a key role in mediating severe placental damage and neurodevelopmental anomalies in offspring [ 15 , 16 ] . however , these studies did not evaluate the social behavior of offspring , so further study is needed to determine whether there is a link between the development of autism and elevated il-1 levels . x - linked interleukin-1 receptor accessory protein like-1 ( il1rapl1 ) is a member of the interleukin-1 receptor family and is similar to the interleukin-1 accessory proteins . pavlowsky and colleagues also reported a novel partner of il1rapl1 , psd-95 , a major scaffold protein of excitatory synapses , and showed that il1rapl1 regulates the dendritic spine number and psd-95 localization to synapses . interleukin-2 ( il-2 ) is a well - known cytokine that plays an important role in multiple immunoregulatory functions related to t - cells in peripheral and cns [ 20 , 21 ] . the il-2 receptor consists of three subunits , il-2r , ( il-2r ) , and ( il-2r ) , which can deliver biochemical signals to the cell interior . they affect different signal pathways , such as the mapk pathway , the phosphoinositide 3-kinase ( pi3k ) pathway , and the jak - stat pathway . there is a large body of literature indicating that il-2 may be involved in cns development , normal brain physiology , and homeostatic repair mechanisms as well as brain dysfunction and neurodegenerative processes . for instance , it has been shown that prolonged exposure to il-2 in cancer patients can result in cognitive dysfunction , altered behavior , and other negative neuropsychiatric side effects . in addition , il-2 levels have been found to be elevated in the postmortem brains of patients with alzheimer 's disease compared with normal control subjects . however , more recent studies have reported that il-2 levels did not differ significantly between subjects with autism and control groups [ 25 , 26 ] . il-6 is characterized as a 26 kda glycoprotein that can trigger cellular responses that mediate inflammation , neurogenesis , gliogenesis , cell growth , cell survival , myelination , and demyelination in the cns [ 28 , 29 ] . ligand binding of this protein complex results in the homodimerization of gp130 and activates multiple signaling mechanisms , including jak / stat , pi3k / akt , and the ras / raf / mapk pathways [ 3134 ] . recently , the role of maternal immune activation ( mia ) has become a hot topic in autism research . several groups have found that the developing fetus 's exposure to maternal cytokines precipitates neurological , immunological , and behavioral abnormalities in the offspring [ 35 , 36 ] . in our laboratory , we found that mice with elevated il-6 in the brain display many autistic features , including impaired cognitive abilities , learning deficits , abnormal anxiety traits , and habituations and decreased social interactions . we also examined the development of synapses and found that il-6 elevation altered excitatory and inhibitory synaptic formations , disrupted the balance of excitatory / inhibitory synaptic transmissions , and resulted in an abnormal change in the shape , length and distributing pattern of dendritic spines . hsiao and patterson confirmed that mia elevates il-6 protein and mrna expression in the placenta and found that maternally derived il-6 mediates jak / stat3 pathway activation specifically in the spongiotrophoblast layer , a fetal compartment of the placenta , which results in the expression of acute - phase genes . furthermore , hsiao and patterson also demonstrated an il-6-dependent dysregulation of the growth hormone / insulin - like growth factor ( gh - igf ) axis in mia placentas that was characterized by decreased levels of gh and igf1 mrna with corresponding decreases in placental igf1 and igf - binding protein 3 ( igfbp3 ) . however , vargas ' group demonstrated that il-6 was increased in autistic brains . in our recent studies , we also found that il-6 was significantly increased in the cerebella of autistic subjects . the cerebellum was suggested as a main focus of neuroinflammation in autism , and the selective vulnerability of the purkinje cells may play a role in the etiopathogenesis of autism . in further studies , we investigated how il-6 affects neural cell development and function by transfecting cultured mouse cerebellar granule cells with an il-6 viral expression vector . these findings suggest that il-6 not only plays an important role in the etiology of autism , but may provide a potential biological marker that enables the early diagnosis of the disorder and earlier therapeutic intervention ( figure 1 ) . because il-6 can attenuate abnormal behavior caused by mia , it might be a potential therapy target in the future . the chemokines are a family of small cytokines that play an important role in the development of lymphocytes , including their recruitment and trafficking to specific tissue compartments . in addition to acting as key chemotactic factors in the immune system , chemokines and their receptors are critical for dictating the movement of leukocytes into the cns in both healthy and diseased individuals . chemokines have been shown to regulate neuronal cell migration , proliferation , and neuronal cell differentiation and are involved in the communication between neurons and microglia [ 43 , 44 ] . in the cns , mcp-1 can modulate the recruitment of myeloid cells to injury or inflammation sites , and it is increased in brain in instances of ischemia , alzheimer 's disease , and experimental autoimmune encephalomyelitis . different groups have measured mcp-1 levels in the brain , csf , and plasma of individuals with autism and found that mcp-1 was elevated in individuals with autism . abdallah and colleagues examined the chemokine levels in amniotic fluid ( af ) samples from individuals diagnosed with autism and controls . osteopontin ( opn ) is a 60 kda phosphoprotein that plays different roles , including initiating inflammation , cell adhesion , chemotaxis , immune responses , and protection against apoptosis , depending on its intracellular or extracellular localization . opn is also involved in regulating chronic inflammatory and autoimmune diseases , including multiple sclerosis ( ms ) , rheumatoid arthritis , inflammatory bowel disease , systemic lupus erythematosus , and type i diabetes [ 5053 ] . recently , studies have demonstrated that opn also plays a role in the development of neurodegenerative diseases , such as parkinson 's and alzheimer 's disease . however , opn can trigger neuronal toxicity and death in some contexts and can function as a neuroprotectant in others . the authors found that the autistic children had significantly higher serum opn levels compared with the healthy controls , and the autistic children 's serum opn levels had significant positive correlations with their childhood autism rating scale ( cars ) scores . at present however , a number of studies have reported abnormal chemokine production in the brain , csf , and/or plasma of autistic individuals , suggesting that chemokines might be involved in aberrant neuronal development that could lead to altered early brain development and function . it is a member of a group of cytokines that stimulate the acute phase reaction . tnf- is produced mainly by activated macrophages ( m1 ) , although it can be produced by other cell types , such as cd4 + lymphocytes and nk cells . their study showed that pbmcs activated by lps produced higher levels of tnf- , il-1 , and/or il-6 in most autistic children ( 83.1% ) compared with the control group . the investigators concluded that a majority of the autistic children in the group , especially those with increased tnf- , exhibited excessive or poorly regulated innate immune responses . ( 2007 ) detected elevated tnf- in the cerebrospinal fluid of autistic children . in 2009 , our lab found that tnf- was significantly increased in the brains of autistic subjects . furthermore , ashwood and colleagues used polyhydroxyalkanoates ( pha ) and tetanus to stimulate pbmcs from autistic subjects and controls to compare group - associated cellular responses . nf-b has also been shown to be aberrantly expressed in the orbitofrontal cortex in patients with autism , which indicates that nf-b could be part of a putative molecular cascade leading to inflammation in brain regions associated with the behavioral and clinical symptoms of autism . however , our laboratory results show that the expression of nf-b ( p65 ) and the phosphorylation / activation of nf-b ( p65 ) at ser536 are not significantly changed in the cerebellum and cortex of both autistic subjects and btbr mice in an autism model . these findings imply that nf-b may be involved in the abnormal inflammatory response processes suggested in autistic brain but do not play an important part . our results suggest that tnf- might affect the progress of autism through another pathway , such as the mapk / jnk pathway . ifn- is critical for innate and adaptive immunity against viral and intracellular bacterial infections , and it plays a role in tumor control . aberrant ifn- expression is associated with a number of autoinflammatory and autoimmune diseases . in 1996 , singh found significantly elevated ifn- levels in the plasma of 20 autistic children compared with 20 healthy controls . recently , our laboratory also demonstrated that the concentration of ifn- was significantly increased in the brains of autistic subjects compared with normal control subjects . ( 2004 ) demonstrated that the plasma levels of ifn- did not differ between the autistic group and control group , but they did correlate positively with plasma nitric oxide measures in the autistic group . these findings support the hypothesis that ifn- is involved in mia and plays a role in the pathologic mechanism that may be responsible for autism . it plays an important role in autoimmune diseases such as myasthenia gravis - related ophthalmoparesis , autoimmune diabetes , and ms [ 6770 ] . considering the importance of tgf1 in immune response and nervous system development , a number of studies have investigated whether tgf-1 alters in autistic subjects . however , decreased tgf-1 levels have been observed in the plasma of autistic individuals [ 67 , 72 ] . in an animal study , depino and colleagues found that overexpression of tgf-1 during postnatal can lead to asd - related behaviors , such as decreased social interaction , increased self - grooming , and depression - related behaviors . however , adult hippocampal levels of murine tgf-1 were reduced in animals that were injected with adtgf at pd14 compared with adbgal - injected mice . in addition , the study found that asd - related behaviors correlate with the long - term downregulation of tgf-1 and il-6 expression in the dentate gyrus and with decreases in the mrna levels of the synaptic protein neuroligin 3 and the number of reelin - positive neurons in the dentate gyrus . conversely , chronic expression of tgf-1 during adulthood leads to transient opposite effects on abnormal social behaviors : it increases social interaction and decreases repetitive behavior . these results indicate that hippocampal tgf-1 plays an important role in the programming and modulation of social interaction , repetitive behavior , and depression - related behavior . egf is a growth factor that stimulates cell growth , proliferation , and differentiation by binding to its receptor egfr . these results suggest a possible role of egf in the pathogenesis of autism . they found that the serum egf levels in the subjects with high - functioning autism were significantly lower compared with those of the normal control subjects . however , there were no correlations between serum egf levels and clinical variables in the subjects with autism . by contrast , other researchers found that the serum egf levels in subjects with autism were significantly higher than those of normal control subjects . bdnf supports neuronal survival , maintains various neuronal activities , modulates neurotransmitter release , participates in neuronal plasticity , and mediates long - term potentiation and memory fixation . nelson and miyazaki reported higher bdnf levels in archived samples of neonatal blood and serum obtained from autistic subjects compared with normal controls [ 83 , 84 ] . however , in a carefully constructed large - sample study , the results demonstrated that the mean levels of bdnf were significantly lower in the serum of 0- to 9-year - old autistic children compared with age - matched healthy controls or teenagers or adults . our study found that the concentrations of bdnf and bcl2 were reduced in the frontal cerebral cortex of autistic subjects compared with age - matched controls . we also found that the expression and activation of akt kinase , which regulates bcl2 , are significantly decreased in the autistic brain . we thus reason that the downregulation of the bdnf - akt - bcl2 antiapoptotic signaling pathway in the autistic brain could be one of the underlying mechanisms responsible for the pathogenesis of autism . granulocyte - macrophage colony - stimulating factor ( gm - csf ) is a cytokine that regulates the survival , proliferation , differentiation , and functional activity of many myeloid hemopoietic cells ; it also regulates dendritic cell and t - cell function . in this study , the in vitro stimulation of pbmc with pha and tetanus was used to compare group - associated cellular responses . the results showed that gm - csf , tnf- , and il-13 production were significantly increased , whereas il-12p40 was decreased following pha stimulation in autism relative to td controls . in addition , the study also found that induced cytokine production was associated with altered behaviors in autistic children : increased proinflammatory or t(h)1 cytokines were associated with greater impairments in the core features of autism and more aberrant behaviors . by contrast , the production of gm - csf and t(h)2 cytokines was associated with better cognitive and adaptive function . our laboratory also found that gm - csf was elevated in the brains of autistic patients compared with controls . however , a study by manzardo 's group showed that the gm - csf levels were significantly lower in the plasma of children with autism compared with unrelated siblings without autism . gm - csf can upregulate neuronal receptors on cd34 + hematopoietic stem progenitor cells to enhance responses to neurotransmitters , stimulate neurite growth , and promote axonal regeneration [ 8991 ] . gm - csf can also promote neuronal differentiation of adult neuronal stem cells and act as a neuronal growth factor in the brain . by contrast , gm - csf receptors are present on microglia , astrocytes , neurons , and oligodendrocytes . the blockade of gm - csf receptors with anti - gm - csf antibodies suppresses microglia activity . these findings suggest that the reduction of microglia activity by gm - csf receptor blockade may have important anti - inflammatory effects and implications for therapy . further investigation of the role of gm - csf in immune responses and neurodevelopment in autism is warranted . the studies published to date that have explored possible cytokines abnormalities in autism have been limited by small sample sizes , study design issues , and conflicting results . although many inconsistencies and controversies exist within these studies , the available data indicate a possible relationship between cytokine alterations and autism . however , systematic investigations of the neuroimmunological factors in autism are needed and will provide a better understanding of the underlying mechanisms responsible for the pathogenesis of autism , thus improving diagnosis and therapy .